2017 Publications



247.Scheller, J.S.; Irvine, G.W.; Wong, D.L.; Hartwig, A.; Stillman, M.J..(2017) "Stepwise copper(I) binding to metallothionein: A mixed cooperative and non-cooperative mechanism for all 20 copper ions" Metallomiccs, in press – DOI: 10.1039/C7MT00041C

 


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Abstract (247)

Scheller, J.S.; Irvine, G.W.; Wong, D.L.; Hartwig, A.; Stillman, M.J..(2017) "Stepwise copper(I) binding to metallothionein: A mixed cooperative and non-cooperative mechanism for all 20 copper ions" Metallomiccs, in press – DOI: 10.1039/C7MT00041C

Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ion chaperone, metallothionein (MT) has been proposed as key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biological function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and emission spectroscopy, we report that the Cu(I) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative Kf-values for the binding of 1 to 20 Cu(I) to the 20 cysteines of MT were obtained from computational simulation of the experimental mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu-SCYS clusters, as a function of Cu(I) loading. These data provide the first overall sequence for Cu(I) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu4SCYS-6, followed by Cu6SCYS-9 (ß), then a second Cu4SCYS-6 (a), and finally Cu7SCYS-x (a) (x= up to 11). Upon further addition of Cu(I), a mixture of species is formed in a non-cooperative mechanism, saturating the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu6SCYS-9 formed solely in the N-terminal ß-domain, as well as confirming the existence of the presumed Cu4SCYS-6 cluster in the a-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MTspeciation that resulted in specific emission and CD spectral properties.

 






246. Wong, D.L.; Zhang, A.; Faponle, A.S.; de Visser, S.P.; Stillman, M.J.(2017) "Glutathione binding to dirhodium tetraacetate: a spectroscopic, mass spectral and computational study of an anti-tumour compound" Metallomics, in press DOI: 10.1039/C7MT00040E

 


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Abstract (243d)

Wong, D.L.; Zhang, A.; Faponle, A.S.; de Visser, S.P.; Stillman, M.J.(2017) "Glutathione binding to dirhodium tetraacetate: a spectroscopic, mass spectral and computational study of an anti-tumour compound" Metallomics, in press DOI: 10.1039/C7MT00040E

Glutathione (?-L-glutamyl-L-cysteinyl-glycine) is a ubiquitous tripeptide found in all plants and animals. Glutathione has key roles as a metallochaperone and as a cellular thiol involved in metabolism. Little is known about how glutathione interacts with organometallic compounds in vivo. Here, we report the reactions of glutathione in vitro with dirhodium(II) tetraacetate (tetrakis(µ-acetato)dirhodium(II), Rh2(OAc)4), a compound with anti-tumour properties. Electrospray ionization mass spectrometry, UV-visible absorption and circular dichroism spectroscopic methods were used to determine the stoichiometries and optical properties of the final conjugate. Computational analyses were used to predict the binding modes of glutathione to the Rh2(OAc)4, and report on the orbital assignments for the resulting products. We explored the competition by GSH for methionine-bound axial sites on Rh2(OAc)4 to investigate the use of weak thioether to protect its cellular-based anti-cancer activity. Our study highlights the important role that axial ligation would play in deactivating or significantly decreasing the efficacy of this bimetallic anti-tumor drug. The computational data explain the stability of the mono-adduct and the appearance of new absorption bands in the UV region including retention of the Rh-Rh single bond. Additionally, these data show that glutathione can effectively disable the potency of these metallo-drugs through orbital overlap of the entire Rh-Rh core as a result of the strong binding. Electronic absorption spectroscopy, mass spectrometry and computational analysis are a powerful combination in understanding possible chemical reactions in vivo and this information can be used to synthetically tune dirhodium complexes for use in the fight against cancer.

 






245. Irvine, G.W.; Stillman, M. J.; “Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins.” Int. J. Mol. Sci. 2017, 18(5), 913; doi:10.3390/ijms18050913

 


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Abstract (243c)

Irvine, G.W.; Stillman, M. J.; “Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins.” Int. J. Mol. Sci. 2017, 18(5), 913; doi:10.3390/ijms18050913

Structural information regarding metallothioneins (MTs) has been hard to come by due to its intrinsic fluxionality in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent ESI-MS and step-wise "snapshot" ESI-MS we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation. Keywords: paper-based biosensor; metallothionein; arsenic; mercury; green chemistry; screen-printed electrode

 






243b. Irvine, G.W.; Tan, S.N.; Stillman, M.J.(2017) "A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury." Biosensors, 7, 14, pp1-11. DOI:10.3390/bios7010014

 


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Abstract (243b)

Irvine, G.W.; Tan, S.N.; Stillman, M.J.(2017) "A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury." Biosensors, 7, 14, pp1-11. DOI:10.3390/bios7010014

Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor. Keywords: paper-based biosensor; metallothionein; arsenic; mercury; green chemistry; screen-printed electrode

 





243a. Irvine, G.W.; Tan, S.N.; Stillman, M.J.(2017) "A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury." Biosensors, 7, 14, pp1-11. DOI:10.3390/bios7010014

 


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Abstract (243)

Irvine, G.W.; Tan, S.N.; Stillman, M.J.(2017) "A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury." Biosensors, 7, 14, pp1-11. DOI:10.3390/bios7010014

Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor. Keywords: paper-based biosensor; metallothionein; arsenic; mercury; green chemistry; screen-printed electrode

 



242. Irvine, G.W.; Santolini, M.; Stillman, M.J. (2017) “Selective cysteine modification of metal-free human metallothionein 1a and its isolated domain fragments: solution structural properties revealed via ESI-MS.” Protein Science DOI: 10.1002/pro.3139

 

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Abstract (242)

Irvine, G.W.; Santolini, M.; Stillman, M.J. (2017) “Selective cysteine modification of metal-free human metallothionein 1a and its isolated domain fragments: solution structural properties revealed via ESI-MS.” Protein Science DOI: 10.1002/pro.3139.

Abstract: Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (a with 11 Cys and ß with 9), was analyzed in its metal-free form by selective, covalent Cys modification coupled with ESI-MS. The modification profiles of the isolated ß- and a-fragments reacted with p-benzoquinone (Bq), N-ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI-mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.

 



241. Takashina, A.; Tiedemann, M.T.; Unno, M; Yamaguchi, T; Stillman, M.J.; Kohzuma, T (2017) "The pH dependent Protein Structure Transitions and Related Spin State Transition of Cytochrome c' from Alcaligenes xylosoxidans NCIMB 11015". Bull. Chemical Society of Japan, 90, #2, 169-177. DOI: 10.1246/bcsj.20160316. "Selected Article" of issue #2.

 

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Takashina, A.; Tiedemann, M.T.; Unno, M; Yamaguchi, T; Stillman, M.J.; Kohzuma, T. (2016) "The pH dependent Protein Structure Transitions and Related Spin State Transition of Cytochrome c' from Alcaligenes xylosoxidans NCIMB 11015". Bull. Chemical Society of Japan, 90, #2, 169-177. DOI: 10.1246/bcsj.20160316. SELECTED ARTICLE OF ISSUE #2.

Abstract: The unusual magnetic/spectroscopic properties of Cytochrome c' (Cyt c') have been discussed, especially about the possibility of a quantum mechanically mixed spin configuration of heme Fe(III). Here, four unique spin species were identified from the magnetic circular dichroism (MCD) spectra of Cyt c' from Alcaligenes xylosoxidans (AxCyt c'). The electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic data showed the overall conformation of AxCyt c' was unchanged, in complete contrast to the drastic changes in the heme MCD spectra over the range of pH 3.5 and 11.8. The pH dependency of ESI-MS, electronic absorption, MCD, and CD spectroscopic properties of AxCyt c' enabled us to reveal the undiscovered correlation between the protein-folding state and the electronic structure of the active site as a function of pH. The mechanism of alkaline spin state transition through the rearrangement of the hydrogen-bonding linkage between Helix C and D is also proposed on the basis of atomic resolution crystal structure analyses (A. Takashina, M. c' from Alcaligenes xylosoxidans (AxCyt c') is 184 mV vs. Unnno, T. Kohzuma, Chem. Lett. 44 (2015) 268-270).

 



2016 Publications

240. Yamamoto, S.; Zhang, A.; Stillman, M.J.; Kobayashi, N.; Kimura, M. (2016) "Low symmetry Omega-shaped zinc phthalocyanine sensitizers having a panchromatic light-harvesting property for dye sensitized solar cells". Chemistry: A European Journal, 2016, 22, 18760-18768. DOI: 10.1002/chem.201603705

 

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Yamamoto, S.; Zhang, A.; Stillman, M.J.; Kobayashi, N.; Kimura, M. (2016) "Low symmetry Omega-shaped zinc phthalocyanine sensitizers having a panchromatic light-harvesting property for dye sensitized solar cells" Chemistry: A European Journal, 2016, 22, 18760-18768. DOI: 10.1002/chem.201603705

Abstract: Two low-symmetry phthalocyanines (Pcs) substituted with thiophene units at the non-peripheral (a) and peripheral (b) positions were synthesized and their optical, electronic-structure, and electrochemical properties were investigated. The substitution of thiophene units at the a positions of the phthalocyanine skeleton resulted in a red shift of the Q band and significantly modified the molecular-orbital electronic distributions just below the HOMO and just above the LUMO, with distortion of the typical Gouterman four-orbital arrangement of MOs. Two amphiphilic W-shaped ZnPcs (aPcS1 and aPcS2) bearing a p-conjugated side chain with an adsorption site at an a position of the Pc macrocycle were synthesized as sensitizers for dye-sensitized solar cells (DSSCs). The absorption spectra of aPcS1 and aPcS2 showed red shifted Q bands and a broad band from 350 to 550 nm assignable to the intramolecular charge-transfer transition from the ZnPc core to the side chains. Time-dependent DFT calculations provided a clear interpretation of the effect of the thiophene conjugation on the typical phthalocyanine core p MOs. Compound aPcS1 was used as a light-harvesting dye on a TiO2 electrode for a DSSC, which showed a panchromatic response in the range 400–800 nm with a power conversion efficiency of 5.5% under one-sun conditions

 



M16. Wong, D.L.; Merrifield-MacRae, M.; Stillman, M.J., (2016) "Lead(II) Binding in Metallothioneins", Metal Ions in Life Sciences (2017), 17 (Lead: Its Effects on Environment and Health) in press.

Abstract (M16)

Wong, D.L.; Merrifield-MacRae, M.; Stillman, M.J., (2016) "Lead(II) Binding in Metallothioneins", Metal Ions in Life Sciences (2017), 17 (Lead: Its Effects on Environment and Health) in press.
Abstract for #M16

Contents ABSTRACT 3

  • 1.0 INTRODUCTION 4
  • 1.1 Essential metals and toxic metals 4
  • 1.2 Metallothioneins – Overview 5
  • 1.3 Mammalian Metallothioneins 6
  • 1.4 Metallothioneins from Non-Mammalian Sources – Snail, Fucus Vesiculosus, and Yeast 7
  • 1.5 Reactions of Metallothioneins with Essential Metals – Zinc and Copper 8
  • 1.5.1 Specific Reactions of Metallothionein with Zinc 10
  • 2.0 METALLOTHIONEIN AND TOXIC METALS 2.1 Reactions of Metallothioneins with Cadmium 11
  • 2.2 Reactions of Metallothioneins with Arsenic 13
  • 2.3 Reactions of Metallothioneins with Mercury 15
  • 3.0 METALLOTHIONEIN AND LEAD 3.1 Overview of Papers and their General Results - a Summary 17
  • 3.2 UV-visible and Circular Dichroism Spectroscopic Data for Pb-MT 18
  • 3.3 ESI-Mass Spectral Data for Pb-MT 19
  • 3.4 Thermodynamic Studies for Pb-MT 20
  • 3.5 X-ray-Absorption Data for Pb-MT 21
  • 3.6 207Pb NMR Data for Pb-MT 22
  • 3.7 Molecular Modelling for Pb-MT 23
  • 4.0 CONCLUSIONS 27
  • List of Common Abbreviations 29
  • Acknowledgements 36
  • References 36 ABSTRACT Heavy metal exposure has long been associated with metallothionein (MT) regulation and its functions. MT is a ubiquitous, cysteine-rich protein that is involved in homeostatic metal response for the essential metals zinc and copper, as well as the detoxification of heavy metals, the most commonly proposed being cadmium. MT binds in vivo to a number of metals in addition to zinc, cadmium and copper, for example, bismuth. In vitro, metallation with a very wide range of metals bind, especially mercury, arsenic, and lead has been reported using a variety of analytical methods. To fully understand MT and its role with lead metabolism, we must explore describe how MT interacts with the wide variety of metals that bind in vitro. In general, affinity to the metal-binding cysteine residues of MT follows that of metal binding to thiols: Zn(II) < Pb(II) < Cd (II) < Cu(I) < Ag(I) < Hg(II) < Bi(III) [1]. To introduce metallothionein metal-binding, we will explore MT’s interactions with metals long known as toxic, particularly, Cd(II), Hg(II), and As(III) and also xenobiotic metals, and how these metal binding studies complement those of lead-binding. Lead’s effects on an organisms’ physiological functions are not fully understood, but it is known that chronic exposure inflicts pernicious anemia and brain developmental issues, especially upon children who are more vulnerable to its toxic effects. Understanding the interaction of lead with metallothioneins throughout the biosphere, from bacteria, to algae, to fish, to humans, is important in determining pathways for lead to enter and damage physiologically significant protein function.

     



  • 239.Visser, SP de, Stillman, MJ (2016) "Challenging Density Functional Theory Calculations with Hemes and Porphyrins", International Journal of Molecular Sciences vol 17 (issue 4), pages 519: 1-25 doi:10.3390/ijms17040519.

     

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    Abstract (239)

    Table of Contents Graphic:"Challenging Density Functional Theory Calculations with Hemes and Porphyrins", International Journal of Molecular Sciences vol 17 (issue 4), pages 519: 1-25 doi:10.3390/ijms17040519 TOC for 239

    Visser, SP de, Stillman, MJ (2016) "Challenging Density Functional Theory Calculations with Hemes and Porphyrins", International Journal of Molecular Sciences vol 17 (issue 4), pages 519: 1-25 doi:10.3390/ijms17040519.
    Abstract for #239

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol-1). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties.

     



    238. Wong, DL, Stillman, MJ. (2016) "Destructive Interactions of Dirhodium(II) Tetraacetate with beta Metallothionein rh1a: Insight into activity associated with drug resistance." Chemical Communication. 52, 5698 - 5701 DOI: 10.1039/C5CC10319C

     

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    Abstract (238)

    Table of Contents Graphic: "Destructive Interactions of Dirhodium(II) Tetraacetate with beta Metallothionein rh1a: Insight into activity associated with drug resistance." Chemical Communication. 52, 5698 - 5701 DOI: 10.1039/C5CC10319C" TOC for 238

    Wong, DL, Stillman, MJ. (2016) "Destructive Interactions of Dirhodium(II) Tetraacetate with beta Metallothionein rh1a: Insight into activity associated with drug resistance." Chemical Communication. 52, 5698 - 5701 DOI: 10.1039/C5CC10319C
    Abstract for #238

    Metal-based therapeutics are vital tools in medicine. Metal-chelating proteins can dramatically decrease drug efficacy. Dirhodium(II) tetraacetate, a potential anticancer compound, binds in vitro to 8 cysteines of the human Metallothionein 1a ß-fragment. Electrospray ionization mass spectrometry shows that the final product is the Rh24+ core encapsulated by the apo-ß-MT protein.

     



    237. Irvine, GW, Stillman, MJ. (2016) Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites. J Inorganic Biochemistry. CanBIC-5 Special Issue, May. doi:10.1016/j.jinorgbio.2016.03.001

     

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    Table of Contents Graphic for " "Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites." J. Inorganic Biochemistry, 158, 115-121doi:10.1016/j.jinorgbio.2016.03.001

    Abstract (237)

    Table of Contents Graphic: " "Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites." J. Inorganic Biochemistry, 158, 115-121; doi:10.1016/j.jinorgbio.2016.03.001" TOC for 237

    Irvine GW, Stillman MJ. (2016) "Cadmium binding mechanisms of isolated domains of human MT isoform 1a: Non-cooperative terminal sites and cooperative cluster sites." J. Inorganic Biochemistry, 158, 115-121; doi:10.1016/j.jinorgbio.2016.03.001
    Abstract for #237

    A number of biological functions have been ascribed to mammalian metallothioneins (MTs) including zinc and copper homeostatic regulation, redox activity and detoxification of heavy metals like cadmium and mercury. It is unclear how these small, fluxional, cysteine rich proteins manage to play each of these vital roles. Using a combination of cadmium and pH titrations of the isolated domains of human MT isoform 1a monitored by electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report the pH dependencies that control metal binding mechanisms of these domains. We report that the a-domain mechanism is driven by the cooperative formation of the Cd4MT cluster at slightly acidic pH (=6.9) switching binding mechanisms over a physiologically relevant pH range, whereas the ß-domain metalation mechanism is dominated by terminal coordination of cadmium in a non-cooperative manner above pH 5.5. These results suggest that, in some acidic sub-cellular compartments, cadmium could be sequestered in the a-domain, leaving zinc or copper bound in the ß-domain and available for donation to other metalloproteins. We propose that these results can be explained by the intrinsic nature of the two domains, the four-metal a-cluster being more resistant to proton attack due to its lower charge-to-metal ratio, compared with the three-metal ß-domain.

     



    236. Irvine GW, Pinter TB, Stillman MJ. (2016) “Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion.” Metallomics (see cover art) Metallomics 8 (1), 71-81. One of the top 20 downloaded papers from Metallomics in 2016.

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    Abstract (236)

    Table of Contents Graphic: "Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion" TOC for 236

    Gordon W. Irvine, Tyler B. J. Pinter and Martin J. Stillman(2016)
    "Defining the metal binding pathways of human metallothionein 1a: balancing zinc availability and cadmium seclusion", Metallomics, 8 (1), 71-81; doi: 10.1021/acs.biochem.5b00912.
    Abstract for #236

    Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are found throughout Nature. This ubiquity highlights their importance in essential metal regulation, heavy metal detoxification and cellular redox chemistry. Missing from the current description of MT function is the underlying mechanism by which MTs achieve their proposed biological functions. To date, there have been conflicting reports on the mechanism of metal binding and the structures of the metal binding intermediates formed during metalation of apoMTs. The form of the metal-bound intermediates dictates the metal sequestering and metal-donating properties of the protein. Through a detailed analysis of spectral data from electrospray ionization mass spectromeric and circular dichroism methods we report that Zn(ii) and Cd(ii) metalation of the human MT1a takes place through two distinct pathways. The first pathway involves formation of beaded structures with up to five metals bound terminally to the 20 cysteines of the protein via a noncooperative mechanism. The second pathway is dominated by the formation of the four-metal domain cluster structure M4SCYS11via a cooperative mechanism. We report that there are different pathway preferences for Zn(ii) and Cd(ii) metalation of apo-hMT1a. Cd(ii) binding follows the beaded pathway above pH 7.1 but beginning below pH 7.1 the clustered (Cd4Scys11) pathway begins to dominate. In contrast, Zn(ii) binding follows the terminal, "beaded", pathway at all physiologically relevant pH (pH = 5.2) only following the clustered pathway below pH 5.1. The results presented here allow us to reconcile the conflicting reports concerning the presence of different metalation intermediates of MTs. The conflict regarding cooperative versus noncooperative binding mechanisms is also reconciled with the experimental results described here. These two metal-specific pathways and the presence of radically different intermediate structures provide insight into the multi-functional nature of MT: binding Zn(ii) terminally for donation to metalloenzymes and sequestering toxic Cd(ii) in a cluster structure.

     

    235. Laakso HA, Marolda CL, Pinter TB, Stillman MJ, Heinrichs DE. (2016) A heme-responsive regulator controls synthesis of staphyloferrin B in Staphylococcus aureus. J Biol Chem.  (2016) 291 (1), 29-40. doi: 10.1074/jbc.M115.696625. This article was selected as a Paper of the Week in January, 2016. .

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    Laakso HA, Marolda CL, Pinter TB, Stillman MJ, Heinrichs DE. (2015) A heme-responsive regulator controls synthesis of staphyloferrin B in Staphylococcus aureus. J Biol Chem. 2015 Nov 3. pii: jbc.M115.696625.
    Abstract for #235

    Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A (SA) and staphyloferrin B (SB), and it expresses transporters for many other siderophores that itself does not synthesize. Expression of genes encoding all of these systems is regulated by the Ferric Uptake Regulator, or Fur, protein. Mechanisms of fine-tuning expression of iron-regulated gene, beyond simple iron-regulation via Fur, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB-like protein that is required for expression of genes in the sbn operon from sbnD onwards. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to a DNA within the sbnC coding region. Moreover, we show that SbnI binds heme, and that heme bound SbnI does not bind DNA. Last, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.

     




    2015 Publications

    234.     Tyler B.J. Pinter and Martin J. Stillman (2015) "Kinetics of Zinc and Cadmium Exchanges between Metallothionein and Carbonic Anhydrase;, Biochemistry. 2015; 54(40): 6284-93. doi: 10.1021/acs.biochem.5b00912

    Table of Contents Graphic for "Kinetics of Zinc and Cadmium Exchanges between Metallothionein and Carbonic Anhydrase - Biochemistry. 2015 54(40): 6284-93. doi: 10.1021/acs.biochem.5b00912.

     

    Abstract (234)

    Table of Contents Graphic: "Kinetics of Zinc and Cadmium Exchanges between Metallothionein and Carbonic Anhydrase." TOC for 234

    Tyler B.J. Pinter and Martin J. Stillman (2015)
    "Kinetics of Zinc and Cadmium Exchanges between Metallothionein and Carbonic Anhydrase", Biochemistry. 2015 54(40):6284-93. doi: 10.1021/acs.biochem.5b00912.
    Abstract for #234

    The flexible coordination stoichiometry of a relatively high number of metal ions is a property unique to the metallothionein (MT) family of proteins. Mammalian MTs, for example, accommodate up to seven divalent metal ions in tetrahedral coordination geometries, using its complement of 20 cysteine ligands. The lability of the metals from these metalloclusters has been used to support the proposal of MTs acting as metal chaperones, by donating to other metal-binding proteins. The metal exchange kinetics between human MT1A and carbonic anhydrase (CA) were examined using time-dependent electrospray ionization mass spectrometry (ESI-MS). The time dependence of three different reaction conditions were studied: (i) zinc donation from partially metalated zinc-MT to apoCA; (ii) metal exchange between zinc saturated MTs and cadmium saturated CA (Cd-CA); and (iii) metal exchange between partially metalated zinc-MTs and Cd-CA. The results show that zinc donation from Zn-MTs to apo-zinc-dependent enzymes is dependent on the metal loading of the Znn-MT (where n = 1-7) and that this is a direct consequence of the increasing metal affinity for smaller values of n. Partially metalated MTs are also shown to extract cadmium from Cd-CA with significantly faster rates than metal saturated MTs and that even under zinc limiting conditions, mammalian Cd-CA would not coexist with MT. On the basis of these and previously published results, we suggest that protein-protein interactions between MT and CA facilitate metal transfers through favorable electrostatic interactions and hypothesize that the metal could be transferred between the MT and the enzyme active site using nearby metal-binding functionalities along the transfer pathway.

     



    233.     Tyler B.J. Pinter and Martin J. Stillman (2015) "Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase", Biochemical Journal Oct 16, 2015, 471 (3) 347-356; DOI: 10.1042/BJ20150676

    TOC for 233

     

    Table of Contents Graphic for "Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase - DOI: 10.1042/BJ20150676

     

    Abstract (233)

    Table of Contents Graphic: "Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase" TOC for 233

    Tyler B.J. Pinter and Martin J. Stillman (2015)
    "Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase", Biochemical Journal, in press. DOI: 10.1042/BJ20150676.
    Abstract for #233

    Mammalian metallothioneins (MTs) bind up to seven Zn2+ using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7-MTs, the seven zinc ions are coordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal ß-domain [(Zn3Cys9)3-] and C-terminal a-domain [(Zn4Cys11)3-]. We report on the competitive zinc metalation of apo-carbonic anhydrase (apo-CA) in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn-ßaMT1A (n = 0-7). The apo-CA is shown to compete effectively only with Zn2-3-ßMT and Zn4-aMT. Detailed modeling of the electrospray ionization mass spectral data has revealed the zinc binding affinities of each of the zinc binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities (log(KF)) of the isolated ß-domain were: 12.2, 11.7, and 11.4 and the four isolated a domain affinities were: 13.5, 13.2, 12.7, and 12.6. These data provide guidance in identification of the location of the strongest bound and weakest bound zinc in the intact two-domain Zn7ßaMT. The ß domain has the weakest zinc binding site and this is where zinc ions are donated from in the Zn7-ßaMT. The a-domain, with the highest affinity, binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apopeptide bundle. We propose that stabilization of the intact Zn6-MT and Zn7-MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.

     



    232.     Tyler B.J. Pinter, Gordon W. Irvine, and Martin J. Stillman (2015) "Domain selection in metallothionein 1A: Affinity controlled mechanisms of zinc binding and cadmium exchange", Biochemistry. 2015 Aug 18;54(32): 5006 - 5016 . doi: 10.1021/acs.biochem.5b00452

    TOC for 230

    Table of Contents Graphic for "Domain selection in metallothionein 1A: Affinity controlled mechanisms of zinc binding and cadmium exchange - DOI: 10.1021/acs.biochem.5b00452;

     

    Abstract (232)

    Table of Contents Graphic: "Metalation kinetics of the human a-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein" TOC for 230

    Tyler B.J. Pinter, Gordon W. Irvine, and Martin J. Stillman (2015)
    "Domain selection in metallothionein 1A: Affinity controlled mechanisms of zinc binding and cadmium exchange", Biochemistry, in press. http://dx.DOI: 10.1021/acs.biochem.5b00452.
    Abstract for #232

    Mammalian metallothioneins (MTs) are small metal-binding proteins implicated in cellular metal ion homeostasis and heavy metal detoxification. Divalent, metal-saturated MTs form two distinct domains; the N-terminal beta domain binds 3 metals using 9 Cys, and the C-terminal a domain binds 4 metals with 11 Cys. Domain selection during zinc binding and cadmium exchange to human MT1A was examined using a series of competition reactions with mixtures of the isolated domain fragments. Equivalents of zinc were added stepwise to equimolar mixtures of the metal-free fragments and monitored with electrospray-ionization mass spectrometry. Once zinc-saturated, cadmium was then added stepwise and the competitive replacement reactions monitored between the domain fragments. These experiments were carried out at two biologically significant pH conditions where MTs exist in vivo. At pH 7.4, zinc binding was essentially randomly distributed between the domains, with no specific selectivity observed. Under acidic conditions, pH 5.8, zinc binding shows increased a domain selectivity due to the preference for clustering of the a domain. There was no domain selection for cadmium exchange at pH 7.4, unlike the data for pH 5.8 where increased a domain selectivity was observed. At both neutral and acidic pHs, the metal distributions and cadmium replacement occurred essentially randomly between the two fragments, and the metal binding affinity distributions between the domain fragments are shown to be comingled and not significantly separated. Extensive and detailed modeling shows that there is little specific domain selectivity for human beta-alpha-MT1A zinc binding and cadmium exchange, rather the binding follows the order of the binding affinities that are distributed across both domains.

     



    231.     Gordon W. Irvine, Kelly E. R. Duncan, Meredith Gullons and Martin J. Stillman (2015) "Metalation kinetics of the human α-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein", Chemistry - A European Journal Volume 21, Issue 3, pages 1269 - 1279, January 12, 2015. . http://dx.doi.org/10.1002/chem.201404283

    Table of Contents Graphic for "Metalation kinetics of the human α-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein"

     

    Abstract (231)

    Table of Contents Graphic: "Metalation kinetics of the human a-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein" TOC for 230

    Gordon W. Irvine, Kelly E. R. Duncan, Meredith Gullons and Martin J. Stillman (2015) "Metalation kinetics of the human α-metallothionein 1a fragment is dependent on the fluxional structure of the apo-protein", Chemistry - A European Journal: Vol 21, Issue 3, 1269-1279, January 12, 2015.
    Abstract for #231... http://dx.doi.org/10.1002/chem.201404283
    Mammalian metallothioneins are cysteine rich metal binding proteins comprising, when fully metalated, two metal binding domains: the alpha-domain binds with M(II)4(Scys)11 stoichiometry and the beta domain binds as M(II)3(Scys)9 stoichiometry. While the fully metalated species have been widely studied, the metalation of the apoprotein is poorly understood. Key to a description of the metalation pathway(s) is the initial conformation of the apoprotein and the arrangement of the metal-coordinating cysteines prior to metalation. We report the effect of the ill-defined, globular structure of apoMT on metalation rates. In order to overcome the experimental limitations inherent in structural determinations of a fluxional protein we used a detailed analysis of the apo-?-metallothionein conformation based on the differential rate of cysteine modification with benzoquinone. The ESI-mass spectral data show the presence of two distinct conformational families: one a folded confomrational family at neutral pH and a second an unfolded family of conformations at low pH. The Cd2+ metalation properties of these two conformationally-distinct families were studied using stopped-flow kinetics. Surprisingly, the unfolded apoprotein metalated significantly slower than the folded apoprotein, a result interpreted as being due to the longer time required to fold into the cluster structure when the 4th Cd2+ binds. These results provide the first evidence for the role of the structure of the apo-aMT in the metalation reaction pathways and show that cysteine modification coupled with ESI-MS can be used to probe structure in cysteine-rich proteins.

     



    2014 Publications

    230.     Pinter, T.; Stillman, M.J. (2014) " The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A", Biochemistry , 2014, 53 (39), pp 6276–6285. DOI: 10.1021/bi5008673.

    TOC Graphic for paper 230

    Table of Contents Graphic for "The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A"

     

    Abstract (230)

    Table of Contents Graphic: "The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A" TOC for 230

    -- Abstract
    "The Zinc Balance: Competitive Zinc Metalation of Carbonic Anhydrase and Metallothionein 1A" Biochemistry , 2014, 53 (39), pp 6276–6285. DOI: 10.1021/bi5008673 The small, cysteine-rich metallothionein family of proteins is currently considered to play a critical role in the provision of metals to metalloenzymes. However, there is limited information available on the mechanisms of these fundamentally important interactions. We report on the competitive zinc metalation of apocarbonic anhydrase in the presence of apometallothionein 1A using electrospray-ionization mass spectrometry. These experiments revealed the relative affinities of zinc to all species in solution. The carbonic anhydrase is shown to compete efficiently only against Zn5-7MT. The calculated equilibrium zinc binding constants of each of the seven zinc metallothionein 1A species ranged from a high of (log(KF)) 12.5 to a low of 11.8. The eight equilibrium constants connecting the 10 active species in competition for the zinc were modeled by fitting the KFs of the eight competitive bimolecular reactions to the ESI-mass spectral data. These modeled Ks are shown to be experimentally connected to the metalation efficiency of the carbonic anhydrase. The series of seven metallothionein binding affinities for zinc highlight the buffering role of zinc metallothioneins that permit simultaneously zinc storage and zinc sensing. Finally, the significance of the multiple zinc binding affinities of zinc metallothionein is discussed in relation to zinc homeostasis.

     



    229.     Lin Jiang, James T. Engle, Ross A. Zaenglein,  Alex Matus,  Christopher J. Ziegler, Hong Wang,* Martin J. Stillman* "Pentacene-Fused Diporphyrins", Chem. Eur. J. 2014, 20, 13865-13870; DOI: 10.1002/chem.201404591; selected as a "hot paper".

    Pentacene porphyrins

    Table of Contents Graphic: "Pentacene-Fused Diporphyrins"






    Abstract (229)

    Table of Contents Graphic: "Pentacene-Fused Diporphyrins"

    Pentacene-Fused Diporphyrins DOI: 10.1002/chem.201404591 In this work, we report the synthesis, spectroscopic characterization and theoretical analysis of a linearly conjugated pentacene-fused porphyrin dimer and cross-conjugated quinone-fused dinaphtho[2,3]porphyrins. These multichromophoric systems display non-typical UV-visible absorptions of either porphyrins or pentacenes/quinones. UV-visible, emission and MCD spectroscopy suggest strong electronic interactions among the multichromophores in the system. DFT calculations revealed the delocalization of the HOMOs and LUMOs spanning the entire dimer and linker assembly. The pentacene-fused porphyrin dimer is significantly more stable than both the corresponding pentacene and the heptacene derivatives. The availability of these huge p-extended and electronically highly interactive multichromophoric systems promises unprecedented electronic and photophysical properties.

     




    228.     Sutherland, D.E.K.; Stillman, M.J. "Challenging Conventional Wisdom: Single Domain Metallothioneins", Metallomics (2014) DOI: 10.1039/C3MT00216K

    Table of Contents Graphic: "Single Domain Metallothioneins"




     

     

     



    Abstract (228)

    Table of Contents Graphic: "Single Domain Metallothioneins"

    Metallothioneins (MT) are a family of small cysteine rich proteins that have been implicated in a range of roles including toxic metal detoxification, protection against oxidative stress, and as metallochaperones are undoubtedly involved in the homeostasis of both essential zinc and copper. While complete details of all possible cellular functions are still unknown, it is clear that they must be directly related to both the accessibility and the metal-binding properties of the many cysteine residues in the protein. The most well studied MTs are of mammalian origin and consist of two domains: a ß-domain with 9 cysteine residues that sequesters 3 Cd2+, 3 Zn2+ or 6 Cu+ ions, and an a-domain with 11 cysteine residues that sequesters 4 Cd2+, 4 Zn2+ or 6 Cu+ ions. The key to understanding the cellular importance of MT in these different roles is in a precise description of the metallation status before and during reactions. An assessment of all possible and all biologically accessible metallation states is necessary before the functional mechanistic details can be fully determined. Conventionally, it has been considered that metal ions bind in a domain-specific and, therefore, cooperative manner, where the apparently isolated domains fill with their full complement of metal ions immediately with no discernible or measurable intermediates. A number of detailed mechanistic studies of the metallation reactions of mammalian MTs have provided significant insight into the metallation reactions. Recent results from electrospray ionization mass spectrometric studies of the stepwise metallation of the two fragments and the whole protein with Zn2+, Cd2+, As3+ and Bi3+ indicate a noncooperative mechanism of a declining series of KF’s. Of particular note are new details about the early stages of the stepwise metallation reactions, specifically the stability of partially metallated species for As3+, Cd2+, and Zn2+ that do not correspond to the two-domain model. In addition, at the other end of the coordination spectrum are the supermetallated species of MT, where supermetallation defines metallation in excess of traditional levels. It has been reported that with metal ion excess the formation of a single ‘super domain’ is possible and again this deviates from the two-domain model of MT. In both cases, these results suggest that the structural view of mammalian MT that is of two essentially isolated domains may be the exceptional case and that under the normal conditions of cellular metal-ion concentrations the two domain structure might coexist in equilibrium with various single domain, multi-metal site structures. This review specifically focuses on providing context for these recent studies and the new ideas concerning metallation prior to the establishment of domain-based clusters that these studies suggest.


    227.      Mack, J., Otaki, T., Durfee, W.S., Kobayashi, N., Stillman, M.J. (2014) "MCD spectroscopy and TD-DFT calculations of low symmetry subnaphthalocyanine analogues", J. Inorg. Biochem. DOI:10.1016/j.jinorgbio.2014.01.001
    226.     Mutter, A.C.; Norman, J.A.; Tiedemann, M.T.;(2014) Singh, S.; Sha, S.; Morsi, S.; Ahmed, I.; Stillman, M.J.; Koder, R.L. "Rational design of a zinc phthalocyanine binding protein", J. Structural Biology,185, 178-185.

    2013 Publications

    225.     Irvine, G.W.; Stillman, M.J. (2013) "Topographical analysis of As-induced folding of alpha-MT1a." Biochem. Biophys. Res. Commun. 441, 208-213. doi: 10.1016/j.bbrc.2013.10.044.

    Table of Contents Graphic: "Topographical analysis of As-induced folding of alpha-MT1a."













    Abstract (225)

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    Table of Contents Graphic: "Topographical analysis of As-induced folding of alpha-MT1a"

    Metallothionein binds multiple metals into two clustered domains. While the structure of the fully metalated protein is well known for the Cd- and Zn- containing protein, there is little known about the structures of the metal-free protein (apo-metallothionein) and even less about the partially metalated forms. However, the partially-metalated species are vitally important intermediates in the passage of the protein from translational synthesis to its homeostatic buffer or metal chaperone roles. Because multiple metals bind to metallothioneins, the partially-metalated species span a wide range depending on the metal bound.. Up to 3 As3+ bind stepwise to the a-domain fragment in a manner that allows measurement of each of the 4 species simultaneously with the number of free cysteines diminishing by 3 for every As3+ bound: apo- (11 Cys), As1- (8 Cys), As2- (5 Cys) and As3-a-MT (2 Cys). The cysteine modifier, benzoquinone (Bq), was used to determine the relative accessibility of the free cysteines in the a-MT fragment as a function of the number of As3+ bound. The effect of each As3+ was to induce folding in the protein. The ESI-MS results show that the whole protein folds significantly even when just one of the three As3+ has bound. The profile of the Bq reacting with the unbound cysteines shows effects of steric hindrance in slowing down the reaction. By freezing the reaction midway to the endpoint, the mass spectral data show the `mid-flight` concentrations of all the key species, 27 in all. Analysis of this mid-flight reaction profile allows an understanding of the topology of partially metalated MT due to varying solvent access to the unbound cysteinyl thiols and consequently, by the Bq. Significantly, the metal-free, apo-a-MT also adopts a folded structure in the presence of the As3+ even though there is no As3+ bound. This can only happen if the apo-protein wraps around other metalated proteins in solution via protein-protein interaction.


    224.     Summers, K.L.; Sutherland, D.E.K.; Stillman, M.J. (2013) "Single-Domain Metallothioneins: Evidence of the Onset of Clustered Metal Binding Domains in Zn-rhMT 1a", Biochemistry, 52, 2461-2471. DOI: 10.1021/bi400021b
    223.     Irvine, G.W.; Summers, K.L.; Stillman, M.J. (2013) "Cysteine accessibility during As3+ metalation of the alpha and beta-domains of recombinant human MT1a.". Biochem. Biophys. Res. Commun., 433, 477-483.
    M14. Book Chapter: K.L. Summers and M.J. Stillman, (2013)"Metallothioneins and Silver", pp 1390-1400, in Encyclopedia of Metalloproteins. Kretsinger, Robert H., Uversky, Vladimir N., Permyakov, Eugene A. (Eds.),Print ISBN 978-1-4614-1532-9

    2012 Publications

    222.     Sutherland, D.E.K.; Summers, K. L.; Stillman M. J. (2012) "Modeling the Zn2+ and Cd2+metalation mechanism in mammalian metallothionein 1a" Biochem. Biophys. Res. Commun. 426, 601-607: DOI 10.1016/j.bbrc.2012.08.136
    221.     Summers K. L.; Mahrok A.K.; Dryden M.D.M.; Stillman M.J. (2012) "Structural properties of metal-free apometallothioneins" Biochem. Biophys. Res. Commun. 425: 485-492. doi: 10.1016/j.bbrc.2012.07.141
    220.     Tiedemann, M.; Heinrichs, D.E.; Stillman, M.J. (2012) "The multi-protein heme shuttle pathway in Staphylococcus aureus: Isd cog-wheel kinetics", J. Am. Chem. Soc. 134, 16578-16585. DOI: 10.1021/ja305115y.

    Table of Contents Graphic: "The multi-protein heme shuttle pathway in Staphylococcus aureus: Isd cog-wheel kinetics"

    219.     Tiedemann, M.T.; Pinter, T.B.J.; Stillman, M.J. (2012) "Insight into blocking heme transfer by exploiting molecular interactions in the core Isd heme transporters IsdA-NEAT, IsdC-NEAT and IsdE of Staphylococcus aureus", Metallomics, 4, 751-760. DOI: 10.1039/C2MT20067H (featured on cover). (TOP 10 downloads in July, 2012)
    218,     Sutherland, D.E.K.; Summers, K.L; Stillman, M.J. (2012) "Noncooperative metalation of metallothionein 1a and its isolated domains", Biochemistry 51: 6690-6700. DOI: /10.1021/bi3004523,
    217.     Bohle, D.S.; Dodd, E.L.;Pinter, T.B.J.; Stillman, M.J. (2012) "Soluble diamagnetic model for malaria pigment: coordination chemistry of gallium(III) protoporphyrin-IX", Inorg. Chem., 51:10747-61. DOI: 10.1021/ic301106g
    216.     Tiedemann, M.T.; Stillman, M.J. (2012) "Heme binding to the IsdE(M78A; H229A) double mutant: Challenging unidirectional heme transfer in the Iron Regulated Surface Determinant (Isd) protein heme transfer pathway of Staphylococcus aureus", J Biol Inorg Chem; 2012 Oct;17(7):995-1007. DOI 10.1007/s00775-012-0914-z
    215.      Pinter, T.B.J.; Dodd, E.L.; Bohle, D.S.; Stillman, M.J. (2012) "Spectroscopic and Theoretical Studies of Ga(III)protoporphyrin-IX and Its Reactions with Myoglobin", Inorg. Chem. 51, 3743-3753. doi: 10.1021/ic202731g
    214.     Sutherland, D.E.K.; Willans, M.J.; Stillman, M.J. (2012) "Single domain metallothioneins: Supermetalation of human MT 1a", J. Am. Chem. Soc., 134, 3290-3299.

    2011 Publications

    213.     Kaluarachchi, H.; Siebel, J.; Kaluarachchi-Duffy, S.; Krecisz, S.; Sutherland, D.; Stillman, M.J.; Zamble, D.B. (2011) "The in vitro selectivity and in vivo specificity of the E. coli nickel metallochaperone, SlyD", Biochemistry, 50, 10666-10677.
    212.     Tiedemann, M.T.; Stillman, M.J. (2011) "Magnetic circular dichroism studies of porphyrins", J. Porphyrins Phthalocyanines, 15, 1134-1149.
    211.     Xue ZL, Mack J, Lu H, Zhang L, You XZ, Kuzuhara D, Stillman M, Yamada H, Yamauchi S, Kobayashi N, Shen Z. (2011) The synthesis and properties of free-base [14]triphyrin(2.1.1) compounds and the formation of subporphyrinoid metal complexes. Chemistry, 17, 4396-4407.
    210.     Sutherland, D.E.K.; Stillman, M.J. (2011) "The magic numbers of metallothionein", Metallomics, 3, 444-463. (Featured on the front cover).

    2010 Publications

    209.     Ngu, T.; Dryden, M.D.M.; Stillman, M.J. (2010) "Arsenic transfer between metallothionein proteins at physiological pH" Biochem. Biophys. Res. Commun. 401, 69-74.
    208.    Ngu, T.; Krecisz, S.; Stillman, M.J. (2010) "Bismuth Binding Studies to the Human Metallothionein using Electrospray Mass Spectrometry", Biochem. Biophys. Res. Commun. 396, 206-212.
    207.     Sutherland, D.E.K; Willans, M.J.; Stillman, M.J. (2010) "Supermetallation of the β domain of human metallothionein 1a", Biochemistry, 49, 3593-3601

    206.     T. Ngu;, J.A. Lee; T.B.J. Pinter; M.J. Stillman (2010) "Arsenic-Metalation of Triple Domain Human Metallothioneins: Support for the Evolutionary Advantage and Interdomain Metalation of Multiple-Metal-Binding Domains", J. Inorg. Biochem., 104, 232-244.

    Abstract (206)

    Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain ba motif, while in lower life forms; MT often consists of only a single domain structure. There are also unusual MTs from American oysters that consist of multiple domains (from one to four ? domains). This report details the study of the As3+-metalation to two different concatenated triple?? and ? domain MTs using time-resolved electrospray ionization mass spectrometry (ESI MS). Analysis of the ESI MS data show thatbbb-human MT and aaa-human MT bind As3+ in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the kinetic reactions for the As3+-metalation of both triple-domain MTs from zero and up to nine (bbb) or ten As3+ ions (aaa). The rate constants for the As3+-metalation are reported for both the bbb and aaa-human MT. At room temperature (298 K) and pH 3.5, the sequential individual rate constants, kn (n = 1-9) for the As3+-metalation of bbb-hMT starting at k1 are 40, 36, 37, 26, 27, 17, 12, 6, and 1 M-1s-1; while at room temperature (298 K) and pH 3.5, the sequential individual rate constants, kn (n = 1-10) for the As3+-metalation of aaa-hMT starting at k1 are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6 M-1s-1. The trend in the rate constant values reported for these two triple domain MT proteins supports our previous proposal that the rate constant values are proportionally related to the total number of equivalent binding sites. The rate of binding for the 1st As3+ is the fastest we have determined for any MT to date. Additionally, we show for the first time for any MT species, that interdomain metalation occurs in the binding of the 10th and 11th As3+ to aaahMT.

    205.     F. Xie; D.E.K. Sutherland; M.J. Stillman; M.Y. Ogawa (2010) "Cu(I) Binding Properties of a Synthetic Electron-transfer Protein", J. Inorg. Biochem., 104, 261-267.

    Abstract (205)

    to come

    204.     Mack, J.; Kobayashi, N.; Stillman, M.J. (2010) "Re-examination of the emission properties of alkoxy- and thioalkyl- substituted phthalocyanines" ,J. Inorg. Biochem., 104, 310-317.

    Abstract (204)

    to come

    2009 Publications

    203.     Suchkova, S.A.; Soldatov, A.; Dziedzik, K.; Stillman, M.J. (2009) "The role of spin state on the local atomic and electronic structures of some metalloporphyrin complexes". J. Phys., 209, 012211.

    Abstract (203)

    Abstract. The porphyrin molecule is an archetypal metalloorganic complex, which shows up in many biochemical molecules like chlorophyll, haemoglobin and cytochrome. The prospect of switching the spin in the metalloporphyrin ring is a particularly interesting one, as this could be used, for example, for spin-dependent electric transport through biomolecular devices. These molecules can be used in various applications like optical switches, information storage and non linear optics. Here, we study the molecular spin state of chloro-hemin, hemin cyanide and hemin carbonyl molecules by ADF code. Chloro-hemin has been studied by analyzing the Fe K-edge X-Ray Absorption Near Edge Structure (XANES) spectra. At first stage of investigation we assume an approximate initial geometry, then we perform the geometry optimization with different molecular spin states and search for configuration with minimal total energy with the use of Density Functional Theory (ADF 2008). The results of geometry optimization of chloro-hemin molecule performed with the GGA OPBE functional showed that configuration with total spin S=5/2 has minimal total energy. This configuration corresponds well with the geometry structure obtained via X-Ray diffraction method (Fe-N-N angle is 13.3°). The similar calculations that were carried out for hemin carbonyl and hemin cyanide molecules showed that for these structures minimal energy is found to be for S=1/2. The experimental Fe K-XANES spectra of the investigated compound have been collected. The theoretical analysis of the experimental data has been performed on the basis of finite difference method (FDMnes2007 program code). Journal of Physics: 190 (2009) 012211

    202.     H. Kaluarachchi; D.E.K. Sutherland; A. Young; I.J. Pickering; M. J. Stillman; D.B. Zamble (2009) "The Ni(II)- binding properties of the metallochaperone SlyD", J. Am. Chem. Soc., 131, 18489-18500.

    Abstract (202)

    Metallochaperones are essential for the safe and targeted delivery of necessary yet toxic metal cofactors to their respective protein partners. In this study we examine the nickel binding properties of the Escherichia coli protein SlyD, a metallochaperone that contributes to optimal nickel accumulation in the organism. This protein is also required for E.coli energy metabolism because it participates in the nickel insertion step during [Ni-Fe]-hydrogenase metallocenter assembly. Our study demonstrates that SlyD is a multiple nickel ion binding protein. The analysis of non-covalent metal- protein complexes via electrospray ionization mass spectrometry revealed that SlyD binds upto seven nickel ions in a non-cooperative manner with submicromolar affinity (< 2 µM, upper limit) and that the protein exists in a dynamic mixture of metalloforms that is dependent on the availability of nickel ions in solution. Structural analysis indicates that this metallochaperone undergoes small but distinct changes in the structure upon metal binding and that the nickel binding sites are assembled through ß-turn formation. Although the C-terminal metal-binding domain is primarily responsible for metal chelation, we find that metal binding also perturbs the structure of the N-terminally located domains. An investigation of the nickel-site structure by using X-ray absorption spectroscopy shows that SlyD binds nickel ions by adapting several different geometries and coordination numbers. Finally, the characterization of SlyD mutants demonstrates that the cysteine residues in the C-terminal domain confer tighter affinity as well as increased binding capacity to SlyD. On the basis of the presented data a model for nickel binding to SlyD as well as its role in nickel homeostasis is discussed.

    201.     Ngu, T., Lee, J., Rushton, M.K., Stillman, M.J. (2009) "Arsenic-Metalation of Seaweed Fucus vesiculosus Metallothionein: The Importance of the Interdomain Linker in Metallothionein", Biochemistry, 48, 8806-8816.

    Abstract (201)

    ABSTRACT: The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for As3þ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As- containing species have been determined following mixing of As3þ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time- resolved mass spectral data for As3þ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M-1 s-1) calculated from the fits for the 7-cysteine ? domain are k1?, 19.8, and k2?, 1.4, and for the 9-cysteine ß domain are k1ß, 16.3, k2ß, 9.1, and k3ß, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3þ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of twodomain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker Biochemistry 2009, 48, 8806–8816

    200.     Ngu, T; Stillman, M.J. (2009) "Metalation of metallothioneins" Dalton Transactions, 5425-5433.

    Abstract (200)

    Metallothionein are small, cysteine-rich, metal-binding proteins that are found ubiquitously in Nature. Most metallothioneins bind multiple metals in two well-defined metal-thiolate clusters. This review discusses the use of optical spectroscopy to study the metalation of metallothioneins and the emergence of electrospray ionization mass spectrometry as a means of studying the mechanism of metalation for metallothioneins. A brief history of past kinetic studies of cadmium metallothioneins and recent kinetic study advances for the arsenic metalation of metallothionein will be presented. Lastly, a possible functional role for the two-domain structure of metallothionein will be briefly discussed.

    199.     Tiedemann, T.M., Muryoi, N., Heinrichs, D.E., Stillman, M.J. (2009) "Characterization of IsdH (NEAT domain 3) and IsdB (NEAT domain 2) in Staphylococcus aureus by magnetic circular dichroism spectroscopy and electrospray ionization mass spectrometry" J. Porph. & Phthal., 13, 1006-1016.

    Abstract (199)

    ABSTRACT: Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the fi rst two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdBN2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits signifi cantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fi fth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme. J. Porphyrins Phthalocyanines 2009; 13: 1006–1016

    198.     , Ngu,T; Stillman, M.J. (2009) "Metalation of metallothioneins". IUBMB Life, 61, 438-446.

    Abstract (198)

    Summary Metalloproteins represent _30% of all proteins known, yet our understanding of the structures of these metalloproteins, the metal content, and the mechanism for metalation are still very limited. One of the most studied metalloproteins is the ubiquitous metallothionein (MT), which in mammals contains two metal-binding domains: a 9-cysteine b domain and a 11-cysteine a domain. Metals are coordinated in MT via the cysteinyl thiols present in the primary amino acid sequence and the geometry is controlled by the metal ion. This short review discusses the use of optical spectroscopy to study the metalation of MT with particular emphasis on the benefits and pitfalls involved. Further, the new properties of MT that have been revealed using electrospray ionization mass spectrometry in recent metalation studies will also be discussed. 2009 IUBMB IUBMB Life, 61(4): 438–446, 2009

    2008 Publications

    197.     Ngu, T.; Easton, A.; Stillman, M.J. (2008) "Kinetic analysis of arsenic-metalation of human metallothionein: Significance of the two-domain structure." J. Am. Chem. Soc., 130, 17016-17028.

    Abstract (197)

    Abstract: Metallothionein (MT) is ubiquitous in Nature, underlying MT’s importance in the cellular chemistry of metals. Mammalian MT consists of two metal-binding domains while microorganisms like cyanobacteria consist of a single metal-binding domain MT. The evolution of a two-domain protein has been speculated on for some time; however, no conclusive evidence explaining the evolutionary necessity of the two-domain structure has been reported. The results presented in this report provide the complete kinetic analysis and subsequent mechanism of the As3+-metalation of the two-domain _RhMT and the isolated single domain fragments using time- and temperature-resolved electrospray ionization mass spectrometry. The mechanism for _RhMT binding As3+ is noncooperative and involves six sequential bimolecular reactions in which the R domain binds As3+ first followed by the _ domain. At room temperature (295 K) and pH 3.5, the sequential individual rate constants, kn (n ) 1-6) for the As3+-metalation of _RhMT starting at k1_R are 25, 24, 19, 14, 8.7, and 3.7 M-1s-1. The six rate constants follow an almost linear trend directly dependent on the number of unoccupied sites for the incoming metal. Analysis of the temperature-dependent kinetic electrospray ionization mass spectra data allowed determination of the activation energy for the formation of As1-H17- _RhMT (14 kJ mol-1) and As2-6-_RhMT (22 kJ mol-1). On the basis of the increased rate of metalation for the two-domain protein when compared with the isolated single-domain, we propose that there is an evolutionary advantage for the two-domain MT structures in higher organism, which allows MT to bind metals faster and, therefore, be a more efficient metal scavenger. J. AM. CHEM. SOC. 9 VOL. 130, NO. 50, 2008

    196.     Chan, J., Huang, Z., Watt, I., Kille, P., Stillman, M.J. (2008) "Metallobiological Necklaces: Mass Spectrometric and Molecular Modeling Study of Metallation in Concatenated Domains of Metallothionein", Chem. Eur. J., 14, 7579-7593.

    Abstract (196)

    Abstract: The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of CuI and ZnII, but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys–cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal–thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd4-containing domain of arhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 CdII ions to the 11, 22, and 33 cysteinyl sulfur atoms in the arhMT1a, aa-rhMT1a, and aaarhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/ time trajectory conformations show for the first time the flexibility of the metal–sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations. Chem. Eur. J. 2008, 14, 7579 –7593

    195.     Muryoi, N., Tiedemann, M.T., Pluym, M., Cheung, J., Heinrichs, D.E., Stillman, M.J. (2008) "Demonstration of the iron-regulated surface determinant (isd) heme transfer pathway in staphylococcus aureus", J. Biol. Chem., 283, 28125-28136.

    Abstract (195)

    In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by S. aureus via the Isd pathway first put forth by Mazmanian et al. 2003 (Mazmanian, SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM and Schneewind, O. Science 2003 299: 906-909). The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A, through NEAT-C, to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 ? NEAT-A ? NEAT-C ? IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 ? NEAT-A ? NEAT-C ? IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, that is in the reverse direction as well, and only with each other. While apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A; heme transfer to IsdE appears to require the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.

    194.     Tiedemann, M.T., Muryoi, N., Heinrichs, D.E., Stillman, M.J. (2008) "Iron acquisition by the heme-binding Isd proteins in Staphylococcus aureus - studies of the mechanism using magnetic circular Dichroism", Biochem. Soc. Trans. 36, 1138-1143.

    Abstract (194)

    Staphylococcus aureus bacteria are responsible for numerous hospital-acquired infections ranging from superficial wound lesions to more severe infections such as pneumonia, osteomyelitis, septicemia and death. The Isd proteins, expressed by S. aureus and select other bacteria, are anchored to the bacterial cell wall and membrane and are involved in extracting heme from hemoglobin as an iron source. There is limited knowledge of the overall heme scavenging mechanism on the bacterial surface. A detailed description of the heme binding properties in the transport pathway is critical in our understanding of the mechanism for heme-iron scavenging in S. aureus. Our work involves using a combination of techniques to characterize both the dynamic and steady state heme binding properties of these proteins. UV-visible absorption and magnetic circular dichroism (MCD) spectroscopy provide diagnostic spectral data sensitive to the axial ligands, the spin state, and oxidation state of the central heme-iron. Electrospray mass spectrometry provides stoichiometric information on the numbers of hemes bound, the effect of heme binding on the overall folding of each protein, and kinetic information about the rate of heme binding. Together, these data allow us to address the outstanding questions regarding the mechanism of heme transport via the Isd protein chain in S. aureus.

    193.     Sutherland, D.E.K., Stillman, M.J. (2008) "Noncooperative cadmium(II) binding to human metallothionein 1a" Biochem. Biophys. Res. Commun., 372, 840-844.

    Abstract (193)

    The two-domain (ßa) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain ßa-rhMT and the isolated ß-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd3-ß-rhMT and Cd7-ßa-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd4-ß- and Cd8-ßa-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.

    192.     Duncan, K.E.R., Kirby, C., Stillman, M.J. (2008) "Metal-exchange in metallothioneins. A novel structurally- significant Cd5 species in the alpha domain of human metallothionein 1a". FEBS Journal 275, 2227–2239.

    Abstract (192)

    Metallothioneins are cysteine-rich, metal binding proteins known to provide protection against cadmium toxicity in mammals. Metal-exchange of Zn2+ ions for Cd2+ ions in metallothioneins is a critical process for which no mechanistic or structural information is currently available. The ? domain of recombinantly-prepared human MT isoform 1a, which characteristically coordinates four Cd2+ ions to 11 cysteinyl sulfurs, is shown to bind an additional Cd2+ ion to form a novel Cd5?-MT species, which is proposed here to represent an intermediate in the metal-exchange mechanism. The ESI mass spectrum shows the appearance of charge state peaks corresponding to a Cd5?? species following the addition of 5.0 mol equivalents of Cd2+ to a solution of Cd4?-MT. Significantly, the structurally-sensitive CD spectrum shows a sharp monophasic peak at 254 nm for the Cd5?? species in contrast to the derivative-shaped spectrum of the Cd4?-MT species, with peak maxima at 260 nm (+) and 240 nm (-) indicating Cd-induced disruption of the exciton coupling between the original four Cd2+ ions in the Cd4? species. The 113Cd chemical shift of the fifth Cd2+ is significantly shielded (~400 ppm) when compared with the data for the Cd2+ ions in the Cd4?-MT by both direct and indirect 113Cd NMR spectroscopy. Three of the four original NMR peaks move significantly upon binding the fifth cadmium. Evidence from the indirect 1H{113Cd} HSQC NMR spectra suggests that the coordination environment of the additional Cd2+ is not tetrahedral to four thiolates, as is the case with the four Cd2+ ions in the Cd4?-MT, but is two thiolate ligands as part of its ligand environment, with additional coordination to either water or anions in solution.

    191.     Mack, J., Bunya, M., Shimizu, Y., Uoyama, H., Komobuchi, N., Tetsuo Okujima, T., Uno, H., Ito, S., Stillman, M.J., Ono, N., and Kobayashi, N., (2008) "The Application of MCD Spectroscopy and TD-DFT to Non-Planar Core Modified Tetrabenzoporphyrins. Effect of Reduced Symmetry on Non-Planar Porphyrinoids". Chem. Eur. J., 14, 5001-5020.

    Abstract (191)

    Abstract: The optical spectra of a series of core modified tetrabenzo-porphyrins have been analyzed to determine the effects of core modification, ligand folding and partial benzo?substitution at the ligand periphery on the electronic structure using magnetic circular dichroism (MCD) and NMR spectroscopy, X?ray crystallography, cyclic and differential pulse voltammetry and TD?DFT calculations. Planar 21?carba?, 21?thia?, 21,23?dithia?, and 21?oxa ?23?thia?tetrabenzo[b,g,l,q]porphyrins reported previously [Y. Shimizu, Z. Shen, T. Okujima, H. Uno, N. Ono, Chem. Commun. 2004, 374?375.] are studied together with the previously unreported 21?oxa? and 21?carba?23?thia? tetrabenzo[b,g,l,q]-porphyrins. The optical properties of these compounds are compared to those of tetrabenzo[b,g,l,q], 5,10,15,20?tetra-phenyl, 5,10,15,20?tetraphenyltetra-benzo [b,g,l,q]?21?thia, 5,10,15,20? tetraphenyltetrabenzodithia, 5,10,15,20 ?tetraphenyldibenzo [g,q]?21,23?dithia, 5,10,15,20?tetraphenyldibenzo[b,l]?21, 23?dithia, 5,10,15,20? tetraphenyltri-benzo[g,q,l]?21?thia and 5,10,15,20? tetraphenylbenzo[b]?21?thiaporphyrins. Michl’s perimeter model and Gouterman’s 4?orbital model are used to conceptualize the results and to account for red shifts commonly observed in the spectral bands of non?planar porphyrinoids.

    190.     Mack, J., Asano, Y., Kobayashi, N. Stillman, M.J. (2008) "Magnetic Circular Dichroism Spectroscopy of Cobalt Tetraacenaphthoporphyrin", J. Inorg. Biochem., 102, 472-479.

    Abstract (190)

    The first MCD spectral data for an open shell first row transition metal complex of tetraphenyltetraacenaphthoporphyrin (TPTANP) are reported. The B (or Soret) band of cobalt tetraphenyltetraacenaphthoporphyrin (CoIITPTANP(_2)) exhibits an anomalous negative Faraday A1 term as was reported previously in the case of ZnTPTANP, while a positive A1 term is observed for the Q band. INDO/1 geometry optimizations predict that the TPTANP ligand is saddled due to steric hindrance at the ligand periphery to a slightly lesser extent than is the case with ZnTPTANP. The Q and B bands of CoTPTANP arising from the p-system are blue shifted relative to those of ZnTPTANP, based on the ‘‘hypso" effect reported previously for planar porphyrin complexes of d6–9 transition metals. Journal of Inorganic Biochemistry 102 (2008) 472–479

    2007 Publications

    189.     Pluym, M., Muryoi, N., Heinrichs, D.E., Stillman, M.J. (2007) “Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus”, J. Inorg. Biochem., in press.

    Abstract (189)

    Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain. Journal of Inorganic Biochemistry 102 (2008) 480–488

    188.     Chan, J., Huang, Z., Watt, I., Kille, P., Stillman, M.J. (2007) “Characterization of the Conformational Changes in Recombinant Human Metallothioneins using ESI-MS and Molecular Modeling”, Can. J. Chem., 85, 898-912.

    Abstract (188)

    Electrospray ionization mass spectrometry (ESI-MS) data and mol. modeling calcns. were used to gain mechanistic, conformational, and domain-specific information from the acid-induced demetallation reactions of human metallothionein. The recombinant proteins studied were the single alpha- and beta-rhMT-1a domains and the beta- alpha- and alpha-beta-rhMT-1a two-domain species, based on the human metallothionein 1a sequence. Complete mol. models (MM3/MD) for all the fully metalated and demetallated species using a modified force field are reported for the first time. Basic residues that contribute to the ESI-MS charge states are identified from the mol. models. Demetallation took place under equil. conditions within a narrow pH range. For the two-domain proteins, these results support a demetallation mechanism involving the initial complete demetallation of one domain followed by the other for both beta-alpha-rhMT and alpha-beta- rhMT. Based on the stability of the sep. domains, the .beta. domain is predicted to demetallate first in the two-domain rhMTs. Both the .alpha. domain and the .beta. domain were obsd. to bind an excess of one Cd2+ ion. The metalated rhMT structures were shown to have very stable conformations, but only when fully metalated. Two or more conformations were obsd. at low pH in the ESI-MS data, which are interpreted as arising from the presence of structure, as opposed to a random coil, in the apo-rhMT. This is the first report of the existence of a structure in the two-domain metal-free apo-MT proteins. Only at extremely low pH does the structure open fully to give the highest charge distribution, which is assocd. with a random coil. Pre-existing structural features in the apo-MT would explain why the metalation reactions occur so rapidly.

    187.     Pluym, M., Vermeiren, C.L., Mack, J., Heinrichs, D.E., Stillman, M.J. (2007) “Heme binding properties of Staphylococcus aureus IsdE”, Biochemistry, 46, 12777-12787.

    Abstract (187)

    Staphylococcus aureus is the source of a large no. of hospital- acquired infections, of which many are serious and can lead to death. Iron is critically important to the survival and growth of the bacterium, and complex, multistep mechanisms are present to fulfill the necessary iron requirement. Isd proteins located on the wall and membrane of S. aureus have been proposed to function in heme acquisition. We report characterization of the S. aureus heme-binding protein IsdE, the lipoprotein component of a membrane-localized ABC transporter that is believed key to receiving heme from cell wall- anchored Isd proteins. Magnetic CD (MCD) data, which greatly extend the results from our initial study of IsdE in bacterial cell lysates, probe the ligand and redox properties of the bound heme. The MCD data show that IsdE, when overexpressed in E. coli, binds either ferric or ferrous heme but that the largest fraction is low spin ferrous heme. Studies of mutants allowed identification and characterization of the ligands in the fifth and sixth position on the heme iron as histidine, proximally, and methionine, distally. This histidine-methionine heme-iron ligation is unique to heme transport proteins. The smaller fraction of ferric heme in the protein is not bound by methionine, allowing for access by strong field ligands, such as cyanide. Electrospray ionization mass spectral data are reported for the first time and show that only one heme ligand binds per IsdE protein mol. These data also show there is little change in the conformation of the protein between the heme-bound and heme-free species, indicating that the heme-free IsdE adopts a structure essentially independent of the heme. The mass spectral data clearly show that IsdE reversibly unwinds under denaturing conditions to form at least two distinct, heme-free conformations.

    186.     Duncan, Rigby, K., Stillman, M.J. (2007) “Evidence for non-cooperative metal binding to the alpha domain of human metallothionein.”  FEBS Journal, 274, 2253- 2261.

    Abstract (186)

    In the present study, we investigated the metal-binding reactivity of the isolated alpha domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metalation reaction of the metal-free alpha domain with Cd2+ was unequivocally shown to proceed by a noncooperative mechanism at physiol. pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of intermediate partially metalated metallothionein species under limiting Cd2+ conditions. Titrn. with four molar equivalents of Cd2+ was required for the formation of the Cd4 alpha species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metalated and metal-free species are stable intermediates, and thus may have a potential role in the currently undefined function of metallothionein.

    185.     Yalovega, G., Smolentsev, G., Soldatiov, A., Chan, J., Stillman, M.J. (2007) ”Cd- metallothionein: Analysis of the local atomic structure”, Nuclear Inst. And Methods in Physical Research A, 575, 162-164.

    Abstract (185)

    Cadmium-metallothioneins (Cd-MT) are small proteins contg. seven cadmium atoms in a tetrahedral arrangement of sulfur atoms. Comparison of exptl. X-ray absorption fine structure (XAFS) with theor. full multiple-scattering XAFS spectra have been used as a tool for verification of the structural models generated using both available exptl. NMR data and theor. simulations on the basis of mol. mechanics/mol. dynamics (MM/MD) and d. functional theory (DFT).

    184.     Pluym, M., Vermeiren, C. L., Mack, J., Heinrichs, D. E., Stillman, M. J. (2007) “Protoporphyrin IX and heme binding properties of Staphylococcus aureus IsdC”, J. Porphyrins Phthalocyanines, 11, 165-171.

    Abstract (184)

    Staphylococcus aureus is a human pathogen that results in numerous infections in hospital settings and recently also in the wider community. Its antibiotic resistant forms are causing considerable alarm. A series of surface-anchored proteins that have heme uptake and transport properties were reported. Through the use of absorption and magnetic CD spectroscopies and mass spectrometry, the iron-free, protoporphyrin IX and the iron-contg., heme-binding characteristics of bacterial rIsdC were obtained. Mass spectrometry showed that following isolation and purifn., the rIsdC is bound predominantly to protoporphyrin IX and to a lesser extent heme, unlike the case of rIsdA, which binds predominantly heme. Magnetic CD anal. provided further information regarding porphyrin binding because the characteristic magnetic CD band envelopes for the iron-free protoporphyrin IX and the iron-contg. heme can be clearly distinguished in the spectrum of the rIsdC. Anal. of these spectral data showed that the minor heme component exists as a high-intermediate spin state ferric heme when bound to rIsdC, similar to the high-spin ferric heme reported for the rIsdA protein.

    183.     Salgado, M. T., Bacher, K. L., Stillman, M. J. (2007) “Probing Coordination Geometry Changes in the alpha and beta Domains of Metallothionein by Emission Spectroscopy”.  J. Biological Inorganic Chemistry, 12, 294-312.

    Abstract (183)

    Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted alpha and beta domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag (I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag (I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted alpha and beta domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag (I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted alpha and beta fragments than when the Ag(I)-MT species were formed from the apo-alpha and apo-beta fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag (I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.

    182.     Mack, J., Stillman, M. J., Kobayashi, N. (2007) “Application of MCD spectroscopy to porphyrinoids”, Coord. Chem,. Rev., 251, 429-453.

    Abstract (182)

    A review. MCD spectroscopy proved invaluable in the assignment of the optical spectra of the porphyrinoids over the last 40 years. Recently, with new power of the DFT theor. treatments available routinely, the assignment of porphyrinoid spectra has become an important factor in the correct interpretation of the electronic structure predicted by the calcns. In this review, the authors have described in detail the formalism required to use the MCD technique to probe the electronic structure of porphyrinoid complexes. The authors begin with a detailed description of the optical background to the technique; continue by describing the current theor. interpretation of the spectral morphol., and end by describing the anal. of the MCD spectral data for a no. of porphyrins and phthalocyanines.

    Older Publications

    Publications in 2006

    181.      Ngu, T. T., S. Sturzenbaum, Stillman, M. J. (2006) "Cadmium Binding Studies to the Earthworm Lumbricus rubellus Metallothionein by Electrospray Mass Spectrometry and Circular Dichroism Spectroscopy”, Biochem. Biophys. Res. Commun., 351, 229-233.


    180.      Duncan, K. E. R., Stillman, M. J. (2006) Metal-dependent protein folding: Metallation of metallothionein, J. Inorg. Biochem. 100, 2101-2107.


    179.     Ngu, T. T. Stillman, M. J. (2006) Arsenic binding to Human Metallothionein. J. Am. Chem. Soc. 128: 12473-12483.


    178.     Vermeiren, C. L., Pluym, M., Mack, J., Heinrichs, D. E., Stillman, M. J. (2006) Characterization of the heme binding properties of Staphylococcus aureus IsdA. Biochemistry, 45, 12867-12875.


    177.     Duncan, K. E. R., Ngu, T., Chan, J., Salgado, M.T., Merrifield, M. E., Stillman, M. J. (2006) “Peptide Folding, Metal-Binding Mechanisms, and Binding Site Structures in Metallothioneins” J. Exp. Biol. & Med., 231, 1488-1499.


    176.     Soldatov, A. V., Smolentsev, G., Yalovega, G., Chan, J., Stillman, M. J. (2006) "The structure of Cd sites in metallothioneins studied by combination of XAFS and molecular dynamics." J. Radiation Physics and Chemistry, 75, 1901-1904.


    175.     Mack, J., Kobayashi, N., Stillman, M.J. (2006) “MCD spectroscopy and TD-DFT calculations of metal phthalocyanine anion and cation radical species”, J. Porphyrins Phthalocyanines, 10, 1219-1237.


    174.     Merrifield, M. E., Chaseley, J., Kille, P., Stillman, M. J. (2006) “Determination of the metallation and structure of Fucus vesiculosus metallothionein”. Am. Chem. Soc. Chem. Res. Toxicol., 19; 365-375.  Kille’s group provided the DNA constructs used to synthesize the protein.


    173.     Rigby, K. E., Chan, J., Mackie, J. Stillman, M. J. (2006) “Molecular dynamics study on the folding and metallation of the individual domains of metallothionein”, Proteins: Structure, Functions and Bioinformatics, 62, 159-172.


    2006 Published abstracts from the 4th International Conference on Porphyrins and Phthalocyanines, Rome, July, 2006: 
    A. Senior, J. Mack, N. Kobayashi and M. J. Stillman (2006) “Π cation radical porphyrinoids: progress towards the assignment of the optical spectrum”, J. Porphyrins Phthalocyanines, 10, 876.


    M. Pluym, C. Vermeiren, J. Mack, D. E. Heinrichs and M. J. Stillman (2006) “Spectroscopic study of the Isd heme-scavenging proteins IsdA and IsdE of Staphylococcus aureus” J. Porphyrins Phthalocyanines, 10, 823.


    J. Mack and M. J. Stillman (2006) “Theoretical aspects of porphyrin and phthalocyanine spectroscopy”, J. Porphyrins Phthalocyanines, 10, 400.


    M. J. Stillman, J. Mack and N. Kobayashi “MCD spectroscopy and TD-DFT calculations of metal phthalocyanine anion and cation radical species”, J. Porphyrins Phthalocyanines, 10, 755.


    J. Mack, Y. Shimizu, M. Bunya, M. J. Stillman, N Ono and N Kobayashi (2006) “MCD spectroscopy and the effect of porphyrinoid ligand saddling: core modified tetrabenzoporphyrins” J. Porphyrins Phthalocyanines, 10, 338.

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    Also from this web page:

    Group Members working in 2013

    Dr. Martin Stillman

    Dr. Maria Salgado

    Dr. Duncan Sutherland

    Dr. Michael Tiedemann

    Tyler Pinter

    Gordon Irvine

    Brit Lewis

    Jasjit Singh

    Brad Reed - from dept of Biology


     

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