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

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.

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.

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.

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).

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.

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.

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.

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.