Staphylococcus aureusA bout one-third of all proteins exploit specific metal ions to assist in macromolecular folding and͞or function at the active site of metalloenzymes (1). All cells restrict the number of bioavailable metal atoms to avoid any excess that would otherwise compete with native metal ion sites that do not support biological activity (2). Essentially all cell types contain intracellular metal sensors that detect surplus metal ions and control the expression of genes encoding proteins that expel or sequester the extra ions (3). For some metals and some cell types, a complementary set of sensors detect deficiency and regulate genes encoding proteins that acquire more of the required ions (4, 5). It is currently poorly understood how such metal-sensing metalloregulators accurately discriminate between various metal ions.SmtB͞ArsR-family regulators are ubiquitous in bacterial genomes and bind to the operator͞promoter (O͞P) regions of gene(s) encoding proteins involved in metal export or sequestration, repressing transcription (for a review, see ref. 6). As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9-11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).Comparative structural and spectroscopic studies of six SmtB͞ ArsR family members reveal that individual members are characterized by one or both of two structurally distinct metal coordination sites (6, 11, 15-20). These two metal sites are designated ␣3N (or ␣3) and ␣5 (or ␣5C), named for the location of the metal-binding ligands within the known or predicted secondary structure of individual family members. The coordination environment and precise ligand set of the ␣3, ␣3N, and͞or ␣5, ␣5C sites in the different SmtB͞ArsR proteins differ and are presumed to contribute toward metal selectivity. A sequence comparison for proteins discussed herein is shown in Fig. 1 and highlights these sites.Here we report insights gained from the study of two additional family members, Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR. CzrA and NmtR share 30% sequence identity and a high degree of similarity (60%) yet respond to distinct but partially overlapping metal profiles in vivo. S. aureus CzrA is a Co(II)͞Zn(II)-specific sensor that regulates the expression of the czr operon, which encodes a Co(II)͞ Zn(II)-facilitated pump, CzrB, that effluxes metal out of the cell (13, 14). Electromobility-shift assays and in vivo expression studies indicate that Zn(II) is the strongest inducer of CzrA regulation, with Co(II) also capable of regulation but only at higher concentrations than Zn(II). Other metals, including Ni(II), have little to no effect on derep...

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.

The Synechococcus PCC7942 SmtB is a zinc-responsive transcriptional repressor and a member of the ArsR superfamily of prokaryotic metalloregulatory transcription factors. The mechanism of negative regulation by Zn(II) and other metals as well as the coordination chemistry (stoichiometry, affinity, and specificity) of SmtB is poorly understood. In contrast to previous results [Kar, S. R., Adams, A. C., Lebowitz, J., Taylor, K. B., and Hall, L. M. (1997) Biochemistry 36, 15343-15348], we find that fully reduced SmtB binds 1 mol equiv of Zn(II) with a very high affinity, K(Zn) in excess of 10(11) M(-1) (pH 7.4, 0.15 M KCl, 22 degrees C). Optical spectroscopic experiments reveal that SmtB binds 1 mol equiv of Co(II) in a tetrahedral or distorted tetrahedral environment with one or two cysteine thiolate ligands in the first coordination shell. Zn(II) and Co(II) EXAFS studies are consistent with the optical spectroscopic data, and further suggest the presence of a mixture of carboxylate and imidazole-containing ligands. K(Co) was determined to be 1.7 (+/-0.1) x 10(9) M(-1) in a chelator (EGTA) competition assay; 1 equiv of Zn(II) results in complete displacement of the bound Co(II). SmtB also binds 1 mol equiv of Ni(II), which, when formed at low Ni(II):SmtB molar ratios, adopts a non-native, six-coordinate complex characterized by at least two histidine and no thiolate ligands. The hierarchy of metal binding affinities is Zn(II) >> Co(II) >> Ni(II).

We heterologously overproduced a hyperthermostable archaeal low potential (E m ‫؍‬ ؊62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys 42 and Cys 61 ) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Riesketype ferredoxin appear to be inequivalent as indicated by much higher stability of the His 64 Proteins containing Rieske-type [2Fe-2S] clusters are widespread in nature from hyperthermophilic Archaea and Bacteria to Eukarya and play critical electron transfer roles in various pathways such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (1-4). In contrast to regular plant-and vertebrate-type ferredoxins having complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron-sulfur core with the S ␥ atom of each of the two cysteine residues coordinated to one iron site and the N ␦ atom of each of the two histidine residues coordinated to the other iron site. This asymmetric ligation results in some unique redox and spectroscopic properties (for reviews, see Refs. 1 and 3-5). This cluster coordination was firmly established by recent x-ray crystal structures of several different Rieske-type protein domains (6 -11).Two different types of Rieske clusters are observed in proteins. One type displays higher reduction potentials (E m ) 1 of approximately ϩ150 to ϩ490 mV and occurs in proton-translocating respiratory complexes (cytochrome bc 1 /b 6 f complexes and their archaeal homologs without c-type cytochromes), being involved in not only electron transfer but also substrate binding and oxidation at the quinol-oxidizing Q o site (2-5, 12-15). The other type displays lower E m values of approximately Ϫ150 to Ϫ50 mV and has been found in a diverse group of bacterial multicomponent terminal oxygenases and soluble Rieske-type ferredoxins (1, 3, 8, 9, 16 -27). However, none of the latter class has been characterized in detail from any archaeal species.We recently found that the genomic DNA sequence of the thermoacidophilic archaeon Sulfolobus solfataricus strain P-1 (DSM 1616T) encodes an archaeal homolog of bacterial small Rieske-type ferredoxins with no consensus disulfide signature (DDBJ accession number AB047031 (27)). This arf gene was found by homology search against the deduced amino acid sequence of Sulfolobus tokodaii sulredoxin, a water-soluble homolog of a high potential Rieske protein (E m,low pH ϳ ϩ190 mV) with a consensus disulfide linkage (DDBJ accession number AB023295) 2 (28 -30) (Fig. 1). Subsequent cloning and heterologous overexpression in Escherichia coli of this S. solfataricus arf gene encoding the archaeal Rieske-type ferredoxin (ARF) (27) have provided an opportunity to define the influence of surrounding amino acid residues on the electronic and s...

Lysine 2,3-aminomutase (KAM) belongs to a class of enzymes that use FeS clusters and S-adenosyl-L-methionine to initiate radical-dependent chemistry. Selenium K-edge X-ray absorption spectroscopic analysis of KAM poised at various stages of catalysis, in the presence of selenomethionine or Se-adenosyl-L-selenomethionine, reveals that the cofactor is cleaved only in the presence of dithionite and the substrate analogue trans-4,5-dehydrolysine. A new Fourier transform peak at 2.7 A, assigned as a Se-Fe interaction, appears concomitant with this cleavage. This is the first demonstration of a direct interaction of S-adenosyl-L-methionine, or its cleavage products, with the FeS cluster in this class of enzymes.

Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH(3)-S-CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F(430) which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme-substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH(3)-S-CoM or the substrate analog coenzyme M (HS-CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F(430) and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH(3)-S-CoM and HS-CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel.

Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism.

The Co and Fe K-edge extended X-ray absorption fine structure (EXAFS) spectra of the methionyl aminopeptidase from Escherichia coli (EcMetAP) have been recorded in the presence of 1 and 2 equiv of either Co(II) or Fe(II) (i.e., [Co(II)_(EcMetAP)], [Co(II)Co(II)(EcMetAP)], [Fe(II)_(EcMetAP)], and [Fe(II)Fe(II)(EcMetAP)]). The Fourier transformed data of both [Co(II)_(EcMetAP)] and [Co(II)Co(II)(EcMetAP)] are dominated by a peak at ca. 2.05 A, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 A. Attempts to include a Co-Co interaction (in the 2.4-4.0 A range) in the curve-fitting parameters were unsuccessful. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f '). These data suggest that a dinuclear Co(II) center does not exist in EcMetAP and that the first Co atom is located in the histidine-ligated side of the active site. The EXAFS data obtained for [Fe(II)_(EcMetAP)] and [Fe(II)Fe(II)(EcMetAP)] indicate that Fe(II) binds to EcMetAP in a similar site to Co(II). Since no X-ray crystallographic data are available for any Fe(II)-substituted EcMetAP enzyme, these data provide the first glimpse at the Fe(II) active site of MetAP enzymes. In addition, the EXAFS data for [Co(II)Co(II)(EcMetAP)] incubated with the antiangiogenesis drug fumagillin are also presented.