Evidence for a ligand CO that is required for catalytic activity of CO dehydrogenase from Rhodospirillum rubrum

Radiolabeling studies support the existence of a nonsubstrate CO ligand (CO(L)) to the Fe atom of the proposed [FeNi] cluster of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified CODH has variable amounts of CO(L) dissociated depending on the extent of handling of the proteins. This dissociated CO(L) can be restored by incubation of CODH with CO, resulting in a 30-40% increase in initial activity relative to as-isolated purified CODH. A similar amount of CO(L) binding is observed when as-isolated purified CODH is incubated with (14)CO: approximately 0.33 mol of CO binds per 1 mol of CODH. Approximately 1 mol of CO was released from CO-preincubated CODH upon denaturation of the protein. No CO could be detected upon denaturation of CODH that had been incubated with cyanide. CO(L) binds to both Ni-containing and Ni-deficient CODH, indicating that CO(L) is liganded to the Fe atom of the proposed [FeNi] center. Furthermore, the Ni in the CO(L)-deficient CODH can be removed by treatment with a Ni-specific chelator, dimethylglyoxime. CO preincubation protects the dimethylglyoxime-labile Ni, indicating that CO(L) is also involved in the stability of Ni in the proposed [FeNi] center.     

Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy

We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed.     

A novel binuclear [CuSMo] cluster at the active site of carbon monoxide dehydrogenase: characterization by X-ray absorption spectroscopy

The structurally characterized molybdoenzyme carbon monoxide dehydrogenase (CODH) catalyzes the oxidation of CO to CO2 in the aerobic bacterium Oligotropha carboxidovorans. The active site of the enzyme was studied by Mo- and Cu-K-edge X-ray absorption spectroscopy. This revealed a bimetallic [Cu(I)SMo(VI)(double bond O)2] cluster in oxidized CODH which was converted into a [Cu(I)SMo(IV)(double bond O)OH2] cluster upon reduction. The Cu...Mo distance is 3.70 A in the oxidized form and is increased to 4.23 A upon reduction. The bacteria contain CODH species with the complete and functional bimetallic cluster along with enzyme species deficient in Cu and/or bridging S. The latter are precursors in the posttranslational biosynthesis of the metal cluster. Cu-deficient CODH is the most prominent precursor and contains a [HSMo(double bond O)OH2] cluster. Se-K-edge X-ray absorption spectroscopy demonstrates that Se is coordinated by two C atoms at 1.94-1.95 A distance. This is interpreted as a replacement of the S in methionine residues. In contrast to a previous report [Dobbek, H., Gremer, L., Meyer, O., and Huber, R. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8884-8889] Se was not identified in the active site of CODH.     

Calcium EXAFS establishes the Mn-Ca cluster in the oxygen-evolving complex of photosystem II

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 A, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is approximately 3.5 A distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster.     

Structural examination of the nickel site in chromatium vinosum hydrogenase: redox state oscillations and structural changes accompanying reductive activation and CO binding

An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI(r), and the carbon monoxide complex, SI-CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI(u); distorted four-coordinate sites in SI(r) and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni-O bond accounts for the change in coordination number from five to four that accompanies formation of SI(r). A shortening of the Ni-Fe distance from 2.85(5) A in form B to 2.60(5) A also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI-CO complex reveals the presence of Ni-CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H(2) or H(-) was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structural differences between the Ni sites were observed.     

Infrared studies of carbon monoxide binding to carbon monoxide dehydrogenase/acetyl-CoA synthase from Moorella thermoacetica

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme that catalyzes the reversible reduction of carbon dioxide into carbon monoxide and the coupled synthesis of acetyl-CoA from the carbon monoxide produced. Exposure of CODH/ACS from Moorella thermoacetica to carbon monoxide gives rise to several infrared bands in the 2100-1900 cm(-1) spectral region that are attributed to the formation of metal-coordinated carbon monoxide species. Infrared bands attributable to M-CO are not detected in the as-isolated enzyme, suggesting that the enzyme does not contain intrinsic metal-coordinated CO ligands. A band detected at 1996 cm(-1) in the CO-flushed enzyme is assigned as arising from CO binding to a metal center in cluster A of the ACS subunit. The frequency of this band is most consistent with it arising from a terminally coordinated Ni(I) carbonyl. Multiple infrared bands at 2078, 2044, 1970, 1959, and 1901 cm(-1) are attributed to CO binding at cluster C of the CODH subunit. All infrared bands attributed to metal carbonyls decay in a time-dependent fashion as CO(2) appears in the solution. These observations are consistent with the enzyme-catalyzed oxidation of carbon monoxide until it is completely depleted from solution during the course of the experiments.     

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO₂. A unique [NiFe₄S₄] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe₃S₄] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.     

Structure of the manganese complex of photosystem II upon removal of the 33-kilodalton extrinsic protein: an X-ray absorption spectroscopy study

The structure of the Mn complex of photosystem II (PSII) was studied by X-ray absorption spectroscopy. Oxygen-evolving spinach PSII membranes containing 4-5 Mn/PSII were treated with 0.8 M CaCl2 to extract the 33-, 24-, and 16-kilodalton (kDa) extrinsic membrane proteins. Mn was not released by this treatment, but subsequent incubation at low Cl- concentration generated preparations containing 2 Mn/PSII. The Mn X-ray absorption K-edge spectrum of the CaCl2-washed preparation containing 4 Mn/PSII is very similar to spectrum of native PSII, indicating that the oxidation states and ligand symmetry of the Mn complex in these preparations are not significantly different. The Mn extended X-ray absorption fine structure (EXAFS) of CaCl2-washed PSII fits to a Mn neighbor at approximately 2.75 A and two shells of N or O at approximately 1.78 and approximately 1.92 A. These distances are similar to those we have previously reported for native PSII preparations [Yachandra, V. K., Guiles, R. D., McDermott, A. E., Cole, J. L., Britt, R. D., Dexheimer, S. L., Sauer, K., & Klein, M. P. (1987) Biochemistry (following paper in this issue)] and are indicative of an oxo-bridged Mn complex. Our results demonstrate that the structure of the Mn complex is largely unaffected by removal of 33-, 24-, and 16-kDa extrinsic proteins, do not provide ligands to Mn. The Mn K-edge spectrum of the CaCl2-washed sample containing 2 Mn/PSII has a dramatically altered shape, and the edge inflection point is shifted to lower energy. The position of the edge is consistent with a Mn oxidation state of +3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H(2)O --> CO(2) + 2 e(-) + 2 H(+). Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C). The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3. 10(9) M(-1) CO s(-1) (CODH I) and 1.7. 10(9) M(-1) CO s(-1) (CODH II). The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.     

X-ray absorption fine structure study of the atomic and electronic structure of molybdenum disulfide intercalation compounds with transition metals

The local structures of ‘host’ and ‘guest’ layers of MoS₂ intercalated with M(OH)₂ (M=Mn, Co and Ni) prepared via interaction of single-layer MoS₂ dispersions and solutions of M²⁺ salts were studied by X-ray absorption spectroscopy. According to M K-edge extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) results, the electronic structure and atomic environment of the M atoms in the intercalates are similar to that of the crystalline hydroxides M(OH)₂. In the Ni intercalate, Mo K-edge EXAFS revealed a structural change of the ‘host’ MoS₂ layers similar to that reported for water dispersions of MoS₂ single layers. S K-edge XANES data indicate that the change is associated with increased electron density on the S atoms in the matrix. SO₄²⁻ and Mo″⁻ (4 < n < 6) were detected in the intercalated materials exposed to air, suggesting that transition metal intercalation may increase the susceptibility of the MoS₂ layers to oxidation.     

Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.     

Effect of cyanide binding on the copper sites of cytochrome c oxidase: an X-ray absorption spectroscopic study

Cu x-ray absorption spectroscopy (XAS) has been used to investigate the effect of cyanide treatment on the structures of the copper sites in beef heart cytochrome c oxidase. The Cu K-edge spectrum changes significantly upon cyanide binding to resting state enzyme, as does the Cu extended x-ray absorption fine structure (EXAFS) spectrum. The Cu EXAFS Fourier transfer (FT) exhibits an enhanced peak for the cyanide-treated enzyme in the region containing the Cu...Fe peak in the resting state FT (at R' approximately equal to 2.6-2.7 A). This peak in the cyanide-treated sample is hypothesized to arise from "outer shell" scattering from a linear Cu-cyanide moiety, suggesting cyanide binding to CuB only (CuB 2+-CN-) or cyanide bridging between the Fe of heme a3 and CuB (Fe3+-(CN-)-CuB 2+).     

Spectroscopic studies of nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: nature of the iron-sulfur clusters

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum utilizes three types of Fe-S clusters to catalyze the reversible oxidation of CO to CO(2): a novel [Ni4Fe5S] active site (C cluster) and two distinct [4Fe4S] electron-transfer sites (B and D clusters). While recent X-ray data show the geometric arrangement of the five metal centers at the C cluster, electronic structures of the various [Ni4Fe5S] oxidation states remain ambiguous. These studies report magnetic circular dichroism (MCD), variable temperature, variable field MCD (VTVH MCD), and resonance Raman (rR) spectroscopic properties of the Fe-S clusters contained in Ni-deficient CODH. Essentially homogeneous sample preparations aided in the resolution of the reduced [4Fe4S](1+) (S = (1)/(2)) B cluster and the reduced Ni-deficient C cluster (denoted C, S > (1)/(2)) by MCD. The three Fe atoms derived from the [Ni3Fe4S] cubane component appear to dominate the reduced C cluster MCD spectrum, while the presence of a fourth Fe center can be inferred from the ground state spin. The same underlying MCD features present in Ni-deficient CODH spectra are also observed for Ni-containing CODH, suggesting that both proteins contain the same C cluster Fe-S component. Overlooked in all spectroscopic studies to date, the D cluster was confirmed by rR to be a typical [4Fe4S] site with cysteinyl coordination. Together, MCD and rR data show that the D cluster remains in the oxidized [4Fe4S](2+) (S = 0) state at potentials > or = -530 mV (versus SHE), thus exhibiting an unusually low redox potential for a standard [4Fe4S](2+/1+) electron-transfer site.     

Sulfur K-edge extended X-ray absorption fine structure spectroscopy of homoleptic thiolato complexes with Zn(II) and Cd(II)

The sulfur K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy is applied to homoleptic thiolato complexes with Zn(II) and Cd(II), (Et(4)N)[Zn(SAd)(3)] (1), (Et(4)N)(2)[{Zn(ScHex)(2)}(2)(mu-ScHex)(2)] (2), (Et(4)N)(2)[{Cd(ScHex)(2)}(2)(mu-ScHex)(2)] (3), (Et(4)N)(2)[{Cd(ScHex)}(4)(mu-ScHex)(6)] (4), [Zn(mu-SAd)(2)](n) (5), and [Cd(mu-SAd)(2)](n) (6) (HSAd=1-adamantanethiol, HScHex=cyclohexanethiol). The EXAFS results are consistent with the X-ray crystal data of 1-4. The structures of 5 and 6, which have not been determined by X-ray crystallography, are proposed to be polynuclear structures on the basis of the sulfur K-edge EXAFS, far-IR spectra, and elemental analysis. Clear evidences of the S...S interactions (between bridging atoms or neighboring sulfur atoms) and the S...C(far) interactions (in which C(far) atom is next to carbon atom directly bonded to sulfur atom) were observed in the EXAFS data for all complexes and thus lead to the reliable determination of the structures of 5 and 6 in combination with conventional zinc K-edge EXAFS analysis for 5. This new methodology, sulfur K-edge EXAFS, could be applied for the structural determination of in vivo metalloproteins as well as inorganic compounds.     

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase

The metal content of bovine NADH-Q oxidoreductase determined by inductively-coupled plasma atomic-emission spectroscopy reveals the presence of about one atom of zinc per molecule of flavin mononucleotide. We applied Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) to investigate the local structure of the bound zinc ion and to identify the nature of the coordinating residues. The EXAFS spectrum is consistent with a structured zinc binding site. By combining information from first-shell analysis and from metalloprotein data bases putative binding clusters have been built and fitted to the experimental spectrum using ab initio simulations. The best fitting binding cluster is formed by two histidine and two cysteine residues arranged in a tetrahedral geometry.     

High-resolution structure of the photosynthetic Mn4Ca catalyst from X-ray spectroscopy

The application of high-resolution X-ray spectroscopy methods to study the photosynthetic water oxidizing complex, which contains a unique hetero-nuclear catalytic Mn4Ca cluster, is described. Issues of X-ray damage, especially at the metal sites in the Mn4Ca cluster, are discussed. The structure of the Mn4Ca catalyst at high resolution, which has so far eluded attempts of determination by X-ray diffraction, X-ray absorption fine structure (EXAFS) and other spectroscopic techniques, has been addressed using polarized EXAFS techniques applied to oriented photosystem II (PSII) membrane preparations and PSII single crystals. A review of how the resolution of traditional EXAFS techniques can be improved, using methods such as range-extended EXAFS, is presented, and the changes that occur in the structure of the cluster as it advances through the catalytic cycle are described. X-ray absorption and emission techniques (XANES and Kbeta emission) have been used earlier to determine the oxidation states of the Mn4Ca cluster, and in this report we review the use of X-ray resonant Raman spectroscopy to understand the electronic structure of the Mn4Ca cluster as it cycles through the intermediate S-states.     

Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. One structure of the Moorella thermoacetica enzyme revealed that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bridged to a binuclear CuNi center with Cu at the proximal metal site (M(p)) and Ni at the distal metal site (M(d)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity and Cu content, we had proposed that the Cu-containing enzyme is active [Seravalli, J., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3689-3694]. Here we have reexamined this proposal. Enzyme preparations with a wider range of Ni (1.6-2.8) and Cu (0.2-1.1) stoichiometries per dimer were studied to reexamine the correlation, if any, between the Ni and Cu content and ACS activity. In addition, the effects of o-phenanthroline (which removes Ni but not Cu) and neocuproine (which removes Cu but not Ni) on ACS activity were determined. EXAFS results indicate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examined to investigate the electronic state of this proposed intermediate in the ACS reaction mechanism. The combined results strongly indicate that the CuNi enzyme is inactive, that the NiNi enzyme is active, and that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](2+) (net S = 0) cluster bridged to a Ni(1+) (S = (1)/(2)) at M(p) that is bridged to planar four-coordinate Ni(2+) (S = 0) at M(d), with the spin predominantly on the Ni(1+). Furthermore, these studies suggest that M(p) is inserted during cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieved with NiFeC signals of approximately 0.8 spin/mol.     

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.     

The structure of the Ni-Fe site in the isolated HoxC subunit of the hydrogen-sensing hydrogenase from Ralstonia eutropha

The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha which forms a [HoxBC]2 complex functions as a hydrogen sensor under aerobic conditions. We have studied a novel Strep-tag isolate of the RH large subunit, HoxC(ST), which lacks the Fe-S clusters of HoxB, allowing for structure determination of the catalytic site by X-ray absorption spectroscopy both at the Ni and, for the first time, also at the Fe K-edge. This technique, together with Fourier-transform infrared spectroscopy, revealed a Ni-Fe site with [O1(CysS)2Ni(II)(mu-SCys)2Fe(II)(CN)2(CO)] structure in about 50% of HoxC(ST) and a [(CysS)2Fe(II)(CN)2(CO)] site lacking Ni in the remainder protein. Possibly both sites may be intermediates in the maturation process of the RH.