Catalysis at a dinuclear [CuSMo(O)OH] cluster in a CO dehydrogenase resolved at 1.1-Å resolution

Abstract

The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa Mo-and Cu-containing iron-sulfur flavoprotein. Here, the enzyme's active site in the oxidized or reduced state, after inactivation with potassium cyanide or with n-butylisocyanide bound to the active site, has been reinvestigated by multiple wavelength anomalous dispersion measurements at atomic resolution, electron spin resonance spectroscopy, and chemical analyses. We present evidence for a dinuclear heterometal [CuSMo(AO)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. C arbon monoxide dehydrogenases (CODHs) of aerobic or anaerobic Bacteria and Archaea, which represent the essential catalyst in the global biogeochemical cycle of atmospheric carbon monoxide (CO), catalyze the oxidation of CO to carbon dioxide (CO 2 ) or the reverse reaction [CO ϩ H 2 O 7 CO 2 ϩ 2 H ϩ ϩ 2 e Ϫ ] (1, 2). The annual removal of CO from the lower atmosphere and earth by microorganisms has been estimated to be Ϸ1 ϫ 10 8 tons (3). Thus, an important role of CODHs is to remove CO from the environment, helping to maintain the toxic gas at subhazardous concentrations.The Ni-containing CODHs from the anaerobic hydrogenogenic bacteria Carboxydothermus hydrogenoformans (4, 5) or Rhodospirillum rubrum (6) and the Mo-containing CODHs from the aerobic carboxidotrophic bacteria Oligotropha carboxidovorans (7-9) or Hydrogenophaga pseudoflava (10) have been structurally characterized. The homodimeric CODHs of C. hydrogenoformans or R. rubrum contain five metal clusters, of which clusters B, BЈ and a subunit-bridging, surface-exposed cluster D are cubane-type [4Fe-4S] clusters (5, 6). The active-site clusters C and CЈ of the C. hydrogenoformans CODH are asymmetric [Ni-4Fe-5S] clusters identified in the enzyme reduced with dithionite. Their integral Ni ion, which is the likely site of CO oxidation, is coordinated by four sulfur ligands with square planar geometry (5). Interestingly, the corresponding cluster of the CODH from R. rubrum has been described as an Fe mononuclear site in combination with an [NiFe 3 S 4 ] cubane (6).CODH from O. carboxidovorans consists of a dimer of LMS heterotrimers (7). Each heterotrimer is composed of a 17.8-kDa iron-sulfur protein (S), which carries two types of [2Fe-2S] clusters, a 30.2-kDa flavoprotein (M), which contains a noncovalently bound FAD cofactor, and an 88.7-kDa molybdoprotein (L), which harbors the active site of the enzyme. In a previous paper (7), a CODH preparation with a specific activity of 6.6 units͞mg was analyzed at a resolution of 2.2 Å. The enzyme's active site was modeled to contain Mo with three oxygen ligands, the molybdopterin cytosine dinucleotide (MCD) cofactor, and an SeH-group bound to the S␥ atom of Cys-388. In the present paper, we have applied multiple wavelength anomalous dispersion methods at up to 1.09-Å resolution to crystals containing fully functional CODH (23.2 units͞mg). The SeH-group could not be confirmed, and a Cu atom was identified instead, at ...

The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

“…The geometry of the molybdenum coordination sphere described here differs from that previously reported for Desulfovibrio gigas aldehyde oxididoreductase (7) and that for XOR, where, due to limited resolution of diffraction data, assignment was based on the aldehyde oxidoreductase precedent (5); the MoAS and the MoAO group have switched positions, making the oxygen the apical ligand. The geometry described here, however, is consistent with a recently refined 1.7-Å structure of the salicylatebound form of XDH (B.T.E., K.O., T.N., and E.F.P., unpublished data) and is also similar to that of a recent high-resolution (1.1-Å) structure of CO dehydrogenase, although the latter contains an additional Cu ion in its active site (28).…”

Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park

“…Genes coding for chemolithotrophic pathways known to be associated with extreme environments (Inskeep et al, 2010;Kozubal et al, 2011), including the oxidation of ferrous iron, hydrogen, arsenic, sulfur, ammonium or methane, were not found in the NAG1 assemblies. However, the NAG1 populations have two separate loci that encode aerobic carbon monoxide (CO) dehydrogenases and associated maturases (Dobbek et al, 2002;King and Weber, 2007). One locus encodes a 'Form I' CO dehydrogenase along with coxFSM and the other contains three coxL 'Form II' CO dehydrogenase sequences along with coxDEFG (Supplementary Figures S5, S6).…”

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

“…Several crystal structures of other members of the family have also been determined [9][10][11][12][13][14]. The combination of structural and spectroscopic data on enzymes of this family has shown that the active site consists, in the Mo(VI) state, of a molybdenum atom in a squarepyramidal geometry that coordinates the two sulfur atoms from one pyranopterin ligand, one oxo ligand, one hydroxyl/water molecule and one sulfido ligand.…”