Publications

• Structural and functional reconstruction in situ of the [CuSMoO2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans.

  Resch M., Dobbek H., Meyer O.

  Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2) at a unique [CuSMoO(2)] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to C ... >> More

  Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2) at a unique [CuSMoO(2)] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to CO dehydrogenase preparations containing the mature and fully functional enzyme along with forms of the enzyme deficient in one or both of these elements. The active sites of mature and immature forms of CO dehydrogenase were converted into a [MoO(3)] centre by treatment with potassium cyanide. We have established a method, which rescues 50% of the CO dehydrogenase activity by in vitro reconstitution of the active site through the supply of sulphide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which were deficient in S and/or Cu at the active site, were similarly activated. X-ray crystallography and electron paramagnetic resonance spectroscopy indicated that the [CuSMoO(2)] cluster was properly reconstructed. However, reconstituted CO dehydrogenase contains mature along with immature forms. The chemical reactions of the reconstitution of CO dehydrogenase are summarized in a model, which assumes resulphuration of the Mo-ion at both equatorial positions at a 1:1 molar ratio. One equatorial Mo-S group reacts with Cu(I) in a productive fashion yielding a mature, functional [CuSMoO(2)] cluster. The other Mo-S group reacts with Cu(I), then Cu(2)S is released and an oxo group is introduced from water, yielding an inactive [MoO(3)] centre. << Less

  J Biol Inorg Chem 10:518-528(2005) [PubMed] [EuropePMC]

• The aerobic CO dehydrogenase from Oligotropha carboxidovorans.

  Hille R., Dingwall S., Wilcoxen J.

  We review here the recent literature dealing with the molybdenum- and copper-dependent CO dehydrogenase, with particular emphasis on the structure of the enzyme and recent advances in our understanding of the reaction mechanism of the enzyme.

  J Biol Inorg Chem 20:243-251(2015) [PubMed] [EuropePMC]

• Catalysis at a dinuclear CuSMo(==O)OH cluster in a CO dehydrogenase resolved at 1.1-A resolution.

  Dobbek H., Gremer L., Kiefersauer R., Huber R., Meyer O.

  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 r ... >> More

  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 [CuSMoO)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. The cluster is coordinated through interactions of the Mo with the dithiolate pyran ring of molybdopterin cytosine dinucleotide and of the Cu with the Sgamma of Cys-388, which is part of the active-site loop VAYRC(388)SFR. The previously reported active-site structure [Dobbek, H., Gremer, L., Meyer, O. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 8884-8889] of an Mo with three oxygen ligands and an SeH-group bound to the Sgamma atom of Cys-388 could not be confirmed. The structure of CO dehydrogenase with the inhibitor n-butylisocyanide bound has led to a model for the catalytic mechanism of CO oxidation which involves a thiocarbonate-like intermediate state. The dinuclear [CuSMo(O)OH] cluster of CO dehydrogenase establishes a previously uncharacterized class of dinuclear molybdoenzymes containing the pterin cofactor. << Less

  Proc. Natl. Acad. Sci. U.S.A. 99:15971-15976(2002) [PubMed] [EuropePMC]

• Reaction of the molybdenum- and copper-containing carbon monoxide dehydrogenase from Oligotropha carboxydovorans with quinones.

  Wilcoxen J., Zhang B., Hille R.

  Carbon monoxide dehydrogenase (CODH) from Oligotropha carboxydovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are ultimately passed to the ... >> More

  Carbon monoxide dehydrogenase (CODH) from Oligotropha carboxydovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are ultimately passed to the electron transport chain of the Gram-negative organism, but the proximal acceptor of reducing equivalents from the enzyme has not been established. Here we investigate the reaction of the reduced enzyme with various quinones and find them to be catalytically competent. Benzoquinone has a k(ox) of 125.1 s(-1) and a K(d) of 48 μM. Ubiquinone-1 has a k(ox)/K(d) value of 2.88 × 10(5) M(-1) s(-1). 1,4-Naphthoquinone has a k(ox) of 38 s(-1) and a K(d) of 140 μM. 1,2-Naphthoquinone-4-sulfonic acid has a k(ox)/K(d) of 1.31 × 10(5) M(-1) s(-1). An extensive effort to identify a cytochrome that could be reduced by CO/CODH was unsuccessful. Steady-state studies with benzoquinone indicate that the rate-limiting step is in the reductive half of the reaction (that is, the reaction of oxidized enzyme with CO). On the basis of the inhibition of CODH by diphenyliodonium chloride, we conclude that quinone substrates interact with CODH at the enzyme's flavin site. Our results strongly suggest that CODH donates reducing equivalents directly to the quinone pool without using a cytochrome as an intermediary. << Less

  Biochemistry 50:1910-1916(2011) [PubMed] [EuropePMC]

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

  Gnida M., Ferner R., Gremer L., Meyer O., Meyer-Klaucke W.

  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( ... >> More

  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. << Less

  Biochemistry 42:222-230(2003) [PubMed] [EuropePMC]

• Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.

  Gremer L., Kellner S., Dobbek H., Huber R., Meyer O.

  The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrog ... >> More

  The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein. << Less

  J Biol Chem 275:1864-1872(2000) [PubMed] [EuropePMC]

• Insights into the posttranslational assembly of the Mo-, S- and Cu-containing cluster in the active site of CO dehydrogenase of Oligotropha carboxidovorans.

  Pelzmann A.M., Mickoleit F., Meyer O.

  Oligotropha carboxidovorans is characterized by the aerobic chemolithoautotrophic utilization of CO. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. Kanamycin insertional mut ... >> More

  Oligotropha carboxidovorans is characterized by the aerobic chemolithoautotrophic utilization of CO. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. Kanamycin insertional mutants in coxE, coxF and coxG were characterized with respect to growth, expression of CO dehydrogenase, and the type of metal center present. These data along with sequence information were taken to delineate a model of metal cluster assembly. Biosynthesis starts with the MgATP-dependent, reductive sulfuration of [Mo(VI)O3] to [Mo(V)O2SH] which entails the AAA+-ATPase chaperone CoxD. Then Mo(V) is reoxidized and Cu(1+)-ion is integrated. Copper is supplied by the soluble CoxF protein which forms a complex with the membrane-bound von Willebrand protein CoxE through RGD-integrin interactions and enables the reduction of CoxF-bound Cu(2+), employing electrons from respiration. Copper appears as Cu(2+)-phytate, is mobilized through the phytase activity of CoxF and then transferred to the CoxF putative copper-binding site. The coxG gene does not participate in the maturation of the bimetallic cluster. Mutants in coxG retained the ability to utilize CO, although at a lower growth rate. They contained a regular CO dehydrogenase with a functional catalytic site. The presence of a pleckstrin homology (PH) domain on CoxG and the observed growth rates suggest a role of the PH domain in recruiting CO dehydrogenase to the cytoplasmic membrane enabling electron transfer from the enzyme to the respiratory chain. CoxD, CoxE and CoxF combine motifs of a DEAD-box RNA helicase which would explain their mutual translation. << Less

  J Biol Inorg Chem 19:1399-1414(2014) [PubMed] [EuropePMC]