Abstract
Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO2 at a unique [CuSMoO2] 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 [MoO3] 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 [CuSMoO2] 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 [CuSMoO2] cluster. The other Mo–S group reacts with Cu(I), then Cu2S is released and an oxo group is introduced from water, yielding an inactive [MoO3] centre.
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Abbreviations
- AAS:
-
Atomic absorption spectroscopy
- EPR:
-
Electron paramagnetic resonance
- MCD:
-
Molybdopterin cytosine dinucleotide
- OD:
-
optical density
- ORF:
-
open reading frame
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulphate
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Acknowledgements
We thank Dr. Lothar Gremer and Jörg Johannes (Microbiology, University of Bayreuth) for the introduction of Cu–thiourea in the reconstitution of CO dehydrogenase, Dr. Lothar Gremer for providing a sample of CO dehydrogenase, and Dr. Reiner Kiefersauer and Professor Robert Huber (Max-Planck-Institut für Biochemie, Martinsried) for the transformation of crystals. This work was supported by grants from by the Deutsche Forschungsgemeinschaft to Ortwin Meyer (ME 732/8-1) and Holger Dobbek (DO 785/1), the Fonds der Chemischen Industrie (Frankfurt am Main, Germany), and contains instrumentation purchased with funds from the Freistaat Bayern, the Bundesministerium für Bildung und Forschung, and the Deutsche Forschungsgemeinschaft.
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Resch, M., Dobbek, H. & Meyer, O. Structural and functional reconstruction in situ of the [CuSMoO2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans . J Biol Inorg Chem 10, 518–528 (2005). https://doi.org/10.1007/s00775-005-0006-4
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DOI: https://doi.org/10.1007/s00775-005-0006-4