J. Bacteriol., 03 1996, 1515-1524, Vol 178, No. 6
Copyright © 1996, American Society for Microbiology

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme

JD Fox, RL Kerby, GP Roberts and PW Ludden
Department of Biochemistry, University of Wisconsin-Madison, Wisconsin 53706, USA.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C- terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.

This article has been cited by other articles:

• Maness, P.-C., Huang, J., Smolinski, S., Tek, V., Vanzin, G. (2005). Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus. Appl. Environ. Microbiol. 71: 2870-2874 [Abstract] [Full Text]  
• Roberts, G. P., Youn, H., Kerby, R. L. (2004). CO-Sensing Mechanisms. Microbiol. Mol. Biol. Rev. 68: 453-473 [Abstract] [Full Text]  
• Youn, H., Kerby, R. L., Conrad, M., Roberts, G. P. (2004). Functionally Critical Elements of CooA-Related CO Sensors. J. Bacteriol. 186: 1320-1329 [Abstract] [Full Text]  
• Sapra, R., Bagramyan, K., Adams, M. W. W. (2003). A simple energy-conserving system: Proton reduction coupled to proton translocation. Proc. Natl. Acad. Sci. USA 100: 7545-7550 [Abstract] [Full Text]  
• Maness, P.-C., Smolinski, S., Dillon, A. C., Heben, M. J., Weaver, P. F. (2002). Characterization of the Oxygen Tolerance of a Hydrogenase Linked to a Carbon Monoxide Oxidation Pathway in Rubrivivax gelatinosus. Appl. Environ. Microbiol. 68: 2633-2636 [Abstract] [Full Text]  
• Meuer, J., Kuettner, H. C., Zhang, J. K., Hedderich, R., Metcalf, W. W. (2002). Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation. Proc. Natl. Acad. Sci. USA 99: 5632-5637 [Abstract] [Full Text]  
• Buhrke, T., Bleijlevens, B., Albracht, S. P. J., Friedrich, B. (2001). Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha. J. Bacteriol. 183: 7087-7093 [Abstract] [Full Text]  
• Jeon, W. B., Cheng, J., Ludden, P. W. (2001). Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum. J. Biol. Chem. 276: 38602-38609 [Abstract] [Full Text]  
• Svetlitchnyi, V., Peschel, C., Acker, G., Meyer, O. (2001). Two Membrane-Associated NiFeS-Carbon Monoxide Dehydrogenases from the Anaerobic Carbon-Monoxide-Utilizing Eubacterium Carboxydothermus hydrogenoformans. J. Bacteriol. 183: 5134-5144 [Abstract] [Full Text]  
• Sapra, R., Verhagen, M. F. J. M., Adams, M. W. W. (2000). Purification and Characterization of a Membrane-Bound Hydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus. J. Bacteriol. 182: 3423-3428 [Abstract] [Full Text]  
• Watt, R. K., Ludden, P. W. (1999). Ni2+ Transport and Accumulation in Rhodospirillum rubrum. J. Bacteriol. 181: 4554-4560 [Abstract] [Full Text]  
• Uchida, T., Ishikawa, H., Takahashi, S., Ishimori, K., Morishima, I., Ohkubo, K., Nakajima, H., Aono, S. (1998). Heme Environmental Structure of CooA Is Modulated by the Target DNA Binding. EVIDENCE FROM RESONANCE RAMAN SPECTROSCOPY AND CO REBINDING KINETICS. J. Biol. Chem. 273: 19988-19992 [Abstract] [Full Text]  
• Watt, R. K., Ludden, P. W. (1998). The Identification, Purification, and Characterization of CooJ. A NICKEL-BINDING PROTEIN THAT IS CO-REGULATED WITH THE Ni-CONTAINING CO DEHYDROGENASE FROM RHODOSPIRILLUM RUBRUM. J. Biol. Chem. 273: 10019-10025 [Abstract] [Full Text]  
• Rakhely, G., Colbeau, A., Garin, J., Vignais, P. M., Kovacs, K. L. (1998). Unusual Organization of the Genes Coding for HydSL, the Stable [NiFe]Hydrogenase in the Photosynthetic Bacterium Thiocapsa roseopersicina BBS. J. Bacteriol. 180: 1460-1465 [Abstract] [Full Text]  
• Shelver, D., Kerby, R. L., He, Y., Roberts, G. P. (1997). CooA, a CO-sensing transcription factor from Rhodospirillum rubrum, is a CO-binding heme protein. Proc. Natl. Acad. Sci. USA 94: 11216-11220 [Abstract] [Full Text]  
• Meuer, J., Kuettner, H. C., Zhang, J. K., Hedderich, R., Metcalf, W. W. (2002). Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation. Proc. Natl. Acad. Sci. USA 99: 5632-5637 [Abstract] [Full Text]