Unleashing hydrogenase activity in carbon monoxide dehydrogenase/acetyl-CoA synthase and pyruvate:ferredoxin oxidoreductase.
These results demonstrate that two well-studied metalloenzymes, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and pyruvate:ferredoxin oxidoreductase (PFOR), can reduce protons to H2 and, at much lower rates, oxidize H2 to protons and electrons. To our knowledge, this if the first time that PFOR has been shown to have hydrogenase activity. CODH/ACS and PFOR evolved H2 at maximum rates when CO and pyruvate were the electron donors, respectively, and when electron acceptors are absent; dithionite was a very poor substitute. PFOR, when purified to greater than 99% homogeneity, exhibited a specific activity for pyruvate-dependent H2 production of 135 nmol min-1 mg-1. The H2 evolution activity divided by the H2 uptake activity was 282:1; the highest ratio previously reported (22:1) was with the membrane-bound hydrogenase from Rhodospirillum rubrum [Fox, J.D., Kerby, R. L., Roberts, G. P., & Ludden, P. W. (1996) J. Bacteriol. 178, 1515-1524]. Highly purified samples of CODH/ACS (> 99% homogeneity) exhibited a specific activity of CO-dependent H2 evolution in the absence of electron carrier of 590 nmol min-1 mg-1. Equivalent rates of CO oxidation and H2 production were observed when determined in the absence of electron acceptor. This level of activity can account for the rate of H2 production that has been observed by growing cultures of Clostridium thermoaceticum and could solve the paradox that the highly CO-sensitive hydrogenases from acetogenic bacteria evolve H2 when grown on CO. The ratio of the rates of (H2 evolution):(H2 uptake) for purified CODH/ACS is between 20:1 and 30:1. H2 evolution and uptake by CODH/ACS were strongly inhibited by cyanide (ki = 1 microM), indicating that these reactions are catalyzed by cluster C, the site of CO oxidation. Our results extend earlier findings that the CODHs from Methanosarcina barkeri [Bhatnagar, L., Krzycki, J. A., & Zeikus, J. G. (1987) FEMS Microbiol. Lett. 41, 337-343] and Oligotropha carboxydovorans [Santiago, B., & Meyer, O. (1996) FEMS Microbiol. Lett. 136, 157-162] exhibit hydrogenase activity. Mechanistic implications of hydrogenase activity are discussed. Several physiological roles for proton reduction by CODH/ACS and PFOR are discussed, including the prevention of radical formation from reduced metal clusters when electron carriers (ferredoxin, flavodoxin, etc.) are limiting.
Menon
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Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln 68588-0664, USA.
Ragsdale
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SW
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D003013:Clostridium / Q000201:enzymology*; D004579:Electron Transport; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D011190:Potassium Cyanide / Q000494:pharmacology; D011522:Protons; D050743:Pyruvate Synthase; D019289:Pyruvic Acid / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D009097:Multienzyme Complexes; D011522:Protons; D000105:Acetyl Coenzyme A; D002248:Carbon Monoxide; D006859:Hydrogen; D019289:Pyruvic Acid; D006864:Hydrogenase; D000445:Aldehyde Oxidoreductases; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; C019823:carbon monoxide dehydrogenase; D011190:Potassium Cyanide; D010269:Paraquat; D013835:Thiamine Pyrophosphate