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 H-2 and, at much lower rates, oxidize H-2 to protons and electrons. To our knowledge, this if the first time that PFOR has been shown to have hydrogenase activity. CODWACS and PFOR evolved H-2 at maximum rates when CO and pyruvate were the electron donors, respectively, and when electron accepters are absent; dithionite was a very poor substitute. PFOR, when purified to greater than 99% homogeneity, exhibited a specific activity for pyruvate-dependent H-2 production of 135 nmol min(-1) mg(-1). The Ha evolution activity divided by the H-2 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 CODWACS (>99% homogeneity) exhibited a specific activity of GO-dependent H-2 evolution in the absence of electron carrier of 590 nmol min(-1) mg(-)1. Equivalent rates of CO oxidation and H-2 production were observed when determined in the absence of electron acceptor. This level of activity can account for the rate of H-2 production that has been observed by growing cultures of Clostridium thermoaceticum and could solve the paradox that the highly GO-sensitive hydrogenases from acetogenic bacteria evolve H-2 when grown on CO. The ratio of the rates of (H-2 evolution):(H-2 uptake) for purified CODH/ACS is between 20:1 and 30:1. H-2 evolution and uptake by CODWACS were strongly inhibited by cyanide (K-i = 1 mu M), 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.