Menon S, Ragsdale S W
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln 68588-0664, USA.
Biochemistry. 1996 Dec 10;35(49):15814-21. doi: 10.1021/bi9615598.
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.
这些结果表明，两种经过充分研究的金属酶，一氧化碳脱氢酶/乙酰辅酶A合酶（CODH/ACS）和丙酮酸：铁氧化还原蛋白氧化还原酶（PFOR），能够将质子还原为H₂，并且以低得多的速率将H₂氧化为质子和电子。据我们所知，这是首次证明PFOR具有氢化酶活性。当分别以CO和丙酮酸作为电子供体且无电子受体时，CODH/ACS和PFOR以最大速率产生H₂；连二亚硫酸盐是一种非常差的替代物。当纯化至同质性大于99%时，PFOR对依赖丙酮酸的H₂产生的比活性为135 nmol min⁻¹ mg⁻¹。H₂产生活性与H₂摄取活性之比为282:1；先前报道的最高比值（22:1）是来自红螺菌的膜结合氢化酶[Fox, J.D., Kerby, R. L., Roberts, G. P., & Ludden, P. W. (1996) J. Bacteriol. 178, 1515 - 1524]。高度纯化的CODH/ACS样品（>99%同质性）在无电子载体时依赖CO的H₂产生的比活性为590 nmol min⁻¹ mg⁻¹。在无电子受体时测定，观察到CO氧化和H₂产生的速率相当。这种活性水平可以解释在热醋梭菌培养物中观察到的H₂产生速率，并可以解决产乙酸细菌中对CO高度敏感的氢化酶在以CO为生长底物时产生H₂这一矛盾现象。纯化的CODH/ACS的（H₂产生）：（H₂摄取）速率之比在20:1至30:1之间。CODH/ACS的H₂产生和摄取受到氰化物的强烈抑制（ki = 1 microM），表明这些反应由簇C催化，簇C是CO氧化的位点。我们的结果扩展了早期的发现，即巴氏甲烷八叠球菌[Bhatnagar, L., Krzycki, J. A., & Zeikus, J. G. (1987) FEMS Microbiol. Lett. 41, 337 - 343]和嗜羧寡养菌[Santiago, B., & Meyer, O. (1996) FEMS Microbiol. Lett. 136, 157 - 162]的CODH表现出氢化酶活性。讨论了氢化酶活性的机制意义。还讨论了CODH/ACS和PFOR还原质子的几种生理作用，包括当电子载体（铁氧化还原蛋白、黄素氧化还原蛋白等）有限时防止还原态金属簇形成自由基。
