Pershad H R, Duff J L, Heering H A, Duin E C, Albracht S P, Armstrong F A
Department of Chemistry, Oxford University, U.K.
Biochemistry. 1999 Jul 13;38(28):8992-9. doi: 10.1021/bi990108v.
The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the 3Fe-4S cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two 4Fe-4S clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the 3Fe-4S cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.
来自嗜酒色杆菌的镍铁氢化酶吸附在热解石墨边缘平面（PGE）电极上，并在半电池反应2H⁺⇌H₂预期的电位下催化H⁺（aq）和H₂的快速相互转化，即无需过电位。伏安法反映了通过传统方法确定的特征，同时具备精确控制和测量电位依赖性活性以及底物-产物质量传输的能力。H₂的氧化极其迅速；在H₂分压为10%时，即使在最高电极旋转速率下，质量传输控制仍然存在。在30℃（pH 5 - 8）时，H₂氧化的周转数在1500 - 9000 s⁻¹范围内，这显著高于以亚甲基蓝作为电子受体时观察到的数值。相比之下，质子还原较慢且受酶中发生的过程控制。一氧化碳以可逆方式结合到活性形式的NiFe位点，抑制电催化作用，并使可归因于氧化还原中心可逆（非周转）氧化和还原的信号定义得到改善。在相对于标准氢电极（SHE）为 - 30 mV（pH 7.0，30℃）处的一个信号，根据电位测量结果被归属于[3Fe - 4S]⁺/⁰簇。第二个信号在 - 301 mV处，幅度大1.5 - 2.5倍，初步归属于两个具有相似还原电位的[4Fe - 4S]²⁺/⁺簇。未观察到其他氧化还原对，这表明这两组中心是CO抑制的氢化酶中仅有的能够进行简单快速氧化还原状态循环的中心。由于埋藏的NiFe活性位点极不可能与电极进行直接电子交换，三个铁硫簇中至少有一个且很可能每个都必须充当中继位点。即使在比[3Fe - 4S]⁺/⁰簇还原电位负近300 mV的电位下H₂氧化仍然迅速，这一事实表明其异常高的平衡还原电位并未损害催化效率。
