Principles of Sustained Enzymatic Hydrogen Oxidation in the Presence of Oxygen 鈥?The Crucial Influence of High Potential Fe鈥揝 Clusters in the Electron Relay of [NiFe]-Hydrogenases

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• 作者：Rhiannon M. Evans ; Alison Parkin ; Maxie M. Roessler ; Bonnie J. Murphy ; Hope Adamson ; Michael J. Lukey ; Frank Sargent ; Anne Volbeda ; Juan C. Fontecilla-Camps ; Fraser A. Armstrong
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：February 20, 2013
• 年：2013
• 卷：135
• 期：7
• 页码：2694-2707
• 全文大小：678K
• 年卷期：v.135,no.7(February 20, 2013)
• ISSN：1520-5126

文摘

鈥淗yd-1鈥? produced by Escherichia coli, exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H₂ oxidation activity in the presence of O₂鈥攁n intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of 鈥淥₂ tolerance鈥?involves a critical role for the Fe鈥揝 clusters of the electron relay, which is to ensure the availability鈥攆or immediate transfer back to the active site鈥攐f all of the electrons required to reduce an attacking O₂ molecule completely to harmless H₂O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 脜 from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H₂ oxidation activity in 1% O₂. The [3Fe-4S] cluster is essential only for the first stage of complete O₂ reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state 鈥淣i鈥揃鈥?or 鈥淩eady鈥?(Ni(III)鈥揙H). Potentiometric titrations show that Ni鈥揃 is easily reduced (E_m 鈮?+0.1 V at pH 6.0); this final stage of the O₂-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme鈥檚 response to O₂, viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H₂ oxidation activity; thus, O₂ behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe鈥揝 clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase.