The [NiFe]-Hydrogenase of Pyrococcus furiosus Exhibits a New Type of Oxygen Tolerance

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• 作者：Patrick Kwan ; Chelsea L. McIntosh ; David P. Jennings ; R. Chris Hopkins ; Sanjeev K. Chandrayan ; Chang-Hao Wu ; Michael W. W. Adams ; Anne K. Jones
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：October 28, 2015
• 年：2015
• 卷：137
• 期：42
• 页码：13556-13565
• 全文大小：489K
• ISSN：1520-5126

文摘

We report the first direct electrochemical characterization of the impact of oxygen on the hydrogen oxidation activity of an oxygen-tolerant, group 3, soluble [NiFe]-hydrogenase: hydrogenase I from Pyrococcus furiosus (PfSHI), which grows optimally near 100 掳C. Chronoamperometric experiments were used to probe the sensitivity of PfSHI hydrogen oxidation activity to both brief and prolonged exposure to oxygen. For experiments between 15 and 80 掳C, following short (<200 s) exposure to 14 渭M O₂ under oxidizing conditions, PfSHI always maintains some fraction of its initial hydrogen oxidation activity; i.e., it is oxygen-tolerant. Reactivation experiments show that two inactive states are formed by interaction with oxygen and both can be quickly (<150 s) reactivated. Analogous experiments, in which the interval of oxygen exposure is extended to 900 s, reveal that the response is highly temperature-dependent. At 25 掳C, under sustained 1% O₂/ 99% H₂ exposure, the H₂oxidation activity drops nearly to zero. However, at 80 掳C, up to 32% of the enzyme鈥檚 oxidation activity is retained. Reactivation of PfSHI following sustained exposure to oxygen occurs on a much longer time scale (tens of minutes), suggesting that a third inactive species predominates under these conditions. These results stand in contrast to the properties of oxygen-tolerant, group 1 [NiFe]-hydrogenases, which form a single state upon reaction with oxygen, and we propose that this new type of hydrogenase should be referred to as oxygen-resilient. Furthermore, PfSHI, like other group 3 [NiFe]-hydrogenases, does not possess the proximal [4Fe3S] cluster associated with the oxygen tolerance of some group 1 enzymes. Thus, a new mechanism is necessary to explain the observed oxygen tolerance in soluble, group 3 [NiFe]-hydrogenases, and we present a model integrating both electrochemical and spectroscopic results to define the relationships of these inactive states.