title: The [NiFe]-Hydrogenase of Pyrococcus furiosus Exhibits a New Type of Oxygen Tolerance.
abstract: 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 °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 °C, following short (<200 s) exposure to 14 μM 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 °C, under sustained 1% O2/ 99% H2 exposure, the H2oxidation activity drops nearly to zero. However, at 80 °C, up to 32% of the enzyme's 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.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kwan
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Chelsea L
authors.initials: CL
authors.lastname: McIntosh
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: David P
authors.initials: DP
authors.lastname: Jennings
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, Georgia 30602, United States.
authors.forename: R Chris
authors.initials: RC
authors.lastname: Hopkins
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Jones
mesh_terms: D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013696:Temperature
chemical_list: D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen