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[Nife]-Hydrogenases James A A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases James A. Cracknella, Annemarie F. Waita, Oliver Lenzb,Ba¨ rbel Friedrichb, and Fraser A. Armstronga,1 aDepartment of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom; and bInstitut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved September 16, 2009 (received for review May 29, 2009) In biology, rapid oxidation and evolution of H2 is catalyzed by attack’’ encompasses its access to the [NiFe]-active site and its metalloenzymes known as hydrogenases. These enzymes have subsequent reaction to form an inactive state. These reactions unusual active sites, consisting of iron complexed by carbonyl, are summarized in Fig. 1. cyanide, and thiolate ligands, often together with nickel, and are In contrast with Df hydrogenase, the membrane-bound typically inhibited or irreversibly damaged by O2. The Knallgas [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 (Re) bacterium Ralstonia eutropha H16 (Re) uses H2 as an energy source can oxidize H2 [and, in vitro, also reduce protons to H2 in the with O2 as a terminal electron acceptor, and its membrane-bound reverse reaction (6)] in the presence of air (7). As a ‘‘Knallgas’’ uptake [NiFe]-hydrogenase (MBH) is an important example of an bacterium, Re uses H2 as an alternative energy source with O2 as ‘‘O2-tolerant’’ hydrogenase. The mechanism of O2 tolerance of Re the terminal electron acceptor. An O2-tolerant hydrogenase is MBH has been probed by measuring H2 oxidation activity in the therefore essential for this metabolic pathway of energy conser- presence of O2 over a range of potential, pH and temperature, and vation and, more generally, would be crucial for other microor- comparing with the same dependencies for individual processes ganisms oxidizing H2 in environments where O2 may be found. involved in the attack by O2 and subsequent reactivation of the Protein film electrochemistry (PFE) has proved especially active site. Most significantly, O2 tolerance increases with increas- useful in probing the reactions of hydrogenases in air (2). In this ing temperature and decreasing potentials. These trends correlate technique, small amounts of enzyme are adsorbed onto an with the trends observed for reactivation kinetics but not for H2 electrode such that they retain native catalytic activity. Catalytic SCIENCES affinity or the kinetics of O2 attack. Clearly, the rate of recovery is activity is proportional to the electrical current, and is directly a crucial factor. We present a kinetic and thermodynamic model to controlled through a precisely applied electrode potential. A key APPLIED BIOLOGICAL account for O2 tolerance in Re MBH that may be more widely advantage of PFE over solution techniques, especially when applied to other [NiFe]-hydrogenases. measuring enzyme activity in air, is that soluble electron donors/ acceptors (which invariably react with O2) are not required. By electrochemistry ͉ hydrogen ͉ hydrogenase ͉ oxygen tolerance ͉ using PFE, we recently showed that the O2 tolerance of the MBH Ralstonia eutropha enzyme from Ralstonia metallidurans CH34 (Rm, closely related to Re) is so effective that the oxidation of even nM levels of H2 CHEMISTRY in air can be detected (8), consistent with the very low threshold ydrogenases play a crucial role in the metabolism of many limits for H uptake determined by Conrad et al. (9). A practical microorganisms, where they catalyze the reversible oxida- 2 H demonstration of O tolerance was provided by a membraneless tion and production of H . Hydrogenases possess active sites 2 2 fuel cell producing power from 3% H in air (10, 11). containing either one Ni and one Fe atom (‘‘[NiFe]- 2 Crystallographic and computational studies have suggested hydrogenases’’), or only Fe atoms (‘‘[FeFe]-hydrogenases’’), that hydrophobic ‘‘gas channels’’ provide a selective filter to coordinated by cysteine thiolates and the biologically unusual restrict the access of small molecules such as O and CO in ligands CO and CNϪ. (A third class, the Hmd or [Fe]- 2 hydrogenases (3). Duche et al. (12) reported how enlarging the hydrogenases (1), will not be discussed here.) Hydrogenases are gas channel in the regulatory hydrogenase from Rhodobacter highly active, with turnover frequencies for H2 oxidation (be- capsulatus increased its O2-sensitivity. We used a similar strategy lieved to occur through a heterolytic cleavage mechanism) in with Re MBH, anticipating that a restricted gas channel would excess of thousands of molecules of H2 per second at 30° C (2). make the enzyme even more O2 tolerant; however, no such effect Hydrogenases are usually reported to be highly O2-sensitive, was observed. Clearly, gas channels alone cannot confer O2 being inactivated or irreversibly damaged by even trace O2.Itis tolerance. Most significantly, recent EPR and FTIR studies on generally considered that [FeFe]-hydrogenases react irreversibly Re MBH showed that reaction with O2 even under electron- with O2, giving rise to as yet poorly characterized inactive deficient conditions produces only the Ready state; no Ni-A products, whereas [NiFe]-hydrogenases react with O2 to give (Unready) was detected (13). products that can be reactivated upon reduction. In this paper, we describe PFE studies on Re MBH that offer The well-characterized ‘‘standard’’ [NiFe]-hydrogenases from valuable insight into the kinetic and thermodynamic aspects of Desulfovibrio species, such as Desulfovibrio fructosovorans (Df), O2 tolerance and support a model that may be generally useful. cannot oxidize H2 in the presence of O2. Exposure to O2 under electron-rich conditions produces mainly the Ready state (also Results known as ‘‘Ni-B’’), in which an HOϪ ligand is bound in a bridging Defining an O2 Tolerance Factor. We measured the inhibitory effect position between the Ni and Fe atoms (3). This state is also of O2 on H2 oxidation activity over a wide range of pH values, produced under anaerobic oxidizing conditions, and can be rapidly recovered by applying a reducing potential under H2. Exposure to O2 under electron-deficient conditions produces Author contributions: J.A.C. designed research; J.A.C. and A.F.W. performed research; mainly the Unready state (also known as ‘‘Ni-A’’), in which a J.A.C. and A.F.W. analyzed data; and J.A.C., A.F.W., O.L., B.F., and F.A.A. wrote the paper. peroxo group is believed to occupy the bridging position, al- The authors declare no conflict of interest. though other modifications may occur, such as the oxidation of This article is a PNAS Direct Submission. sulfur ligands (3–5). Although the Unready state can be activated 1To whom correspondence should be addressed. E-mail: [email protected]. by applying a reducing potential under H2, its recovery is many This article contains supporting information online at www.pnas.org/cgi/content/full/ orders of magnitude slower than that of Ready. The term ‘‘O2 0905959106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905959106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 27, 2021 H2 Table 2. Values of KM under a range of conditions H2 Variable KM , ␮M Potential, V vs. SHE* Ϫ0.158 8 Ϫ0.058 6 ϩ0.042 14 ϩ0.142 Ϸ100 ϩ0.242 Ϸ130 pH† 4.5 6 5.0 8 5.5 6 6.0 15 6.5 10 Temperature, ° C‡ Fig. 1. A simplified scheme showing the reactions of standard [NiFe]- 10 0.4 ϩ hydrogenases. Active hydrogenase molecules catalyze H2/H cycling, and are 20 1 subject to aerobic inactivation, forming either the Ready or Unready forms, 30 6 depending on the availability of electrons and protons. The Ready form can 40 24 also be formed anaerobically at oxidizing potentials. (Adapted from ref. 20 with permission from the Royal Society of Chemistry.) *All values were recorded at pH 5.5, 30° C. †All values were recorded at 30° C, at a constant overpotential (driving force) ϩ of ϩ273 mV relative to the thermodynamic H /H2 cell potential at each pH. ‡ O2 concentrations, electrode potentials, and temperatures. From All values were recorded at pH 5.5, at a constant overpotential (driving force) of ϩ273 mV relative to the thermodynamic Hϩ/H cell potential at each these measurements, we obtained values for the ‘‘O2 tolerance 2 O2,app temperature. factor,’’ KI , by using the method described previously (14, 15). The O2 tolerance factor is the O2 concentration required to attenuate H oxidation activity by 50%; therefore, a high value 2 that O tolerance does not rely on a selective filter, such as a indicates a high level of O tolerance. The H oxidation current 2 2 2 restrictive gas channel. at a pyrolytic graphite ‘‘edge’’ (PGE) electrode modified with Re MBH was monitored following a succession of injections of O 2 Michaelis Constants for H . The O tolerance of a hydrogenase in gas into the electrochemical cell headspace. After correcting for 2 2 O2,app H2 oxidation is expected to depend on the enzyme’s affinity for film loss over time, KI was obtained by analyzing the catalytic H2 because of competition for binding at the active site. We current that stabilizes at each O2 concentration. Details are H2 provided in Methods and values are summarized in Table 1. define KM as the Michaelis constant for H2. We used the H2 method described previously (14, 15) to determine KM for Re Significantly, O2 tolerance increases with decreasing electrode potential (i.e., more reducing conditions) and with increasing MBH over a range of pH, potentials, and temperatures (Table O2,app 2). A known volume of H -saturated buffer solution was intro- temperature. It appears that KI may decrease at high pH, but 2 we were unable to extend the pH range studied as the enzyme duced into the electrochemical cell, and the H2 was then activity became too low.
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