A kinetic and thermodynamic understanding of O₂ tolerance in [NiFe]-hydrogenases

Abstract

In biology, rapid oxidation and evolution of H₂ is catalyzed by metalloenzymes known as hydrogenases. These enzymes have unusual active sites, consisting of iron complexed by carbonyl, cyanide, and thiolate ligands, often together with nickel, and are typically inhibited or irreversibly damaged by O₂. The Knallgas bacterium Ralstonia eutropha H16 (Re) uses H₂ as an energy source with O₂ as a terminal electron acceptor, and its membrane-bound uptake [NiFe]-hydrogenase (MBH) is an important example of an “O₂-tolerant” hydrogenase. The mechanism of O₂ tolerance of Re MBH has been probed by measuring H₂ oxidation activity in the presence of O₂ over a range of potential, pH and temperature, and comparing with the same dependencies for individual processes involved in the attack by O₂ and subsequent reactivation of the active site. Most significantly, O₂ tolerance increases with increasing temperature and decreasing potentials. These trends correlate with the trends observed for reactivation kinetics but not for H₂ affinity or the kinetics of O₂ attack. Clearly, the rate of recovery is a crucial factor. We present a kinetic and thermodynamic model to account for O₂ tolerance in Re MBH that may be more widely applied to other [NiFe]-hydrogenases.

Hydrogenases play a crucial role in the metabolism of many microorganisms, where they catalyze the reversible oxidation and production of H₂. Hydrogenases possess active sites containing either one Ni and one Fe atom (“[NiFe]-hydrogenases”), or only Fe atoms (“[FeFe]-hydrogenases”), coordinated by cysteine thiolates and the biologically unusual ligands CO and CN⁻. (A third class, the Hmd or [Fe]-hydrogenases (1), will not be discussed here.) Hydrogenases are highly active, with turnover frequencies for H₂ oxidation (believed to occur through a heterolytic cleavage mechanism) in excess of thousands of molecules of H₂ per second at 30° C (2). Hydrogenases are usually reported to be highly O₂-sensitive, being inactivated or irreversibly damaged by even trace O₂. It is generally considered that [FeFe]-hydrogenases react irreversibly with O₂, giving rise to as yet poorly characterized inactive products, whereas [NiFe]-hydrogenases react with O₂ to give products that can be reactivated upon reduction.

The well-characterized “standard” [NiFe]-hydrogenases from Desulfovibrio species, such as Desulfovibrio fructosovorans (Df), cannot oxidize H₂ in the presence of O₂. Exposure to O₂ under electron-rich conditions produces mainly the Ready state (also known as “Ni-B”), in which an HO⁻ ligand is bound in a bridging position between the Ni and Fe atoms (3). This state is also 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 mainly the Unready state (also known as “Ni-A”), in which a peroxo group is believed to occupy the bridging position, although other modifications may occur, such as the oxidation of sulfur ligands (3–5). Although the Unready state can be activated by applying a reducing potential under H₂, its recovery is many orders of magnitude slower than that of Ready. The term “O₂ attack” encompasses its access to the [NiFe]-active site and its subsequent reaction to form an inactive state. These reactions are summarized in Fig. 1.

Fig. 1.

A simplified scheme showing the reactions of standard [NiFe]-hydrogenases. Active hydrogenase molecules catalyze H₂/H⁺cycling, and are subject to aerobic inactivation, forming either the Ready or Unready forms, depending on the availability of electrons and protons. The Ready form can also be formed anaerobically at oxidizing potentials. (Adapted from ref. 20 with permission from the Royal Society of Chemistry.)

In contrast with Df hydrogenase, the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 (Re) can oxidize H₂ [and, in vitro, also reduce protons to H₂ in the reverse reaction (6)] in the presence of air (7). As a “Knallgas” bacterium, Re uses H₂ as an alternative energy source with O₂ as the terminal electron acceptor. An O₂-tolerant hydrogenase is therefore essential for this metabolic pathway of energy conservation and, more generally, would be crucial for other microorganisms oxidizing H₂ in environments where O₂ may be found.

Protein film electrochemistry (PFE) has proved especially useful in probing the reactions of hydrogenases in air (2). In this technique, small amounts of enzyme are adsorbed onto an electrode such that they retain native catalytic activity. Catalytic activity is proportional to the electrical current, and is directly controlled through a precisely applied electrode potential. A key advantage of PFE over solution techniques, especially when measuring enzyme activity in air, is that soluble electron donors/acceptors (which invariably react with O₂) are not required. By using PFE, we recently showed that the O₂ tolerance of the MBH enzyme from Ralstonia metallidurans CH34 (Rm, closely related to Re) is so effective that the oxidation of even nM levels of H₂ in air can be detected (8), consistent with the very low threshold limits for H₂ uptake determined by Conrad et al. (9). A practical demonstration of O₂ tolerance was provided by a membraneless fuel cell producing power from 3% H₂ in air (10, 11).

Crystallographic and computational studies have suggested that hydrophobic “gas channels” provide a selective filter to restrict the access of small molecules such as O₂ and CO in hydrogenases (3). Duche et al. (12) reported how enlarging the gas channel in the regulatory hydrogenase from Rhodobacter capsulatus increased its O₂-sensitivity. We used a similar strategy with Re MBH, anticipating that a restricted gas channel would make the enzyme even more O₂ tolerant; however, no such effect was observed. Clearly, gas channels alone cannot confer O₂ tolerance. Most significantly, recent EPR and FTIR studies on Re MBH showed that reaction with O₂ even under electron-deficient conditions produces only the Ready state; no Ni-A (Unready) was detected (13).

In this paper, we describe PFE studies on Re MBH that offer valuable insight into the kinetic and thermodynamic aspects of O₂ tolerance and support a model that may be generally useful.

Results

Defining an O₂ Tolerance Factor.

We measured the inhibitory effect of O₂ on H₂ oxidation activity over a wide range of pH values, O₂ concentrations, electrode potentials, and temperatures. From these measurements, we obtained values for the “O₂ tolerance factor,” K_I^O_2,app, by using the method described previously (14, 15). The O₂ tolerance factor is the O₂ concentration required to attenuate H₂ oxidation activity by 50%; therefore, a high value indicates a high level of O₂ tolerance. The H₂ oxidation current at a pyrolytic graphite “edge” (PGE) electrode modified with Re MBH was monitored following a succession of injections of O₂ gas into the electrochemical cell headspace. After correcting for film loss over time, K_I^O_2,app was obtained by analyzing the catalytic current that stabilizes at each O₂ concentration. Details are provided in Methods and values are summarized in Table 1. Significantly, O₂ tolerance increases with decreasing electrode potential (i.e., more reducing conditions) and with increasing temperature. It appears that K_I^O_2,app may decrease at high pH, but we were unable to extend the pH range studied as the enzyme activity became too low. These observations suggest immediately that O₂ tolerance does not rely on a selective filter, such as a restrictive gas channel.

Table 1.

Values of K_I^O_2,app under a range of conditions

Variable	KIO2,app, μM
Potential, V vs. SHE*
−0.018	1,630
+0.052	390
+0.122	110
+0.192	40
+0.262	8
pH†
4.5	70
5.0	80
5.5	110
6.0	90
6.5	70
Temperature, ° C‡
10	70
20	70
30	110
40	140

*All values were recorded at pH 5.5, 30° C.

^†All values were recorded at 30° C, at a constant overpotential (driving force) of +453 mV relative to the thermodynamic H⁺/H₂ cell potential at each pH.

^‡ All values were recorded at pH 5.5, at a constant overpotential (driving force) of +453 mV relative to the thermodynamic H⁺/H₂ cell potential at each temperature.

Michaelis Constants for H₂.

The O₂ tolerance of a hydrogenase in H₂ oxidation is expected to depend on the enzyme's affinity for H₂ because of competition for binding at the active site. We define K_M^H₂ as the Michaelis constant for H₂. We used the method described previously (14, 15) to determine K_M^H₂ for Re MBH over a range of pH, potentials, and temperatures (Table 2). A known volume of H₂-saturated buffer solution was introduced into the electrochemical cell, and the H₂ was then removed by a constant flow of inert gas, typically N₂. Values of K_M^H₂ were determined by analyzing the current vs. time trace as described in refs. 14 and 15.

Table 2.

Values of K_M^H₂ under a range of conditions

Variable	KMH2, μM
Potential, V vs. SHE*
−0.158	8
−0.058	6
+0.042	14
+0.142	≈100
+0.242	≈130
pH†
4.5	6
5.0	8
5.5	6
6.0	15
6.5	10
Temperature, ° C‡
10	0.4
20	1
30	6
40	24

*All values were recorded at pH 5.5, 30° C.

^†All values were recorded at 30° C, at a constant overpotential (driving force) of +273 mV relative to the thermodynamic H⁺/H₂ cell potential at each pH.

^‡All values were recorded at pH 5.5, at a constant overpotential (driving force) of +273 mV relative to the thermodynamic H⁺/H₂ cell potential at each temperature.

Rates of O₂ Reaction.

The rate of reaction of O₂ at the active site of Re MBH was probed by analyzing the current decay following O₂ introduction at a range of potentials, pH, and temperatures. A typical experiment is shown in Fig. 2A. An electrode modified with Re MBH was placed in a gas-tight electrochemical cell under an atmosphere of 50% H₂ and 50% N₂. The electrode was initially polarized at −0.508 V to ensure that all of the enzyme was active. After 120 s, the potential was stepped to the value at which the rate of O₂ reaction was to be determined. After allowing a background current to be determined, the atmosphere was changed to 50% H₂, 25% O₂, and 25% N₂; simultaneously, an aliquot of temperature-equilibrated buffer solution, saturated with 50% H₂ and 50% O₂ was injected into the cell solution. Under the experimental conditions, the mixing time is ≈0.1 s and therefore insignificant on the experimental timescale. The decrease in current that begins immediately is purely due to O₂ reaction at the active site; at the potentials used, direct O₂ reduction at the electrode surface is negligible, and, crucially, following the introduction of O₂, neither H₂ nor O₂ concentrations change over the course of the measurement. After correcting data for anaerobic inactivation and film loss (Fig. 2A), the rate of O₂ reaction was determined (Fig. 2B). Typically, the reaction was first order for more than two half-lives, and rate constants are presented in Table 3 (see also Table S1 of the SI Appendix; trends within sets of experiments are independent of the method used to determine the rates).

Fig. 2.

A typical experiment to determine the rate of O₂ reaction with Re MBH. (A) The current vs. time trace (black) with the fit to film loss/anaerobic inactivation (gray). (B) The corrected data (black), with the fit to the initial attack by O₂ (gray). Experimental conditions were pH 5.5, 10° C, +0.192 V, electrode rotation rate = 4,500 rpm.

Table 3.

Calculated rates of reaction of O₂ (25%) with Re MBH under a range of conditions, electrode rotation rate = 4500 rpm

Variable	Rate of O2 reaction, s−1
Potential, V vs. SHE*
+0.192	0.11
+0.262	0.10
+0.332	0.10
+0.402	0.11
pH†
4.5	0.12
5.5	0.11
6.5	0.11
Temperature, ° C‡
0	0.06
10	0.11
20	0.19
30	0.31

*All values were recorded at pH 5.5, 10° C.

^†All values were recorded at 10° C, at a constant overpotential (driving force) of +523 mV relative to the thermodynamic H⁺/H₂ cell potential at each pH.

^‡All values were recorded at pH 5.5, at a constant overpotential (driving force) of +523 mV relative to the thermodynamic H⁺/H₂ cell potential at each temperature.

The temperature dependence of the rate constants gave a linear fit to the Eyring equation with an activation enthalpy of ≈34 kJ mol⁻¹. We also measured the rate of O₂ reaction with the O₂-sensitive [NiFe]-hydrogenase from Desulfovibrio gigas by using the same method; a rate of 0.73 s⁻¹ was calculated at pH 6.5, 10° C, +0.192 V.

Rates of Reactivation.

Rate and potential dependencies of recovery from both anaerobic and aerobic inactivation were measured at 10° C (the rate of recovery at 30° C is so rapid that Re MBH regains full activity within the “dead time” caused by the release of electrode capacitive charge). To measure the rate of recovery after anaerobic inactivation, an electrode modified with Re MBH was placed in a gas-tight cell under a constant flow of H₂. After 120 s at −0.508 V, the electrode was stepped to a high potential (+0.442 V), causing the enzyme to anaerobically inactivate. After 900 s, the electrode was stepped to the potential at which the rate of recovery was to be measured. A single exponential was fitted to the current vs. time trace (Fig. 3A; data from the first 10 s following the potential step were within the dead time and thus not included).

Fig. 3.

Reactivation of aerobically and anaerobically inactivated states of Re MBH. (A and B) Typical experiments to measure the rate of reactivation of Re MBH under 100% H₂ following anaerobic (A) and aerobic (B) inactivation. In these examples, reactivation was measured at +0.192 V. (C) Rates of reactivation as a function of potential with a fit to the data overlaid. Data for the reactivation of Av MBH from the Unready state (filled squares, taken from reference 16, pH 6, 45° C) are also shown, and have been multiplied by 10 for clarity (unfilled squares). Sigmoidal fits are also shown. (D) Voltammograms (100% H₂, 0.1 mV s⁻¹) of Re MBH (bold) and Av MBH (dashed), showing recovery after aerobic inactivation at 392 mV under N₂. All at pH 5.5, 10° C, electrode rotation rate = 2,500 rpm.

Experiments to measure the rate of reactivation following aerobic inactivation were more complex (Fig. 3B). Again, the electrode was held, initially, for 120 s at −0.508 V under 100% H₂. The potential was then stepped to +0.092 V. (At this potential, anaerobic inactivation is negligible.) The cell headgas was then changed from 100% H₂ to 80% H₂ and 20% O₂, causing the current to decrease as the enzyme becomes aerobically inactivated. After 900 s, the current reaches a plateau level, at which point the electrode potential was stepped to +0.442 V and the headgas changed back to 100% H₂. At this potential, the enzyme does not reactivate, thus allowing a clear time window for gas exchange. After 900 s (to ensure that all O₂ was removed from solution), the electrode was stepped to the potential at which the rate of recovery was to be measured, and the rate of reactivation was determined by fitting a single exponential as before.

Fig. 3C compiles the rates of reactivation following both anaerobic and aerobic inactivation, plotted as a function of potential. The rate of recovery is similar in each case, and a limiting rate of ca. 0.1 s⁻¹ is reached at potentials below 0 V. Fitting the data to a sigmoid gives the number of electrons involved in the reactivation process, n (16). The best fit to the data yields n = 0.96 (overlaid in Fig. 3C), consistent with a single electron transfer. Also overlaid in Fig. 3C are the rates of recovery at 45° C of the Unready state of the O₂-sensitive MBH from Allochromatium vinosum (Av) (data from ref. 16). The Unready state is produced in large amounts under similar conditions in Av MBH. Reactivation of Re MBH following aerobic inactivation is clearly much faster than that of Av MBH. (Note that Ready and Unready reactivate at similar potentials in Av MBH.)

Potentials of reactivation are most easily compared by using voltammetry. Fig. 3D shows reactivation of both Re MBH and Av MBH after aerobic inactivation, recorded under the same conditions. The MBH-modified electrode was initially held at −0.558 V under 100% N₂. The potential was then stepped to +0.392 V and an aliquot of O₂-saturated buffer was simultaneously injected into the cell solution, causing the enzyme to inactivate aerobically. After 300 s, the headgas was changed to 100% H₂; then, after 900 s, the potential was slowly (0.1 mV s⁻¹) swept back to −0.558 V, allowing the recovery to be monitored. Reactivation of Av MBH occurs at a much lower potential, requiring ≈200 mV extra driving force compared to Re MBH.

Discussion

PFE allows the detailed measurement of reactions crucial to understanding O₂-tolerant H₂ oxidation in [NiFe]-hydrogenases. The O₂ tolerance factor, K_I^O_2,app, depends strongly on electrode potential and temperature but is not affected by pH in the region 4.5–6.5. Greatest O₂ tolerance is observed at low potentials and, significantly, at high temperatures. Standard O₂-sensitive [NiFe]-hydrogenases typically have values of K_I^O_2,app that are very low. For example, under one bar H₂, Av MBH is completely inhibited by just 4 μM O₂ (+0.142 V, pH 5.6, 30° C) (7), and the soluble hydrogenase from Df is completely inhibited by 5 μM O₂ (+0.190 V, pH 7, 40° C) (14)—thus, even micromolar O₂ concentrations are well in excess of K_I^O_2,app for these enzymes. By contrast, for Re MBH, K_I^O_2,app is ≈50 μM at these conditions and exceeds 1.6 mM at −0.018 V. To deconvolute the O₂ tolerance factor, it is necessary to probe the potential, pH, and temperature dependencies of the various contributory reactions and incorporate these within a model. Specifically, we are inspecting whether the individual reactions that must form the basis of O₂ tolerance either support, oppose, or are indifferent to the potential and temperature dependencies of K_I^O_2,app.

We first consider H₂ cycling in the absence of O₂, where both the affinity for H₂ and the catalytic turnover frequency (k_cat) are crucial. Although it has not yet proved possible to determine k_cat for Re MBH electrochemically, it is likely, by analogy with other [NiFe]-hydrogenases, that Re MBH has a turnover frequency for H₂ oxidation in the order of 10³ s⁻¹ at 30° C (17).

The Michaelis constant for H₂, K_M^H₂, increases with increasing electrode potential and increasing temperature. The smaller is K_M^H₂, the more long-lived is the Michaelis complex (E_act−H₂, where E_act represents Active hydrogenase), and hence the more likely the hydrogenase should be protected against O₂ binding at the active site. This is indeed the case for the potential dependence but not for the temperature dependence, showing that K_M^H₂ cannot be the dominating factor in determining O₂ tolerance. Consistent with this, K_M^H₂ values for Re MBH at low potentials are similar to those determined for some O₂-sensitive hydrogenases, such as Av MBH and the soluble enzyme from Df [which has K_M^H₂ ≈5 μM at −0.16 V (14)]. In addition, some mutants of Re MBH exhibit a very high K_M^H₂ but retain substantial O₂ tolerance (15).

There is a natural potential dependence in K_M^H₂, because the rate of catalysis (k_cat, cf. Eq. 2, below) increases with increasing driving force (a consequence of the inherent high activity of the active site and the limit on how fast electrons can be supplied from the electrode). This dependence is evident in voltammograms for numerous hydrogenases catalyzing H₂ oxidation (2, 18). (Interestingly, low H₂ concentrations also increase the extent of anaerobic inactivation, potentially leading to small overestimates in K_M^H₂.) An increase in K_M with increasing temperature is a typical trend and has origins in the increase in k_cat and, in this case, the increasing rate of anaerobic inactivation with increasing temperature.

The rates of reaction of Re MBH with O₂ (Fig. 2) are unaffected by changing potential or pH but increase strongly with increasing temperature. If the rate of O₂ attack (which includes its transport through the protein) were a controlling factor in determining K_I^O_2,app, then at higher temperatures the hydrogenase would be less O₂-tolerant, which is not the case. The lack of a pH- or potential-dependence in the rate of reaction with O₂ is in excellent agreement with the findings of Léger et al. (14) in their studies on Df hydrogenase. The potential dependence of K_I^O_2,app does not therefore arise from a dependence in the rate of O₂ reaction. It is worth noting, however, that the rates of O₂ reaction for Re MBH are considerably lower than for the periplasmic hydrogenase from D. gigas, whose catalytic subunit shares 67% sequence identity with the large subunit of Df hydrogenase.

We finally turn to the properties of the reactivation reaction. Our electrochemical results support the spectroscopic evidence that, in contrast with standard O₂-sensitive hydrogenases, Re MBH makes only the Ready state on reaction with O₂: No detectable amounts of Unready are made. All data fitted well to a single exponential, showing that only one inactive state is being recovered (recovery of Av MBH, which produces both Ready and Unready states, is biphasic). The rates of recovery of Re MBH following both anaerobic and aerobic inactivation cannot be distinguished, suggesting strongly that the same state is formed regardless of whether the active site is oxidized by O₂ or anaerobically at high potential. As expected, rates increase with increasing temperature (18). The value of n (the number of electrons involved in the reductive reactivation of inactive Ni^III to an active Ni^II state) is 0.96, implying a single electron transfer consistent with reactivation of Ready (Fig. 1). The potential of reactivation of Re MBH is more than 200 mV more positive than for the O₂-sensitive [NiFe]-hydrogenases from Av and D. gigas (18), and reactivation is therefore thermodynamically more favorable. [Interestingly, in the soluble hexameric NAD⁺-reducing [NiFe]-hydrogenase from Re, the inactive Ready state can be reactivated by using reducing equivalents from added NAD(P)H (19).]

To bring these factors together, the catalytic cycle shown in Fig. 1B was modeled by using the kinetic cycle shown in Fig. 4(Re MBH is represented by the symbol E). The steady-state approximation was applied to all intermediate species (E_act−H₂, E_act−O₂, E_Ready, and E_Unready). The following expression (Eq. 1) was obtained (see SI Appendix for derivation), in which K_M^H₂ and K_I^O₂ are as defined in Eqs. 2 and 3, respectively, and i is the catalytic current.

The term K_I^O₂ is the inhibition constant for O₂, and would vary from K_I^O_2,app by a factor of {[H₂]/K_M^H₂ × (1+K_M^H₂/[H₂])}, if O₂ were to be considered simply as a competitive inhibitor.

Fig. 4.

The kinetic scheme used to model O₂ tolerance. Re MBH is abbreviated as “E” and is shown in the reduced active form (E_act), as an adduct with H₂ or O₂ (E_act−H₂ and E_act−O₂, respectively) or in the Ready (E_Ready) or Unready (E_Unready) state.

Eq. 1 was fitted to the data to determine K_I^O_2,app as described above at 10° C (pH 5.5, +0.122 V). Values of k_cat (≈250 s⁻¹) and k_−A, the rate of reactivation of Unready (≈0.00025 s⁻¹) were used as “best guesses”, based on analogy with standard [NiFe]-hydrogenases. The value of k_P, the rate of anaerobic inactivation, is ≈0.003 s⁻¹ at 10° C and is potential independent (see Fig. S2 of the SI Appendix). The value of k_−B, the rate of reactivation of Ready (≈0.03 s⁻¹) was determined under these conditions (Fig. 3). To simplify, the approximation was made that the rates of access of H₂ and O₂ (k₁ and k₂, respectively) depend only on their masses according to the kinetic theory of gases (v_H2/v_O2 = $\sqrt{\left[{\text{mass}}_{{\text{O}}_{\text{2}}}\right]/\left[{\text{mass}}_{{\text{H}}_{\text{2}}}\right]}$), and hence k₁ = 4 × k₂, and, for the reverse reaction, k₋₁ = 4 × k₋₂. Taking typical values of K_M^H₂ ≈4 μM and K_I^O_2,app ≈60 μM at these conditions, the best fit to the experimental data (Fig. 5A) yielded k₁ = 68 μM⁻¹ s⁻¹, k₋₁ = 22 s⁻¹ and the rate of formation of Ready from the initial O₂ adduct, k_B = 0.01 s⁻¹.

Fig. 5.

Simulations obtained by applying Eq. 1 to Re MBH. (A) Limiting H₂ oxidation currents at varying O₂ concentrations, determined experimentally (black) and calculated from the model (gray). (B–D) show the effect of altering the percentage O₂ (B), the electrode potential (C), and k_A (D) on the catalytic currents calculated from the model. All conditions are pH 5.5, 10° C. B and D are at 0.122 V, and C and D are at 10% O₂.

The model allows an understanding of the roles of the different rate constants in affecting O₂ tolerance. Fig. 5B shows the effect of varying the O₂ concentration on the H₂ oxidation activity under the conditions above. Fig. 5C shows the effect of changing the electrode potential on the H₂ oxidation activity at 10% O₂; the potential dependence of k_−B is modeled by using Eq. 4. Fig. 5D stresses the importance, for O₂ tolerance, of not producing the Unready state, i.e., of ensuring that k_A is very small. It is unclear as yet why Re MBH does not produce Unready, but formation of the Ready state (k_B) requires a greater immediate availability of electrons (and protons) than for producing the Unready state (k_A). This requirement could be accommodated by some property of Re MBH, such as an increase in the number of redox sites able to donate an electron. Analogously, being embedded in the cytoplasmic membrane or immobilized on an electrode with a ready supply of electrons may protect the enzyme against O₂ inactivation.

In essence, both the potential and temperature dependencies of K_I^O_2,app emphasize the importance of enhancing the rate of recovery as opposed to limiting the rate of O₂ attack. Reactivation from Ready is faster at low potential and at high temperature, and this matches the characteristics of the O₂-tolerance factor. Interestingly, this means that Re MBH can be viewed as acting not only in H₂ cycling, but also as a very slow oxidase (20), catalyzing the four-electron reduction of O₂ to water with a rate constant in the order of 10⁻¹–10⁻² s⁻¹. The positive temperature dependence could have particular significance for O₂-tolerant H₂ cycling in microorganisms such as Aquifex aeolicus (21), Thermococcus litoralis (22), and Pyrococcus furiosus (23) that live at high temperatures and could encounter O₂.

Methods

The Re MBH enzyme was isolated and purified as described previously (15). All experiments were performed in an N₂-filled anaerobic glove box (M. Braun, <2 ppm O₂). A PGE rotating disk electrode (area 0.03 cm²) was used with an electrode rotator (EcoChemie Autolab Rotator), which fitted tightly above a gas-tight, glass, electrochemical cell. A three-electrode configuration was used, with a Pt wire counter electrode and a saturated calomel reference electrode (SCE) located in a reference arm containing aqueous 0.10 M NaCl and connected to the working electrode compartment via a Luggin capillary. The temperature of the working compartment was controlled by using a water jacket, whereas the reference electrode remained at ≈25° C. The potential (E) was corrected with respect to the standard hydrogen electrode (SHE) by using the following formula: E_SHE = E_SCE + 0.242 V at 298 K (24): All potentials are quoted relative to SHE. An Autolab PGSTAT 10 electrochemical analyzer was used with an electrochemical detection module and GPES software (EcoChemie).

All experiments were carried out in 0.05 M sodium phosphate buffer with 0.10 M NaCl as supporting electrolyte (prepared by using purified water, 18.2 MΩ cm, Millipore) and titrated to the desired pH at the experimental temperature. Gases were of Premier/Research Grade and supplied by Air Products or BOC. Mass-flow controllers (Smart-Trak, Sierra Instruments) were used to supply precise mixtures of gases to the cell headspace at a constant flow rate.

To prepare films of MBH, the PGE electrode was polished by using an aqueous slurry of 1 μm α-alumina (Buehler) on cotton wool, then sonicated for ca. 5 s and rinsed with purified water, before 1.5 μl of enzyme solution (0.1–0.2 mg/mL) was successively applied and withdrawn from the electrode surface over a period of ca. 30 s. In all experiments, the electrode was rotated at a constant rate, typically 2,500 rpm, to provide efficient supply of substrate and removal of product from the electrode surface.

To measure K_I^O_2,app and the rate of O₂ reaction, data were corrected for “film loss” over the course of the experiment by fitting anaerobic data points to a single exponential. A good fit was usually obtained (see Fig. 2) and by dividing the raw data by the exponential curve, normalized/corrected data were obtained. To obtain K_I^O_2,app, currents were corrected for film loss, and the inverse of the limiting value of the corrected current at each O₂ concentration was plotted against O₂ concentration, with K_I^2,app given by dividing the y intercept by the gradient. Values of the Henry's constants for H₂ and O₂, and the temperature dependence thereof, were taken from ref. 25.

Supporting Information.

Analysis of the rates of O₂ reaction, determination of the rate of anaerobic inactivation, and the derivation of Eq. 1, are available in the SI Appendix.