How Escherichia coli is equipped to oxidize hydrogen under different redox conditions

Abstract

The enterobacterium Escherichia coli synthesizes two H(2) uptake enzymes, Hyd-1 and Hyd-2. We show using precise electrochemical kinetic measurements that the properties of Hyd-1 and Hyd-2 contrast strikingly, and may be individually optimized to function under distinct environmental conditions. Hyd-2 is well suited for fast and efficient catalysis in more reducing environments, to the extent that in vitro it behaves as a bidirectional hydrogenase. In contrast, Hyd-1 is active for H(2) oxidation under more oxidizing conditions and cannot function in reverse. Importantly, Hyd-1 is O(2) tolerant and can oxidize H(2) in the presence of air, whereas Hyd-2 is ineffective for H(2) oxidation under aerobic conditions. The results have direct relevance for physiological roles of Hyd-1 and Hyd-2, which are expressed in different phases of growth. The properties that we report suggest distinct technological applications of these contrasting enzymes.

FIGURE 1.

The activity of E. coli hydrogenases-1 and -2 on a pyrolytic graphite edge electrode. Cyclic voltammograms were recorded at 30 °C, pH 6, scan rate, 1 mV s⁻¹; total gas flow rate, 1000 standard cubic centimeters min⁻¹; electrode rotation rate, 8500 rpm, except under 0.3% H₂, when it was 9500 rpm. Hyd-1 results are indicated by dark gray traces and Hyd-2 results are indicated by light gray traces. Top panel shows voltammograms recorded under 10% H₂ in argon; middle panel shows voltammograms recorded under 3% H₂ in argon; and bottom panel shows voltammograms recorded under 0.3% H₂ in argon. Asterisks in the top panel indicate the potential E_switch, and single-headed arrows in the middle panel indicate the scan direction. As indicated in the top panel, the negative current is a direct measurement of enzyme-catalyzed H₂ production, and the positive current is a direct measure of enzyme-catalyzed H₂ oxidation. Maximum currents were normalized to those of the same sample under 100% H₂, which was measured prior to each voltammogram. Blank voltammograms, not shown, recorded using a bare pyrolytic graphite edge electrode with no enzyme adsorbed on the surface, showed that no H₂ oxidation or H⁺ reduction occurs directly on the electrode surface. SHE, standard hydrogen electrode.

FIGURE 2.

X-band continuous wave EPR spectra of as-isolated E. coli Hyd-1 and Hyd-2. 10 K, microwave power 0.13 milliwatt; modulation amplitude 0.6 millitesla; 80 K, microwave power 2.0 milliwatts; modulation amplitude 1.0 millitesla (Hyd-1)/0.5 millitesla(Hyd-2). * and ^○ denote presently unexplained peaks (see text). g values (Hyd-1 and Hyd-2): Ni-A, g_x = 2.31, g_y = 2.24, and g_z = 2.01; Ni-B, g_x = 2.31, g_y = 2.16, and g_z = 2.01; [3Fe-4S]⁺(Hyd-1/Hyd-2), g_x = 2.03/2.03, g_y = 2.01/2.02, and g_z = 2.00/2.01. The overall simulations, and the individual simulations of the components Ni-A, Ni-B, and [3Fe-4S]⁺, are shown as dotted lines. When necessary, spectra were baseline subtracted using a baseline spectrum recorded under the same conditions as the spectrum of the enzyme sample.

FIGURE 3.

Chronoamperometry traces showing the activation of as-isolated Hyd-1 and Hyd-2. Chronoamperometry traces show activation of as-isolated samples of Hyd-1 and Hyd-2 during their first exposure to an atmosphere of 100% H₂ following aerobic purification. Measurements were made at pH 6.0, 30 °C, 100% H₂, at a potential of −0.060 V in the case of Hyd-1, and −0.200 V in the case of Hyd-2 (both potentials below E_switch of the respective enzyme).

FIGURE 4.

A comparison of the O₂ tolerance of hydrogenases-1 and -2. Cyclic voltammograms in A recorded at 30 °C, pH 6.0, scan rate 1 mV s⁻¹, under 100% H₂. Black arrows mark scan direction, and labeled gray arrows mark the point of O₂ injection. Chronoamperometry traces in B and C were recorded at pH 6.0, 30 °C, under either 100% H₂ (white background) or 100% argon (gray background). Potentials are as indicated. The cyclic voltammograms in A show the response of Hyd-1 and Hyd-2 to O₂ at high potential. In the case of Hyd-1, at 0.03 V on the forward sweep, O₂-saturated buffer is injected into the cell solution to give an O₂ concentration of 144 μm. The O₂ concentration immediately begins to fall due to continued flushing of H₂; by the time the potential reaches +0.25 V and the scan direction reverses, most O₂ has been flushed from the cell. A similar experiment on Hyd-2 is shown, with all conditions the same except that 16 μm O₂ is injected at 0.03 V, instead of 144 μm. B, left-hand side: a chronoamperometry experiment to investigate the inhibited active site states of Hyd-1 after exposure to O₂ at high potential in the absence of H₂. An initial measure of the H₂ oxidation activity of the film is made under 100% H₂ (white background), and the system is then equilibrated with 100% argon (gray background). For the first 1200 s, the potential is held at −0.060 V. Immediately on stepping the potential to +0.344 V, O₂-saturated buffer is injected into the cell solution to give an O₂ concentration of 325 μm. After re-equilibrating the electrochemical cell with H₂ (white background), the potential is stepped back to −0.060 V. B, right-hand side: a similar experiment on Hyd-2, in which all conditions are the same, except that the initial and final potential is −0.175 V instead of −0.06 V. C, similar chronoamperometry experiments to those in B, but this time O₂ is injected under 100% H₂.

FIGURE 5.

Aerobic H₂ oxidation by Hyd-1. Cyclic voltammograms recorded at 1 mV s⁻¹, pH 6.0, 37 °C, under three different gas mixtures: 10% H₂, 90% argon; 10% H₂, 20% O₂, 70% argon; and 20% O₂, 80% argon, a total flow rate 500 standard cubic centimeters min⁻¹ in each case. Prior to each measurement, the cell was allowed to equilibrate for 15 min with the appropriate gas mixture. Note that an O₂ reduction current arises from direct reaction of O₂ with the electrode.

FIGURE 6.

Sensitivity of Hyd-1 and Hyd-2 to CO. Chronoamperometry traces of Hyd-1 (A) and Hyd-2 (B and C) during exposure to varying CO concentrations, as indicated. Experimental conditions were: pH 6.0, 20 °C, electrode rotation rate 3500 rpm, and total gas flow rate 1000 standard cubic centimeters min⁻¹. Panel A (Hyd-1), electrode potential −0.060 V, carrier gas 20% H₂ in argon with increasing partial pressures of CO (the resulting concentration of CO in the electrochemical cell solution is indicated). Panel B (Hyd-2), electrode potential −0.175 V, carrier gas 20% H₂ in argon with increasing partial pressures of CO. Panel C (Hyd-2), electrode potential −0.560 V, carrier gas argon with increasing partial pressures of CO. In each case, CO is flushed from solution at the end of the experiment, to demonstrate reversibility of inhibition. Insets show plots used to determine K_i(CO) in each case (see “Results”). K_i(CO/H₂) for H₂ oxidation by Hyd-1 (A) was measured to be 51 ± 6 μm; K_i(CO/H₂) for H₂ oxidation by Hyd-2 (B) was 3 ± 1 μm; and K_i^app(CO/H⁺) for H₂ production by Hyd-2 (C) was 11 ± 3 μm.

FIGURE 7.

O₂-tolerant H₂ production catalyzed by Hyd-2. Experimental conditions were: pH 6.0, 25 °C, electrode potential −0.560 V, electrode rotation rate of 3500 rpm. Argon is the carrier gas, and CO and O₂ are introduced into the headspace at the times and concentrations indicated. Double-headed arrows indicate the magnitude of CO-inhibited H₂ production catalyzed by Hyd-2.

FIGURE 8.

Comparisons of the in vitro capabilities (represented as potential activity windows) of Hyd-1 and Hyd-2. This is valid both under anaerobic conditions and upon exposure to O₂, provided that the product of inhibition is the Ni-B state and not Ni-A. E_switch values, as measured under 10% H₂, pH 6.0, 30 °C, are indicated.