Catalytic Electron Transport in Chromatium vinosum [NiFe]-Hydrogenase:  Application of Voltammetry in Detecting Redox-Active Centers and Establishing That Hydrogen Oxidation Is Very Fast Even at Potentials Close to the Reversible H⁺/H₂ Value

Abstract

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

“…Our model predicts the following: Electrons leave or enter the catalytic cycle of CaHydA at a reduction potential of approximately −0.4 V -i.e., a more negative potential than the H þ ∕H 2 couple at pH 6.0 (−0.36 V). Electrons enter and leave the catalytic cycle of EcHyd2 at −0.30 V, which is similar to the reduction potential of the distal [4Fe-4S] cluster in some other standard (non-O 2 tolerant) [NiFe]-hydrogenases (34,35 can account for why that enzyme does not evolve H 2 and oxidizes H 2 only once an overpotential has been applied. In summary, this simple model reproduces key features of the catalytic voltammograms of different hydrogenases.…”

The Role of Pyruvate Ferredoxin Oxidoreductase in Pyruvate Synthesis during Autotrophic Growth by the Wood-Ljungdahl Pathway

“…For example, fumarate reductase and succinate dehydrogenase preferentially catalyze opposing reactions (30,31) and are separately regulated and distinct gene products (32,33). In many organisms, H ϩ reduction and H 2 oxidation are catalyzed by separate enzymes with the converse catalytic biases (34,35). Can PFOR serve as an efficient pyruvate synthase or is it prejudiced toward oxidative decarboxylation?…”

Distal [FeS]-Cluster Coordination in [NiFe]-Hydrogenase Facilitates Intermolecular Electron Transfer

“…Turn-over-frequencies (TOFs) of up to 50.000 s -1 have been measured on electrodes. The activity of the adsorbed enzyme is greater than the catalytic activity with electron acceptors and donors [6]. This demonstrates that both the reaction with H2 and electron transfer are very efficient and do not require any overpotential as excessive driving force.…”

“…This indicates (i) a direct contact between enzyme and electrode surface so that mediator molecules cannot interfere with electron transfer, (ii) the distal iron-sulfur cluster [FeS]d in close proximity to the electron acceptor and (iii) a uniform distribution of enzymes on the electrode surface (see Figure 2). When using an artificial soluble electron acceptor (methyl viologen) the measured rates of electron transfer are even slower than those of electrode associated [NiFe]-hydrogenases [16].…”

“…The nickel-iron hydrogenase absorbs directly on a graphite electrode [6] and also on carbonnanotube-coated pyrolytic graphite electrodes [7] to, here, catalyze the consumption of H2 (see Figure 2). When adsorbed on an electrode, the hydrogenase enzyme produces high catalytic current and occurs at potentials expected for this half-cell reaction [2].…”