Protein film electrochemistry (PFE) is providing cutting-edge insight into the chemical principles underpinning biological hydrogen. Attached to an electrode, many enzymes exhibit "reversible" electrocatalytic behavior, meaning that a catalyzed redox reaction appears reversible or quasi-reversible when viewed by cyclic voltammetry. This efficiency is most relevant for enzymes that are inspiring advances in renewable energy, such as hydrogen-activating and CO2-reducing enzymes. Exploiting the rich repertoire of available instrumental methods, PFE experiments yield both a general snapshot and fine detail, all from tiny samples of enzyme. The dynamic electrochemical investigations blaze new trails and add exquisite detail to the information gained from structural and spectroscopic studies. This Account describes recent investigations of hydrogenases carried out in Oxford, including ideas initiated with PFE and followed through with complementary techniques, all contributing to an eventual complete picture of fast and efficient H2 activation without Pt. By immobilization of an enzyme on an electrode, catalytic electron flow and the chemistry controlling it can be addressed at the touch of a button. The buried nature of the active site means that structures that have been determined by crystallography or spectroscopy are likely to be protected, retained, and fully relevant in a PFE experiment. An electrocatalysis model formulated for the PFE of immobilized enzymes predicts interesting behavior and gives insight into why some hydrogenases are H2 producers and others are H2 oxidizers. Immobilization also allows for easy addition and removal of inhibitors along with precise potential control, one interesting outcome being that formaldehyde forms a reversible complex with reduced [FeFe]-hydrogenases, thereby providing insight into the order of electron and proton transfers. Experiments on O2-tolerant [NiFe]-hydrogenases show that O2 behaves like a reversible inhibitor: it is also a substrate, and implicit in the description of some hydrogenases as "H2/O2 oxidoreductases" is the hypothesis that fast and efficient multielectron transfer is a key to O2 tolerance because it promotes complete reduction of O2 to harmless water. Not only is a novel [4Fe-3S] cluster (able to transfer two electrons consecutively) an important component, but connections to additional electron sources (other Fe-S clusters, an electrode, another quaternary structure unit, or the physiological membrane itself) ensure that H2 oxidation can be sustained in the presence of O2, as demonstrated with enzyme fuel cells able to operate on a H2/air mixture. Manipulating the H-H bond in the active site is the simplest proton-coupled electron-transfer reaction to be catalyzed by an enzyme. Unlike small molecular catalysts or the surfaces of materials, metalloenzymes are far better suited to engineering the all-important outer-coordination shell. Hence, recent successful site-directed mutagenesis of the conserved outer-shell "canopy" residues in a [NiFe]-hydrogen...

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H 2 + O 2 = 2 H 2 O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H 2 O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H 2 and 10% O 2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O 2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H 2 oxidation. The oxidase activity is too slow to be useful in removing O 2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation.hydrogen | mass spectrometry | Fe-S cluster H ydrogenases are enzymes that catalyze the interconversion of H 2 and H + with great efficiency. Containing Fe or Fe and Ni as active metals, they are not only important in biohydrogen production (by fermentative and photosynthetic means) but also provide inspiration for detailed understanding and development of optimal molecular electrocatalysts. The minimal active site motif, common to all hydrogenases, is a low-spin Fe atom coordinated by CO, CN − , and thiolate ligands, a combination expected to be unstable under aerobic conditions. Indeed, most hydrogenases suffer long-term or permanent inactivation when exposed to even traces of O 2 . It is therefore of special interest that certain [NiFe]-hydrogenases have evolved to sustain H 2 oxidation in the continued presence of O 2 , without inactivation: these enzymes are known as O 2 -tolerant [NiFe]-hydrogenases.Most of our current insight into the mechanism of O 2 tolerance stems from studies on respiratory membrane-bound [NiFe]-hydrogenases that couple H 2 oxidation to reduction of quinones (1-3). These enzymes are localized at the cytoplasmic membrane and project into the periplasmic space. A model proposed for the O 2 -tolerance mechanism of these [NiFe]-hydrogenases ( Fig. 1) is based on the following evidence. Oxygen reacts with O 2 -tolerant membrane-bound [NiFe]-hydrogenases to form, exclusively, an inactive state known as Ni-B or "ready," formulated as a Ni(III)-OH species, which is rapidly reactivated by one-electron transfer to rejoin the catalytic cycle of H 2 oxidation. Provided Ni-B is the sole product of O 2 attack, the presence of O 2 merely attenuates the steady-state rate of H 2 oxidation. In contrast, standard (O 2 sensitive) [NiFe]-hydrogenases react with O 2 to give a mixture of states, including ones variously known as "unready" or Ni-A, in which O 2 is either only partially reduced (possibly trapped as a peroxide) or has oxygenated atoms of the active site (3-7). The unready states are only reactivated very slowly; consequently, t...

‘Oxygen-tolerant’ [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.