Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.

Electrochemical experiments define potentials associated with binding of substrates and inhibitors to nitrogenase MoFe protein.

Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction. The molybdenum-iron nitrogenase is the most well-studied, but redox potentials associated with proton-coupled transformations between the redox levels of the catalytic MoFe protein have proved difficult to pin down, in part due to a complex electron-transfer pathway from the partner Fe protein, linked to ATP-hydrolysis. Here, we apply electrochemical control to the MoFe protein of Azotobacter vinelandii nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators. We combine insight from the electrochemical current response with data from gas chromatography and in situ infrared spectroscopy, in order to define potentials for the binding of a series of inhibitors (carbon monoxide, methyl isocyanide) to the metallo-catalytic site of the MoFe protein, and the onset of catalytic transformation of alternative substrates (protons and acetylene) by the enzyme. Thus, we associate potentials with the redox levels for inhibition and catalysis by nitrogenase, with relevance to the elusive mechanism of biological nitrogen fixation.

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase.

Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.

Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultaneously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN- ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH+, the super-reduced HsredH+ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates.

Redox state and photoreduction control using X-ray spectroelectrochemical techniques - advances in design and fabrication through additive engineering.

The design and performance of an electrochemical cell and solution flow system optimized for the collection of X-ray absorption spectra from solutions of species sensitive to photodamage is described. A combination of 3D CAD and 3D printing techniques facilitates highly optimized design with low unit cost and short production time. Precise control of the solution flow is critical to both minimizing the volume of solution needed and minimizing the photodamage that occurs during data acquisition. The details of an integrated four-syringe stepper-motor-driven pump and associated software are described. It is shown that combined electrochemical and flow control can allow repeated measurement of a defined volume of solution, 100 µl, of samples sensitive to photoreduction without significant change to the X-ray absorption near-edge structure and is demonstrated by measurements of copper(II) complexes. The flow in situ electrochemical cell allows the collection of high-quality X-ray spectral measurements both in the near-edge region and over an extended energy region as is needed for structural analysis from solution samples. This approach provides control over photodamage at a level at least comparable with that achieved using cryogenic techniques and at the same time eliminates problems associated with interference due to Bragg peaks.

Unifying Activity, Structure, and Spectroscopy of [NiFe] Hydrogenases: Combining Techniques To Clarify Mechanistic Understanding.

Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic sampling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic "bottlenecks" introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN- ligands, linked to the protein via bridging and terminal cysteine sulfur ligands, as well as an electron relay chain of iron sulfur clusters. Infrared spectroscopy is ideal for probing hydrogenases because the CO and CN- ligands are strong IR absorbers, but the suite of IR-based approaches we describe here will be equally valuable in studying substrate- or intermediate-bound states of other metalloenzymes where key mechanistic questions remain open, such as nitrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase. We therefore hope that this Account will encourage future studies which unify information from different techniques across bioinorganic chemistry.

Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster.

Iron-sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress. Both iron-sulfur proteins and synthetic model clusters are extremely sensitive to nitrosylation, tending towards rapid multi-step reaction and cluster degradation. Reaction of protein-bound iron-sulfur clusters with nitric oxide can be stopped at partial nitrosylation in vivo, and repair of protein-bound nitrosylated clusters is possible in the cellular environment. We have used a combination of infrared, EPR, and UV-visible spectroscopies to show that a model [4Fe4S] cluster-containing protein, A. ferroxidans high potential iron-sulfur protein (HiPIP), reacts with NO to give a product mixture dominated by Roussin's Black Salt (RBS) and Roussin's Red Ester (RRE) species. We have shown that O2 plays a critical role in controlling the major product of nitrosylation, with RBS-like products favoured under strictly anaerobic conditions and RRE favoured in the presence of trace O2. Moreover, addition of trace O2 to anaerobically nitrosylated samples induces conversion of RBS-like products to RRE. These findings may have implications for mechanisms of iron-sulfur cluster repair following nitrosative stress, suggest a crucial role for trace O2, and provide an important link between nitrosylation chemistry of iron-sulfur proteins and the well-established reactivity of synthetic iron-sulfur clusters.

Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase.

Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Nia-R" and "Nia-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase.

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Electrochemical CO Oxidation at Platinum on Carbon Studied through Analysis of Anomalous in Situ IR Spectra.

The oxidation of adsorbed CO is a key reaction in electrocatalysis. It has been studied extensively on both extended model surfaces and on nanoparticles; however, correlation between the two is far from simple. Molecular insight into the reaction is often provided using in situ IR spectroscopy; however, practical challenges mean in situ studies on nanoparticles have yet to provide the same level of detail as those on model surfaces. Here we use a new approach to in situ IR spectroscopy to study the mechanism of CO adlayer oxidation on a commercial carbon-supported Pt catalyst. We observe bipolar IR absorption bands but develop a simple model to enable fitting. Quantitative analysis of band behavior during the oxidation prepeak using the model agrees well with previous analysis based on conventional absorption bands. A second linear CO band is observed during the main oxidation region and is assigned to the distinct contribution of CO on step as opposed to terrace sites. Analysis of the step and terrace CO bands during oxidation shows that oxidation begins on the terraces of the nanoparticles before CO on steps is removed. Further correlation of this behavior with the current shows that step CO is only lost in the first of the two main oxidation peaks.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy.

Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases.

Enzymes as modular catalysts for redox half-reactions in H2-powered chemical synthesis: from biology to technology.

The present study considers the ways in which redox enzyme modules are coupled in living cells for linking reductive and oxidative half-reactions, and then reviews examples in which this concept can be exploited technologically in applications of coupled enzyme pairs. We discuss many examples in which enzymes are interfaced with electronically conductive particles to build up heterogeneous catalytic systems in an approach which could be termed synthetic biochemistry We focus on reactions involving the H+/H2 redox couple catalysed by NiFe hydrogenase moieties in conjunction with other biocatalysed reactions to assemble systems directed towards synthesis of specialised chemicals, chemical building blocks or bio-derived fuel molecules. We review our work in which this approach is applied in designing enzyme-modified particles for H2-driven recycling of the nicotinamide cofactor NADH to provide a clean cofactor source for applications of NADH-dependent enzymes in chemical synthesis, presenting a combination of published and new work on these systems. We also consider related photobiocatalytic approaches for light-driven production of chemicals or H2 as a fuel. We emphasise the techniques available for understanding detailed catalytic properties of the enzymes responsible for individual redox half-reactions, and the importance of a fundamental understanding of the enzyme characteristics in enabling effective applications of redox biocatalysis.

Formate adsorption on Pt nanoparticles during formic acid electro-oxidation: insights from in situ infrared spectroscopy.

Adsorbed formate is observed on a supported Pt nanoparticle for the first time during formic acid electro-oxidation. Bands assigned to OCO stretching and CH bending reveal some OCO but little CH bond weakening on adsorption compared to the free anion. The formate potential dependence is similar to polycrystalline electrodes while adsorbed CO persists up to +1.2 V, 0.5 V higher than on polycrystalline Pt.

Vibrational Spectroscopic Techniques for Probing Bioelectrochemical Systems.

A more complete understanding of bioelectrochemical interfaces is of increasing importance in both fundamental studies and biotechnological applications of proteins. Bioelectrochemical methods provide detailed information about the activity or rate of a process, but in situ spectroscopic methods are needed to gain direct structural insight into functionally relevant states. A number of methods have been reported that allow electrochemical and spectroscopic data to be collected from the same electrode, providing direct spectroscopic 'snapshots' of protein function, and here we focus on the application of infrared and Raman spectroscopies to the study of electrode-immobilised species. The ability to probe coordination at metal centres, protonation changes in amino acid side chains, reaction-induced changes in organic cofactors or substrates, protein orientation and subtle changes in protein secondary structure simultaneously, rapidly and at room temperature means that vibrational spectroscopic approaches are almost uniquely applicable to answering a wide range of questions in bioelectrochemistry.

Synchrotron-Based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control.

We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s(-1), and therefore, fast experimental triggers are needed in order to study subturnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment, this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 µm(2). We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control.

Discovery of Dark pH-Dependent H(+) Migration in a [NiFe]-Hydrogenase and Its Mechanistic Relevance: Mobilizing the Hydrido Ligand of the Ni-C Intermediate.

Despite extensive studies on [NiFe]-hydrogenases, the mechanism by which these enzymes produce and activate H2 so efficiently remains unclear. A well-known EPR-active state produced under H2 and known as Ni-C is assigned as a Ni(III)-Fe(II) species with a hydrido ligand in the bridging position between the two metals. It has long been known that low-temperature photolysis of Ni-C yields distinctive EPR-active states, collectively termed Ni-L, that are attributed to migration of the bridging-H species as a proton; however, Ni-L has mainly been regarded as an artifact with no mechanistic relevance. It is now demonstrated, based on EPR and infrared spectroscopic studies, that the Ni-C to Ni-L interconversion in Hydrogenase-1 (Hyd-1) from Escherichia coli is a pH-dependent process that proceeds readily in the dark-proton migration from Ni-C being favored as the pH is increased. The persistence of Ni-L in Hyd-1 must relate to unassigned differences in proton affinities of metal and adjacent amino acid sites, although the unusually high reduction potentials of the adjacent Fe-S centers in this O2-tolerant hydrogenase might also be a contributory factor, impeding elementary electron transfer off the [NiFe] site after proton departure. The results provide compelling evidence that Ni-L is a true, albeit elusive, catalytic intermediate of [NiFe]-hydrogenases.

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO.

The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.