Philip A. Ash

The noncanonical amino acid, para‐cyanophenylalanine (CNF), when incorporated into metalloproteins, functions as an infrared spectroscopic probe for the redox state of iron‐sulfur clusters, offering a strategy for determining electron occupancy in the electron transport chains of complex metalloenzymes. A redshift of ≈1–2 cm⁻¹ in the nitrile (NC) stretching frequency is observed, following reduction of spinach ferredoxin modified to contain CNF close to its [2Fe–2S] center, and this shift is reversed on re‐oxidation. We extend this to CNF positioned near to the proximal [4Fe–4S] cluster of the [FeFe] hydrogenase from Desulfovibrio desulfuricans. In combination with a distal [4Fe–4S] cluster and the [4Fe–4S] cluster of the active site ‘H‐cluster’ ([4Fe–4S]_H), the proximal cluster forms an electron relay connecting the active site to the surface of the protein. Again, a reversible shift in wavenumber for CNF is observed, following cluster reduction in either apo‐protein (containing the iron‐sulfur clusters but lacking the active site) or holo‐protein with intact active site, demonstrating the general applicability of this approach to studying complex metalloenzymes.

Redox enzymes capable of carrying out multi-electron reactions serve as blueprints for the rational design of bio-inspired catalysts for future green technologies. During catalysis, enzymes transition through multiple ‘active’ intermediate states. Movement of both electrons and substrate to/from the active site is precisely controlled to achieve the equivalent of high Faradaic efficiencies, with minimal electrons wasted in detrimental side reactions. High turnover frequencies in metalloenzymes such as the nickel-iron (NiFe) hydrogenases require mechanisms for highly-choreographed movement of two quantum particles, protons and electrons. According to Marcus theory, structural rigidity is key to a low reorganisation energy barrier for rapid, outer-sphere electron transfer. However, this is at odds with the requirement for a degree of conformational flexibility to enable rapid proton tunnelling between residues separated by distances greater than ~2.7 Å. Here we exploit the specific redox poising possible with electrochemical control of protein crystals and characterise, structurally and spectroscopically, [NiFe]-hydrogenase in each of the key states of the catalytic cycle as well as its CO-bound and oxidised states. These structures confirm extraordinarily fixed metal coordination at the active site, conducive to fast multi-electron catalysis, and reveal a subtle carboxylate ‘flick’ that provides molecular detail of how glutamate acts as a proton shuttle.

Changing the conserved active-site arginine of [NiFe]-hydrogenases into a lysine greatly lowers the rates of catalytic H₂ activation in each direction and results in the extremely tight binding of a diatomic ligand.

We associate potentials with the binding of substrates and inhibitors, H⁺, C₂H₂, CO and MeNC, to nitrogenase MoFe protein by coupling electrochemical control with gas chromatography analysis and in situ infrared spectroscopy.

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.

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.

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.

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.

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.

The activation and redox chemistry of small molecules by metalloenzymes play important roles in vital processes such as the global carbon and nitrogen cycles, and as sources of energy or low-potential reducing equivalents in cellular environments (for example bacteria such as R. eutropha have evolved the capability to use CO₂ as sole carbon source and H₂ as sole energy source). Despite the current urgent need for sustainable fuels and greener routes to chemical synthesis, detailed mechanistic understanding of how small molecule activation reactions are carried out in nature is often lacking. In part this is due to the high turnover frequencies achieved by metalloenzymes such as carbon monoxide dehydrogenase (CO₂/CO, >2000 s^-1), formate dehydrogenase (CO₂/HCOO^-, >112 s^-1), and hydrogenases (H⁺/H₂, >9000 s^-1). By combining electrochemical control with microspectroscopic imaging we are able to take advantage of high protein concentrations in the crystalline state, and apparently retarded reactivity, to probe bioelectrocatalytic processes at the sub-single crystal level and gain fresh mechanistic insight. X-ray crystallography is almost ubiquitous in contributing structural insight into the mechanism of redox proteins. At present, significant challenges arise in preparing (and maintaining) protein crystals in all redox states,¹ there is often no means of controlling and verifying the state or uniformity of the crystallised sample, and the possibilities for kinetic study are limited. There is a growing trend towards implementing optical spectroscopic techniques which are sensitive to both electronic structure and radiation-induced effects on crystallography beamlines.² However, these are mainly used to assess the extent of photodamage and photoreduction during X-ray data collection, or confirm that crystallisation maintains the protein in a physiologically relevant state. Even in cases where microspectroscopy is used to gain information to complement the crystal structure, the ability to precisely control and maintain a desired redox state remains largely elusive. Recently we have developed an approach that allows precise electrochemical control over the redox state of a crystallised protein at a carbon electrode, while monitoring the redox state in situ using diffraction-limited synchrotron infrared microspectroscopic imaging (Figure 1).³ Using NiFe hydrogenase 1 from E. coli as a case study we show that the redox state of a crystal can be rapidly and reversibly manipulated, and that the whole crystal sample responds uniformly to the applied potential. The crystals are stable for several hours over multiple potential cycles. Intriguingly, retarded reactivity in the crystalline state gives insight into proton transfer during H⁺ reduction by NiFe hydrogenases, adding missing chemical detail to the hydrogenase catalytic cycle.⁴ The addition of electrochemical control will allow us to ‘trap’ NiFe hydrogenase crystals in elusive (or otherwise short-lived) redox states, enabling structural information to be obtained in catalytically-relevant states that have to date been crystallographically inaccessible. The new electrochemical method presented here can be readily coupled with other spectroscopies, thereby addressing a variety of chromophores in crystallo and greatly broadening the range of bioelectrocatalytic processes that can be studied at the sub-single crystal level. References Bowman SEJ et al., Acc. Chem. Res. 2016, 49, 695. von Stetten D et al., Acta Crystallogr. 2015, 71, 15. Ash PA et al., Chem. Commun. 2017, 53, 5858. Ash PA et al., ACS Catal. 2017, 7, 2471. Figure 1. Combined infrared microspectroscopy and electrochemistry of NiFe hydrogenase single crystals. (A) Schematic view of the reflection-absorbtion cell developed for this work (WE, working electrode; CE, counter electrode; RE, reference electrode). (B) Synchrotron IR microspectroscopy allows mapping at the sub-crystal level during transitions between well-defined redox states, as evidenced by the stretching vibration of the intrinsic CO ligand at the hydrogenase active site (inset and structure). Figure 1