Kylie Alison Vincent

We report X-ray crystallographic structures of the Azotobacter vinelandii nitrogenase MoFe protein showing the 8Fe-7S electron-transfer P-cluster in four redox states. Using electrochemical poising of protein crystals, together with in crystallo EPR spectroscopic verification of the redox state, we obtain structures showing the P-cluster at PN, P1+, P2+, and P3+ levels. This provides a detailed structural characterization of P-cluster rearrangement between the catalytically relevant PN and P1+ levels and the first experimental confirmation that the S = 7/2 P3+ state is structurally similar to P2+. These studies pave the way for future understanding of the structure–function relationship in nitrogenase catalysis.

Iron-sulfur (Fe-S) clusters are ubiquitous as redox-active protein cofactors, but it is often difficult to collect protein structures in which redox centres are in uniform and well-defined oxidation states. Using spinach ferredoxin I (Fdx) as a model redox protein, we demonstrate an integrated methodological pathway for electrochemical modulation of redox state in protein crystals coupled with in crystallo EPR and online-UV-visible spectroscopy to verify oxidation state. We show that Fdx crystals can be electrochemically reduced, reversibly, without compromising lattice integrity or X-ray diffraction quality. We show that redox levels can be precisely ascertained in crystallo via EPR and UV-visible spectroscopy, enabling a direct correlation between protein structure and electronic state of the metal cluster. In this way, we generate and compare 'oxidised', 'reduced' and 're-oxidised' structures of Fdx. Overall, our approach demonstrates a pipeline which will be applicable to structure-function studies of a wide range of electron-transfer proteins and redox enzymes.

We report a simplified, H ₂ ‐driven method for operating biocatalytic halogenation by a flavin‐dependent halogenase, PyrH, which enables intensification of biocatalytic conversion of L‐tryptophan to its 5‐halo product. Flavin‐dependent halogenases are gaining traction in biotechnology as their substrate scope is expanded by enzyme discovery and engineering, but their application remains impeded by a particularly complex electron transfer chain and the fact that full conversion is generally only achieved at sub‐millimolar substrate concentrations. Here, we apply nickel‐iron hydrogenase and ambient pressure H ₂ in place of the NAD(P) ⁺ , glucose, glucose dehydrogenase and reductase which are normally used to supply reduced FAD to halogenases. Together with controlled delivery of O ₂ , which is needed for generating a hypohalous acid intermediate, we achieve full conversion of 5.5 mM tryptophan, with a PyrH total turnover number of 275, and PyrH turnover frequency of 0.76 min ⁻¹ over a 6 h reaction, comparable with rates sustained only for short reaction times using the conventional glucose‐driven system. This should help to facilitate application of flavin‐dependent halogenases in fine chemical synthesis.

H₂ is an ideal energy vector, but catalysts for its clean production from water are inefficient or expensive. [FeFe]-hydrogenases are the most active H₂-converting catalysts in nature, using a unique organometallic active site finely tuned by the protein matrix. M3 type [FeFe]-hydrogenases from Clostridium pasteurianum and Clostridium acetobutylicum are exceptionally active for H₂ production, and less O₂ sensitive than most other types of [FeFe]-hydrogenases, making them attractive targets for biotechnology. However, they are more challenging to work with because of their large size and the number of iron–sulfur clusters. Here, the [FeFe]-hydrogenase from C. acetobutylicum was systematically engineered to truncate each iron–sulfur-containing region of the F-domain, yielding smaller and easier-to-produce catalytic systems. Detailed characterization revealed that these variants retain high electrocatalytic performance and other essential properties of the natural enzyme.

We show that europium trisbipyridyl cryptate exhibits an electrochemically reversible Eu^III/II redox couple at -360 mV vs the standard hydrogen electrode (SHE) and is susceptible to reduction or oxidation by a range of redox enzymes or biological small molecule redox agents. Modulation of the europium oxidation state can be used to achieve dramatic changes in the T₁ relaxation time of bulk water in solutions containing these complexes: in the reduced form, the complex exhibits high relaxivity (3.08 mM^-1 s^-1 at 499.9 MHz) comparable with that of commercial MRI contrast media, while the oxidized form has negligible relaxivity, illustrating the scope for application in imaging reducing environments in biological systems.

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.

We report a family of lanthanide(III) complexes that act as redox probes via both magnetic resonance (MR) and luminescence outputs. The ligands are functionalized with nitro, azobenzene and azide groups which are reduced to a common aniline product, and each responds to both chemical and biocatalytic reductive conditions at different cathodic onset potentials. By judicious choice of complexed Ln(III), the probes can be optimized either for use in MR imaging (Ln = Gd), or as highly efficient turn‐on luminescence probes (Ln = Tb). The Tb(III) analogues are essentially nonemissive, until reductive generation of the aniline affords a complex which when excited by visible light (488 nm) emits green light with a quantum yield of 45% and millisecond long luminescent lifetimes (ms). The tunable redox response and imaging modalities of these versatile complexes have the potential to open up new approaches to redox sensing, such as the imaging of hypoxic environments in biology.

Lanthanide complexes of DOTA monoesters bearing nitrobenzyl and nitroimidazole groups are shown to be converted to the corresponding DOTA complexes under chemical and enzymatic conditions, giving rise to favorable changes in the luminescence properties of the europium and terbium complexes and relaxometric properties of the gadolinium complexes. The nitroimidazole complexes are converted more rapidly than their nitrobenzyl and benzyl analogues. We propose that activation of these complexes may occur by ester cleavage rather than nitro reduction and fragmentation since complexes bearing a simple benzyl group may also be cleaved under the same conditions, albeit more slowly.

This study shows that the organic cofactor riboflavin provides a cheap and atom‐efficient source of reducing equivalents to sustain biocatalytic alkene reductions by ene‐reductase enzymes, when coupled with a H₂‐driven or electrochemical recycling system. We employ the robust NiFe hydrogenase, Escherichia coli Hyd1, for H₂‐driven riboflavin reduction and show the feasibility of unmediated electrochemical recycling of reduced riboflavin at a simple carbon electrode. We show that H₂‐driven reduction of riboflavin can be extended to continuous flow with a packed bed reactor comprising of Hyd1 immobilized on a carbon support. These findings demonstrate that there is scope for replacing the expensive nicotinamide cofactors—NADH or NADPH with riboflavin, for applications of ene‐reductases in biotechnology in either batch or continuous flow and in electrosynthesis.

The ability of hydrogenase enzymes to activate H ₂ with excellent selectivity leads to many interesting possibilities for biotechnology driven by H ₂ as a clean reductant.