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Biocatalysis for Sustainable Chemistry – Understanding Oxidation/Reduction of Small Molecules by Redox Metalloenzymes via a Suite of Steady State and Transient Infrared Electrochemical Methods

Periodic Reporting for period 1 - BiocatSusChem (Biocatalysis for Sustainable Chemistry – Understanding Oxidation/Reduction of Small Molecules by Redox Metalloenzymes via a Suite of Steady State and Transient Infrared Electrochemical Methods)

Reporting period: 2019-03-01 to 2020-08-31

This project addresses the significant global challenges in catalysis for energy and sustainable chemistry via learning from, and exploiting, nature’s enzyme catalysts. Metal-containing enzymes within microorganisms catalyse the transformation of carbon dioxide into simple carbon building blocks or fuels, the reduction of dinitrogen to ammonia under ambient conditions and the production and utilisation of dihydrogen. Catalytic sites for these reactions within the enzymes are necessarily based on metals that are abundant in the environment, including iron, nickel and molybdenum. However, attempts to generate biomimetic catalysts have largely failed to reproduce the high activity, stability and selectivity of enzymes. One important key to the exquisite catalysis of enzymes is the choreography of proton and electron transfer and substrate binding during catalytic turnover, and this project sets out to establish and utilise a suite of spectroscopic and structural techniques to understand proton-coupled electron transfer and how this is timed with substrate binding and transformation in enzymes. Success in this work offers the promise of new insight to feed into the creation of catalysts for energy technologies: clean, sustainable fuel production, fuel cells, and more energy-economical industrial chemical processes, thus benefitting society by contributing to the urgent challenges posed by global climate change and energy/climate inequality. Key objectives of the action are (i) to establish operando techniques which can be used to understand the intricately controlled chemistry of enzymes; (ii) to apply these to hydrogenases to see what catalytic lessons we can glean from understanding how hydrogen gas is activated cleanly in biology, and how we can exploit hydrogenases themselves in cleaner chemical production; (iii) to apply similar approaches to nitrogenase to understand how natural nitrogen fixation to ammonia can be conducted under ambient conditions in contrast to the high temperature/pressure Haber Bosch process; (iv) to understand the selectivity and efficiency of biological CO2 reduction; and (v) to disseminate these lessons to the wider catalysis community to contribution new catalyst design principles.
In the first period of the project:
* We have set up equipment, including an IR microscope with focal plane array and bolometer detectors for study of metalloenzyme electrodes and single crystals, and an IR matrix gas analyser for detection of products of enzyme catalytic reactions.
* We have successfully demonstrated incorporation of the non-natural amino acid cyanophenyl alanine into a model protein (spinach ferredoxin, which has a [2Fe2S] cluster) as an infrared probe for the iron-sulfur cluster redox state. A small but reproducible shift in the absorption frequency of the CN group of cyanophenyl alanine was observed following either chemical or electrochemical reduction of the protein. Fortuitously, we were able to obtain diffraction-quality crystals from a small quantity (<10 microlitres) of the labelled protein, and hence have crystal structures available to prove the location of the cyanophenyl alanine and to show that there is minimal structural change between the oxidised and reduced protein. Preparation of a manuscript to describe this work is well underway.
* Further work has successfully established a recombinant system in E. coli for expressing FeFe hydrogenase, ready to extend the cyanophenylalanine labelling to this enzyme. We have also established an artificial maturation protocol with synthetic cofactors for overproducing [FeFe] hydrogenases in our labs, following procedures developed elsewhere. This sets the groundwork for studying the redox state of the FeS clusters in hydrogenase in next Period.
* We have extended our electrochemical control of NiFe hydrogenases to FeFe hydrogenase crystals.
* We have discovered a new non-natural activity of hydrogenase in reducing flavin cofactors, and applied this in biotechnology for supply of reducing equivalents to ene reductases. This has interesting fundamental mechanistic consequences, in terms how the hydrogenases supply 2 single electrons in rapid succession for the 2 electron flavin reduction, but also substantial biotech relevance, and has been protected by a patent filing.
* Experiments have been conducted to assess the possibility of exploiting E. coli hydrogenase I in other areas of reductive catalysis, and interesting chemistry towards hydrogenation of unsaturated organic aromatic nitrogen-containing compounds has been uncovered.
* We have shown potential-dependent electrocatalysis and inhibition for MoFe nitrogenase
* We have successfully crystallised carbon monoxide dehydrogenase I (CODH-I) from Carboxydothermus hydrogenoformans thermophilic bacterium, in collaboration with Prof Steve Ragsdale (U. Michigan), and have carried out preliminary experiments to evaluate electrochemical control over single crystals of this enzyme and structural measurements in the presence of substrate and inhibitors.
Taking as models the enzymes nitrogenase, hydrogenase, carbon monoxide dehydrogenase and formate dehydrogenase, the project sets out to establish a unified understanding of central concepts in small molecule activation in biology. It will reveal precise ways in which chemical events are coordinated inside complex multicentre metalloenzymes, propelling a new generation of bio-inspired catalysts and uncovering new chemistry of enzymes. The techniques we have set up in period 1 go well beyond the state of the art, allowing, for the first time, electrochemical control over single crystals of metalloenzymes, and elucidation of the electron loading of electron-relay clusters in metalloenzymes. During the next stages of the action, we will be able to explore chemical steps which are slowed down in the crystalline state, understanding intermediates in proton transfer and how their formation is regulated and coupled to electron transfer. The exciting new reactivity we have discovered for NiFe hydrogenase I of E. coli in flavin cofactor reduction opens doors to many applications in biotechnology, in the H2-driven supply of reducing equivalents to ene reductases, nitro reductases, halogenases and monooxygenases. Potentially even more exciting are our preliminary results on non-natural reduction of unsaturated nitrogen-containing molecules by hydrogenase, opening up completely new reactivities for biotechnology. We will keep in close contact with the host University’s tech. transfer company, OUI, to ensure appropriate exploitation of these results. In the latter stage of the project we will ensure that all of our results are disseminated effectively into the catalysis community by hosting a major international workshop. Overall, we anticipate substantial progress in understanding metalloenzyme catalysis and unifying understanding across different metalloenzymes which will translate into new catalyst design.