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
* 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.