Probing iron-sulfur clusters redox states by incorporating an unnatural amino acid

Iron-sulfur clusters (FeS) are crucial redox cofactors in nature. They are involved in complex enzyme catalysis, acting as electron-relays. Techniques focusing on studying the electron loading on FeS-cluster relays during enzyme catalysis are rather scarce. Here, a novel approach is described to probe the redox state of FeS clusters by infrared (IR) spectroscopy, exploiting the versatility of this technique across a range of sample conditions and during turnover. In this work, an unnatural amino acid p-cyano-L-phenylalanine (pCNF) was incorporated into the model protein spinach ferredoxin, which contains a [2Fe-2S] cluster. It is shown that pCNF can be used as infrared probe to prove the redox states of the [2Fe-2S] cluster. The integrity and correct functioning of the [2Fe-2S] cluster upon pCNF incorporation was confirmed by UV-Vis spectroscopy and electrochemistry. A small but reproducible shift in the streching frequency of the CN group of pCNF upon chemical or electrochemical reduction of the protein was observed by IR spectroscopy. Furthermore, detailed crystallographic characterisation was performed to compare the structures of the oxidised and reduced pCNF-containing ferredoxin, suggesting that the IR shift does not arise from a structural change but rather from the reduction of the [2Fe-2S] cluster. Then this method was applied to more complex metalloenzymes containing FeS clusters. Experiments were then conducted on E. coli [NiFe] hydrogenase I to test whether an overexpression system could be suitable for extending the pCNF incorporation to Hyd-1. Although the overexpressed protein was found to behave normally comparing to the chromasomally encoded enzyme, it was decided not to continue with this enzyme due to difficulties in crystallising the overexpressed protein, unusual IR active site spectra features and limitation in yield. Instead, the method was applied to [FeFe] hydrogenases from the sulfate reducing bacterium Desulfovibrio desulfuricans (DdHydAB), to investigate how the FeS clusters interact with the diiron prosthetic group to allow efficient catalysis. pCNF was incorporated successfully close to the proximal cluster in DdHydAB. From the preliminary IR results, there is a detectable difference in the band position of the cyano group in the F27CNF variant between the oxidised and reduced states on both apo- (lacking the binuclear site) and holo- (containing the full active site H-cluster) enzymes. Overall, the research work in this thesis demonstrated a novel approach that applying an unnatural amino acid pCNF to metalloenzymes which contain FeS clusters to study the redox state of the FeS cluster by IR spectroscopy.