High-Field Electron Paramagnetic Resonance (EPR) and Double Resonance Methods for Mechanistic Studies of Ribonucleotide Reductase and Heterodisulfide Reductase

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Progress in microwave technology has rendered pulsed and high frequency EPR a unique tool for research directed at the understanding of enzymatic catalysis based on radical chemistry. This research project is for the application of advanced EPR methods such as 180 GHz pulse EPR, Electron Nuclear Double Resonance (ENDOR) and Pulsed Electron Double Resonance (PELDOR), to representative problems as found in the investigation of ribonucleotide reductase (RNR) and heterodisulfide reductase (Hdr). We propose the use of PELDOR methodology to monitor distances in the nanometer range between radicals generated during the catalytic cycle. In studies of class I RNRs, the method has allowed to detect two tyrosyl radicals on one homodimeric R2 subunit of the enzyme from E.coli. We are extending the investigation to the R1:R2 activated complex to obtain information about the docking sites of the two subunits during turnover, which plays an essential role for the postulated electron transfer mechanism of radical initiation in R1. Furthermore, we employ high frequency pulse EPR amd ENDOR to characterise the structure of radical intermediates. In Hdr from methanognic archaea, reaction of the enzyme with the substrate gives rise to a FeS-cluster based intermediate with very unusual EPR spectroscopic features. High frequency ENDOR in combination with selective isotopic labelling will allow to investigate in detail the interaction of the substrate with the proposed 4Fe4S cluster.