Ribonucleotide reductase (RNR) catalyzes the penultimate step in de novo deoxyribonucleic acid biosynthesis via a radical mediated mechanism involving a cystine thiyl radical. In the class 1a RNR, such as those found in E. coli and H. sapiens, the thiyl radical in one subunit is transiently generated by intra- and inter-protein radical transfer from a stable diiron-tyrosyl radical cofactor in a second subunit through a pathway of redox active amino acid residues. The radical transfer process is highly regulated on many levels including allosteric and thermodynamic regulation, both of which remain poorly understood due to the complexities of RNR. We have developed two methodologies which allow us to yield unprecidented insight into the radical transfer process: Incorporation of non-canonical tyrosine analogues with altered reduction potentials and/or pKas, and phototriggering radical transfer by photosensitizing RNR with a covalently linked photooxidant adjacent to the radical transfer pathway. Using these methodologies and the E. coli class 1a RNR as a paradigm, we have uncovered conformational dynamics with the radical transfer pathway that mediate the obligate proton-coupled electron transfer (PCET) involved in radical propagation. In addition, by examining conserved residues at the subunit interface know to correlate with activity, we revealed a mechanism of allosteric regulation occuring over 35 angstroms and across a protein-protein interface. The implications of our findings on RNR activity and biological PCET are discussed.

Conformational dynamics affecting PCET during radical transfer in ribonucleotide reductase

256th National Meeting (2018)

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