The Development and Analysis of Model Systems to Probe Proton-Coupled Electron Transfer in Ribonucleotide Reductase Ia of E. Coli

Abstract

Proton-coupled electron transfer (PCET) combines proton transfer with electron transfer to bypass high-energy intermediates. The ribonucleotide reductase (RNR) family of enzymes catalyzes the conversion of ribonucleotides to deoxynucleotides using amino acid radicals. The enzyme contains an efficient PCET pathway that transfers an electron and proton over a 35 Å distance across two subunits, the longest PCET pathway known in biology. The enzyme func-tions with very high fidelity, performing >105 turnovers before radical loss.

This thesis explores PCET in model systems to understand the radical transport pathway in E. coli RNR type Ia. First, amino acid radical generation was explored by laser spectroscopy. Small molecule systems, where photooxidants were appended with amino acids, provide a plat-form for probing both the pH and driving force dependence of PCET. Analysis of the emission kinetics of these systems indicate that amino acid radicals were generated by sequential electron and proton transfers as a result of the strong driving force of the photooxidant. Next, the dyad model with cofacial aromatic units was developed to investigate the PCET radical transport mechanism between two adjacent amino acids. Inspired by the unique redox cooperativity ob-served in Y730-Y731 tyrosine dyads at the α2β2 interface of RNR, electrochemical and computa-tional approaches were used to probe the correlation between the structural and redox properties of the dyad model. Using electrochemical and computational analysis, it was determined that the presence of two phenol units in the dyad lowered the reduction potential by ~ 60 mV. This result demonstrates that the tyrosine dyad behaves as a discrete redox unit, consistent with the observed radical transport pathway observed in RNR. Finally, the bidirectional PCET pathway in the β2 subunit of RNR (Y122-W48-Y356) was modeled using a polyproline-based peptide. The long-distance electron transfer between tyrosine analogs and tryptophan was investigated by pulse ra-diolysis to determine the radical transfer rate in these peptide models. Since efficient electron transfer was observed between tetrafluorotyrosine and tryptophan at a 10 Å distance in the model system, Y122 and W48 at a 7.4 Å distance in RNR likely serve as the radical transport pathway in a bidirectional, sequential electron and proton transfer mechanism.