Photochemical ribonucleotide reductase for the study of proton-coupled electron transfer

Abstract

Charge transport and catalysis in enzymes often rely on amino acid radicals as intermediates. The generation and transport of these radicals are synonymous with proton-coupled electron transfer (PCET), which intrinsically is a quantum mechanical effect as both the electron and proton tunnel. The caveat to PCET is that proton transfer (PT) is fundamentally limited to short distances relative to electron transfer (ET). This predicament is resolved in biology by the evolution of enzymes to control PT and ET coordinates on very different length scales. In doing so, the enzyme imparts exquisite thermodynamic and kinetic control over radical transport and radical-based catalysis at cofactor active sites. New tools are needed to study PCET reactions of amino acid radical in biology. This thesis describes methods for photogeneration of amino acid radicals, with particular emphasis on tyrosine. Unnatural fluorotyrosine amino acids are developed to vary the driving force for proton and electron transfer in PCET reactions of tyrosyl radical (Ye), and to provide unique spectroscopic handles to study enzymes utilizing multiple Yes. These tools allow for an in-depth study of the PCET mechanism of tyrosyl radical generation, both in solution and within the ribonucleotide reductase enzyme. Enzymatic acitivity of class I E. coli ribonucleotide reductase requires the transport of charge from an assembled diiron-tyrosyl radical cofactor to the enzyme active site over 35 A away via an amino acid radical hopping pathway spanning two protein subunits.

(cont.) To study the mechanism of this radical transport, we have developed photochemical RNRs wherein radical generation, transport, and enzymatic turnover can be initiated by UV-vis excitation of a peptide bound to the subunit containing the enzyme active site. This method allows us to observe Y*s competent for initiating turnover on the peptide bound to the protein subunit. Turnover assays with the wild-type and mutant proteins show that both the electron and proton move along a unidirectional pathway to affect radical transport in this subunit.