Escherichia coli class Ia ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2′-deoxynucleotides using a radical mechanism. Each turnover requires radical transfer from an assembled diferric tyrosyl radical (Y•) cofactor to the enzyme active site over 35 Å away. This unprecedented reaction occurs via an amino acid radical hopping pathway spanning two protein subunits. To study the mechanism of radical transport in RNR, a suite of biochemical approaches have been developed, such as site-directed incorporation of unnatural amino acids with altered electronic properties and photochemical generation of radical intermediates. The resulting variant RNRs have been investigated using a variety of time-resolved physical techniques, including transient absorption and stopped-flow UV-Vis spectroscopy, as well as rapid freeze-quench EPR, ENDOR, and PELDOR spectroscopic methods. The data suggest that radical transport occurs via proton-coupled electron transfer (PCET) and that the protein structure has evolved to manage the proton and electron transfer co-ordinates in order to prevent ‘off-pathway’ reactivity and build-up of oxidised intermediates. Thus, precise design and control over the factors that govern PCET is key to enabling reversible and long-range charge transport by amino acid radicals in RNR.

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