The class Ia ribonucleotide reductases require an unprecedented oxidation across the interface of the two protein subunits (α and β in the α2β2 active complex) to initiate the complex free radical chemistry of the nucleotide reduction process. The oxidation involves a pathway (Y122 → [W48] → Y356 in β2 to Y731→Y730 → C439 in α2) where Y122● in β catalyzes oxidation of C439 in α over 35 Å. Fluorinated tyrosines (FnY where n = 2, 3) have pKas that range from 6.4 and 7.8 and reduction potentials that vary from 50 mv easier to 250 mv harder to oxidize than tyrosine. Our ability to site-specifically incorporated them in place of any of the four pathway Ys in conjunction with high field paramagnetic resonance methods (EPR and ENDOR) and kinetic analyzes have provided us with otherwise inaccessible insight about the mechanism of this unprecedented reaction. Under various perturbative conditions, all of the transient Y● pathway intermediates are detected, allowing us to observe their equilibration. These studies in conjunction with kinetic studies of FnY122●-β2α2/CDP/ATP have provided us with insight about the thermodynamic and kinetic landscape of this radical transfer pathway. We propose that the pathway is uphill (&gt; 200 mv) (Y122● to C439) and is driven to the right by a rapid irreversible step in the nucleotide reduction process (loss of water). A rationale for this design of the RT landscape is discussed.

Unnatural fluorinated tyrosines allow mapping of the thermodynamic and kinetic landscape of the E. coli class Ia ribonucleotide reductases

252nd National Meeting (2016)

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