pH Rate Profiles of FnY356−R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-Active Amino Acid along the Radical Propagation Pathway

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Abstract
The Escherichiacoli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y122•) in R2 generate a transient cysteinyl radical (C439•) in R1 through a pathway thought to involve amino acid radical intermediates [Y122• → W48 → Y356 within R2 to Y731 → Y730 → C439 within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y356 with a variety of fluorinated tyrosine analogues (2,3-F2Y, 3,5-F2Y, 2,3,5-F3Y, 2,3,6-F3Y, and F4Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivatives have potentials that vary from −50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pKas that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these FnY356−R2s are reported. The results suggest that the rate-determining step can be changed from a physical step to the radical propagation step by altering the reduction potential of Y356• using these analogues. As the difference in potential of the FnY• relative to Y• becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y356 is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO2Y356−R2, we assume that 2,3,5-F3Y356, 2,3,6-F3Y356, and F4Y356−R2s are all deprotonated at pH > 7.5. We show that they all efficiently initiate nucleotide reduction. If this assumption is correct, then a hydrogen-bonding pathway between W48 and Y356 of R2 and Y731 of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.