pH Rate Profiles of F_nY₃₅₆−R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase:  Evidence that Y₃₅₆ Is a Redox-Active Amino Acid along the Radical Propagation Pathway

Abstract

The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y(122)*) in R2 generate a transient cysteinyl radical (C(439)*) in R1 through a pathway thought to involve amino acid radical intermediates [Y(122)* --> W(48) --> Y(356) within R2 to Y(731) --> Y(730) --> C(439) within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y(356) with a variety of fluorinated tyrosine analogues (2,3-F(2)Y, 3,5-F(2)Y, 2,3,5-F(3)Y, 2,3,6-F(3)Y, and F(4)Y) 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 pK(a)s that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these F(n)()Y(356)-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 Y(356)* using these analogues. As the difference in potential of the F(n)()Y* 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 Y(356) is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO(2)Y(356)-R2, we assume that 2,3,5-F(3)Y(356), 2,3,6-F(3)Y(356), and F(4)Y(356)-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 W(48) and Y(356) of R2 and Y(731) of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.

Engineering Upper Hinge Improves Stability and Effector Function of a Human IgG1

“…2B), suggesting that Tyr does not function like His to facilitate the hinge degradation, rather inhibits the degradation. Similar observations that Tyr functions as a redox-active on or off switch in the ET reactions to mediate protein function were reported previously (13,21,22).…”

The prototypic class Ia ribonucleotide reductase from Escherichia coli: still surprising after all these years

“…Earlier studies demonstrated that C-terminal residues of β 2 were critical for its interaction with α 2 [22, 23]. Just upstream from this C-terminal sequence within the unstructured tail is the absolutely conserved Tyr356 that subsequent studies have shown to be part of the radical relay between the RNR subunits [19, 24, 25]. …”

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

“…Thus, F n Ys are excellent probes for examining proton-coupled electron transfer mechanisms in radical propagation and should be generally useful for studying proteins that require stable or transient tyrosyl radicals for catalysis 15,16 . We next developed an inteinmediated semisynthesis of R2 that allows site-specific replacement of residue Y 356 in R2 with F n Ys 17,18 . Saccharomyces cerevisiae, was discovered in the early 1990s (ref.…”