pH Rate Profiles of F_nY_(356)-R2s (n = 2,3,4) in Escherichia coli Ribonucleotide Reductase:Evidence that Y_(356) Is a Redox-Active Amino Acid along the Radical Propagation Pathway

摘要

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 centre dot) in R1 through a pathway thought to involve amino acid radical intermediates [Y_(122 centre dot)-> 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_2Y,3,5-F_2Y,2,3,5-F_3Y,2,3,6-F_3Y,and F_4Y) 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_as that range from 5.6 to 7.8.The pH rate profiles of deoxynucleotide production by these F_nY_(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 centre dot) using these analogues.As the difference in potential of the F_nY centre dot relative to Y centre dot 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_2Y_(356)-R2,we assume that 2,3,5-F_3Y_(356),2,3,6-F_3Y_(356),and F_4Y_(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.