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

Mohammad R. Seyedsayamdost, Cyril S. Yee, Steven Y. Reece, Daniel G. Nocera, Jo Anne Stubbe

Research output: Contribution to journalArticle

91 Scopus citations

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 [Y122• → W48 → Y 356 within R2 to Y731 → Y730 → 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-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 Y 356 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.

Original languageEnglish (US)
Pages (from-to)1562-1568
Number of pages7
JournalJournal of the American Chemical Society
Volume128
Issue number5
DOIs
StatePublished - Feb 8 2006

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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