pH Rate Profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-

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• 作者：Mohammad R. Seyedsayamdost ; Cyril S. Yee ; Steven Y. Reece ; Daniel G. Nocera ; and JoAnne Stubbe
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：February 8, 2006
• 年：2006
• 卷：128
• 期：5
• 页码：1562 - 1568
• 全文大小：168K
• 年卷期：v.128,no.5(February 8, 2006)
• ISSN：1520-5126

文摘

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 tyrosylradical (Y₁₂₂s/entities/bull.gif">) in R2 generate a transient cysteinyl radical (C₄₃₉s/entities/bull.gif">) in R1 through a pathway thought to involveamino acid radical intermediates [Y₁₂₂s/entities/bull.gif"> s/entities/rarr.gif"> W₄₈ s/entities/rarr.gif"> Y₃₅₆ within R2 to Y₇₃₁ s/entities/rarr.gif"> Y₇₃₀ s/entities/rarr.gif"> C₄₃₉ within R1]. To studythis radical propagation process, we have synthesized R2 semisynthetically using intein technology andreplaced Y₃₅₆ with a variety of fluorinated tyrosine analogues (2,3-F₂Y, 3,5-F₂Y, 2,3,5-F₃Y, 2,3,6-F₃Y, andF₄Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosinederivatives have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pHrange for RNR and pK_as that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production bythese F_nY₃₅₆-R2s are reported. The results suggest that the rate-determining step can be changed froma physical step to the radical propagation step by altering the reduction potential of Y₃₅₆s/entities/bull.gif"> using theseanalogues. As the difference in potential of the F_nYs/entities/bull.gif"> relative to Ys/entities/bull.gif"> becomes >80 mV, the activity of RNRbecomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the modelthat Y₃₅₆ is a redox-active amino acid on the radical-propagation pathway. On the basis of our previousstudies with 3-NO₂Y₃₅₆-R2, we assume that 2,3,5-F₃Y₃₅₆, 2,3,6-F₃Y₃₅₆, and F₄Y₃₅₆-R2s are all deprotonatedat 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 rolein triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radicalpropagation.