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Publication Abstract from PubMed

Escherichia coli ribonucleotide reductase is an alpha2beta2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(*)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 A between the two subunits by a specific pathway (Y(122)(*)-->W(48)-->Y(356) within beta to Y(731)-->Y(730)-->C(439) within alpha). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in beta2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(*)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-beta2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(*) that has a half-life of 40 s at 25 degrees C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(*)/beta2 and then mixed with alpha2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(*) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(*) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(*) is predominantly located at 356 in beta2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-beta2, a mechanism for PCET uncoupling in NO(2)Y(*)-RNR is proposed.

A Hot Oxidant, 3-NO(2)Y(122) Radical, Unmasks Conformational Gating in Ribonucleotide Reductase.,Yokoyama K, Uhlin U, Stubbe J J Am Chem Soc. 2010 Oct 7. PMID:20929229^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.