Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase.

Biochemistry
Ellen C MinnihanJoAnne Stubbe

Abstract

Escherichia coli ribonucleotide reductase (RNR), an alpha2beta2 complex, catalyzes the conversion of nucleoside 5'-diphosphate substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). beta2 contains the essential diferric tyrosyl radical (Y(122)(*)) cofactor which, in the presence of S and E, oxidizes C(439) in alpha to a thiyl radical, C(439)(*), to initiate nucleotide reduction. This oxidation occurs over 35 A and is proposed to involve a specific pathway: Y(122)(*) --> W(48) --> Y(356) in beta2 to Y(731) --> Y(730) --> C(439) in alpha2. 3-Aminotyrosine (NH(2)Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH(2)Y(*)) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y(356)F-beta2:Y(731)NH(2)Y-alpha2, Y(356)F-beta2:Y(730)NH(2)Y-alpha2, and wt-beta2:Y(731)F/Y(730)NH(2)Y-alpha2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH(2)Y(*) was detected. To study off-pathway oxidation, Y(413), located...Continue Reading

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Citations

Feb 11, 2012·Analytical and Bioanalytical Chemistry·Hui-wang Ai
Jun 5, 2013·Accounts of Chemical Research·Ellen C MinnihanJoanne Stubbe
Nov 15, 2007·Chemical Reviews·My Hang V Huynh, Thomas J Meyer
Jun 19, 2012·Chemical Reviews·David R WeinbergThomas J Meyer
Aug 10, 2011·Journal of the American Chemical Society·Berta M MartinsThorsten Selmer
Nov 30, 2011·Journal of the American Chemical Society·Patrick G HolderDaniel G Nocera
Oct 5, 2011·Proceedings of the National Academy of Sciences of the United States of America·Nafez Abu TarboushVictor L Davidson
Feb 23, 2013·Proceedings of the National Academy of Sciences of the United States of America·Ellen C MinnihanJoanne Stubbe
Aug 3, 2014·Bioorganic Chemistry·Heather R WilliamsonVictor L Davidson
May 11, 2017·Essays in Biochemistry·Steven Y Reece, Mohammad R Seyedsayamdost
Oct 19, 2017·Journal of the American Chemical Society·Brandon L GreeneDaniel G Nocera

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