Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.

ABSTRACT: Escherichia coli ribonucleotide reductase is an ?2?2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in ?2 to initiate catalysis in ?2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•) ? [W(48)?] ? Y(356) within ? to Y(731) ? Y(730) ? C(439) within ?). Previously, we reported that a ?2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/?2) in place of Y(122)(•) in the presence of ?2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(•) proposed to be located on Y(356) in ?2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) in ?2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in ?2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-?2, which revealed F(2)Y(•), studies using globally incorporated [?-(2)H(2)]Y-?2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(•) delocalized in ?2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y(•) radicals in the PCET pathway and their conformations.

SUBMITTER: Yokoyama K 

PROVIDER: S-EPMC3236566 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature