title: 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 α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in β2 to initiate catalysis in α2. 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 β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) in the presence of α2, 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 β2. 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 α2. 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 α2 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)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(•) delocalized in α2 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, United States.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Albert A
authors.initials: AA
authors.lastname: Smith
authors.forename: Björn
authors.initials: B
authors.lastname: Corzilius
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
authors.forename: Joanne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D014443:Tyrosine; D012264:Ribonucleotide Reductases