title: Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase.
abstract: The Escherichia coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ∼35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ET/PT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731 and Y730 in α. This construct displays an ∼3-fold enhancement in photochemical yield of W(•) relative to F3Y(•) and a ∼7-fold enhancement relative to Y(•). Photogeneration of the W(•) radical occurs with a rate constant of (4.4 ± 0.2) × 10(5) s(-1), which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W(•) within the subunit interface results in 20% activity for turnover relative to wild-type RNR under the same conditions.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138-2902, United States.|Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Olshansky
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138-2902, United States.
authors.forename: Brandon L
authors.initials: BL
authors.lastname: Greene
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, United States.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Finkbeiner
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, United States.
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138-2902, United States.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Nocera
mesh_terms: D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010777:Photochemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D005609:Free Radicals; D014443:Tyrosine; D014364:Tryptophan; D012264:Ribonucleotide Reductases