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Research article2010Peer reviewedOpen access

Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK(a) Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase

Yokoyama, K; Uhlin, Ulla; Stubbe, J


E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5'-diphosphates into 2'-deoxynucleotides and is composed of two subunits: alpha 2 and beta 2. During turnover, a stable tyrosyl radical (Y center dot) at Y-122-beta 2 reversibly oxidizes C-439 in the active site of alpha 2. This radical propagation step is proposed to occur over 35 angstrom, to use specific redox-active tyrosines (Y-122 and Y-356 in beta 2, Y-731 and Y-730 in alpha 2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO2Y, pK(a) 7.1) has been incorporated in place of Y-122, Y-731, and Y-730 to probe how the protein environment perturbs each pK(a) in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 x 10(-3) that of the wild-type (wt) subunit. The [NO2Y730]-alpha 2 and [NO2Y731]-alpha 2 each exhibit a pKa of 7.8-8.0 with E and E/beta 2. The pK(a) of [NO2Y730]-alpha 2 is elevated to 8.2-8.3 in the S/E/beta 2 complex, whereas no further perturbation is observed for [NO2Y731]-alpha 2. Mutations in pathway residues adjacent to the NO2Y that disrupt H-bonding minimally perturb its pK(a). The pK(a) of NO2Y122-beta 2 alone or with alpha 2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO2Y]-alpha 2 mutants (2.1-3.1 angstrom resolution), which show minimal structural perturbation compared to wt-alpha 2. Together with the pK(a) of the previously reported NO2Y356-beta 2 (7.5 in the alpha 2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541-14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway.

Published in

Journal of the American Chemical Society
2010, Volume: 132, number: 24, pages: 8385-8397

      SLU Authors

    • Uhlin, Ulla

      • Department of Molecular Biology, Swedish University of Agricultural Sciences

    UKÄ Subject classification

    Biochemistry and Molecular Biology

    Publication identifier


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