Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase
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Author(s)
Yokoyama, Kenichi
Uhlin, Ulla
Stubbe, JoAnne
Date Issued
June 2010
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Citation
Yokoyama, Kenichi, Ulla Uhlin, and JoAnne Stubbe. “Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a]Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase.” Journal of the American Chemical Society 132.24 (2010): 8385–8397.
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Author's final manuscript
Abstract
E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y[subscript 122] and Y[subscript 356] in β2, Y[subscript 731] and Y[subscript 730] in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO[subscript 2]Y, pK[subscript a] 7.1) has been incorporated in place of Y[subscript 122], Y[subscript 731], and Y[subscript 730] to probe how the protein environment perturbs each pK[subscript a] in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 × 10[subscript −3] that of the wild-type (wt) subunit. The [NO[subscript 2]Y[subscript 730]]-α2 and [NO[subscript 2]Y[subscript 731]]-α2 each exhibit a pK[subscript a] of 7.8−8.0 with E and E/β2. The pK[subscript a] of [NO[subscript 2]Y[subscript 730]]-α2 is elevated to 8.2−8.3 in the S/E/β2 complex, whereas no further perturbation is observed for [NO[subscript 2]Y[subscript 731]]-α2. Mutations in pathway residues adjacent to the NO[subscript 2]Y that disrupt H-bonding minimally perturb its pK[subscript a]. The pK[subscript a] of NO[subscript 2]Y[subscript 122-]β2 alone or with α2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO[subscript 2]Y]-α2 mutants (2.1−3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK[subscript a] of the previously reported NO[subscript 2]Y[subscript 356-]β2 (7.5 in the α2/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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
http://dx.doi.org/10.1021/ja101097p
