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

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 122 -β2 reversibly oxidizes C 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 122 Mutations in pathway residues adjacent to the NO 2 Y that disrut H bonding minimally perturb its Ka. The pK a of NO 2 Y 122 -β2 alone or with α2/S/E is > 9.6. X-ray crystal structures have been obtained for all NO 2 Y-α2 mutants (2.1 -3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK a of the previously reported NO 2 Y 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.

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

“…The choice of site, residue 355, for labeling β2 was guided by our previous work using PO-Y-βC19 peptides in place of β2. This peptide, representing the C terminus (355-375 of β2), contains the elements in large part responsible not only for subunit interactions but also activity; Y356 mediates radical transport between α2 and β2 (28,29). The S355 residue was thus targeted as the site of labeling because it is directly adjacent to Y356 and it occupies a position analogous to that of the photooxidant in PO-Y-βC19, a site that has been shown to allow •Y356 generation, radical injection into α2, and subsequent substrate turnover (19)(20)(21).…”

A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

“…Conformational changes thus mask the PCET process. Recently we have incorporated 3-nitrotyrosine (NO 2 Y) site-specifically in place of each Y in the pathway 10. Incorporation of NO 2 Y in place of Y 122 in β revealed an assembled diferric cluster with no 3-nitrotyrosine radical (NO 2 Y•).…”

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects

“…less polarity will increase the pKa value) (91,107,122); ii) changes the amino acid size by the incorporation of a substituent (30 Å 3 larger) (124); iii) changes light absorption with the appearance of a band centered at 360 nm at acidic pH and 420 nm at alkaline pH (107) (i.e. ionization of the phenol group in 3-nitrotyrosine causes a large red shift in absorptivity); iv) influences the ionization process of 3-nitrotyrosine under certain mass spectrometer circumstances (4); and v) increases the hydrophobicity of the nitrated peptide (14,68).…”