Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase

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 Y122-β2 reversibly oxidizes C439 in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y122 and Y356 in β2, Y731 and Y730 in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO2Y, pKa 7.1) has been incorporated in place of Y122, Y731 and Y730 to probe how the protein environment perturbs each pKa in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is < 4 × 10−3 that of the wt subunit. The [NO2Y730]-α2 and [NO2Y731]-α2 each exhibits a pKa of 7.8 – 8.0 with E and E/β2. The pKa of [NO2Y730]-α2 is elevated to 8.2 8.3 in the S/E/β2 complex, while no further perturbation is observed for [NO2Y731]-α2. Mutations in pathway residues adjacent to the NO2Y that disrut H bonding minimally perturb its Ka. The pKa of NO2Y122-β2 alone or with α2/S/E is > 9.6. X-ray crystal structures have been obtained for all NO2Y-α2 mutants (2.1 – 3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pKa of the previously reported NO2Y356-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. *To whom correspondence should be addressed. Tel: (617) 253-1814. Fax: (617) 324-0505. [email protected] . AUTHOR EMAIL ADDRESS: [email protected] SUPPORTING INFORMATION PARAGRAPH (Word Style “TE_Supporting_Information”). SDS-PAGE of purified [NO2Y]-αs; absorption spectra of [NO2Y]-α2s in their denatured state; absorption spectra of N-acetyl-3-nitrotyrosine amide (0.2 mM) in organic solvent; SDS-PAGE and ESI-QTOF-MS of β expressed in E. coli TOP10/pBAD-NrdB-CS(Y356Z)/pEVOL-NO2Y; UV-vis absorption spectra of [NO2Y122]-β2 with α2/ATP/CDP; pH titration curves of [NO2Y]-α2s; the environment of Y122 in the crystal structure of wt-β2; pKas of o-, mand p-nitrohenol in solution; primers used for cloning and site-directed mutagenesis; crystallographic data collection and refinement statistics; the purity of [NO2Y]-α2s and β2; λmax of N-acetyl-3-nitrotyrosine in organic solvent with 1% triethylamine; Stability of diferric cluster of met-[NO2Y122]-β2 in basic conditions. This material is available free of charge via the Internet at http://ubs.acs.org. NIH Public Access Author Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 June 23. Published in final edited form as: J Am Chem Soc. 2010 June 23; 132(24): 8385–8397. doi:10.1021/ja101097p. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript