Site-specific incorporation of 3-nitrotyrosine as a probe of pKa 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: alpha2 and beta2. During turnover, a stable tyrosyl radical (Y*) at Y(122)-beta2 reversibly oxidizes C(439) in the active site of alpha2. This radical propagation step is proposed to occur over 35 A, to use specific redox-active tyrosines (Y(122) and Y(356) in beta2, Y(731) and Y(730) in alpha2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO(2)Y, 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 [NO(2)Y(730)]-alpha2 and [NO(2)Y(731)]-alpha2 each exhibit a pK(a) of 7.8-8.0 with E and E/beta2. The pK(a) of [NO(2)Y(730)]-alpha2 is elevated to 8.2-8.3 in the S/E/beta2 complex, whereas no further perturbation is observed for [NO(2)Y(731)]-alpha2. Mutations in pathway residues adjacent to the NO(2)Y that disrupt H-bonding minimally perturb its pK(a). The pK(a) of NO(2)Y(122)-beta2 alone or with alpha2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO(2)Y]-alpha2 mutants (2.1-3.1 A resolution), which show minimal structural perturbation compared to wt-alpha2. Together with the pK(a) of the previously reported NO(2)Y(356)-beta2 (7.5 in the alpha2/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.

Figure 1

Tyrosines responsible for the PCET in E. coli class I RNR. (a) Proposed PCET pathway. Red and blue arrows indicate orthogonal transfer of the electron and proton, respectively. The purple arrow indicates co-linear movement of the electron and proton. (b) Structure of the tyrosine-diferric-cluster. Distances in parentheses are those of the Mn₂-β2.

Figure 2

SDS-PAGE of [NO₂Y₇₃₀]-α and [NO₂Y₁₂₂]-β. (a) SDS-PAGE of purified [NO₂Y₇₃₀]-α and whole cell lysate of E. coli expressing [NO₂Y₇₃₀]-α using pSUP-3NT/8 (left) and pEVOL-NO₂Y (right). Cells were grown in the presence or absence of NO₂Y as indicated. The position of protein bands for full-length α (85.6 kDa) and truncated α (82.2 kDa) are denoted by arrows. (b) SDS-PAGE of purified [NO₂Y₁₂₂]-β (45.0 kDa) and a whole cell lysate of E. coli TOP10/pEVOL-NO₂Y/pBAD-nrdB-NS5-Y₁₂₂Z grown in the absence and presence of NO₂Y as indicated. The truncated protein is 15.9 kDa and thus not observable in this gel (10 % acrylamide).

Figure 3

Absorption spectra of nitrophenolate feature of [NO₂Y]-α2s and nitrophenol feature of [NO₂Y₁₂₂]-β2. (a) [NO₂Y₇₃₀]-α2 (blue), [NO₂Y₇₃₁]-α2 (green) and N-acetyl-3-nitrotyrosine amide (yellow) in 50 mM TAPS (pH 9.0), 1 mM EDTA, 15 mM MgSO₄. Spectral intensities were normalized to NO₂Y concentration (15 μM) according to the purity of [NO₂Y₇₃₀]-α2 (92%), [NO₂Y₇₃₁]-α2 (79%) determined from A_435nm in 6 M guanidine. (b) Red trace, absorption spectra of [NO₂Y₁₂₂]-β2 (2.9 Fe/β2, 15 μM) in HEPES (pH 7.6); blue trace, absorption spectrum after subtraction of the met-β2 spectrum (3.2 Fe/β2, 13.5 μM, the concentration of β2 was normalized for the iron content); black trace, absorption spectrum of N-acetyl-3-nitrotyrosine amide in 50 mM MES (pH 5.0) buffer.

Figure 4

UV-vis absorption spectra of (a) [NO₂Y₇₃₀]-α2 (7.5 μM) and (b) [NO₂Y₇₃₁]-α2 (7.5 μM) at pH 6.0 (the pink trace), 7.4, 8.0, 8.6 and 9.2 (the blue trace) in the presence of 1 mM ATP. Loss of the phenol feature (360 nm) occurs concomitant with the formation of the phenolate feature (442 and 437 nm for [NO₂Y₇₃₀]-α2 and [NO₂Y₇₃₁]-α2, respectively) with increasing pH (pink to blue).

Figure 5

Titration curves of (a) [NO₂Y₇₃₀]-α2 and (b) [NO₂Y₇₃₁]-α2 in the presence of 1 mM ATP (yellow diamonds), 1 mM ATP and 7.5 μM β2 (blue squares) and 1 mM ATP, 7.5 μM β2 and 1 mM CDP (red circles). Absorption at 442 nm and 437 nm were monitored for [NO₂Y₇₃₀]-α2 and [NO₂Y₇₃₁]-α2, respectively. Each data point prepresents an average of three replicates. Lines are from fits to eq. 2.

Figure 6

UV-vis absorption spectra of (a) met-[NO₂Y₁₂₂]-β2 at pH 7.1 (red), 7.6 (orange), 8.2 (yellow), 8.6 (dark green), 9.0 (light green), 9.2 (light blue), 9.5 (blue) and 10.0 (dark blue), and (b) apo-[NO₂Y₁₂₂]-β2 at pH 7.6 (the pink trace), 8.4, 9.0, 9.6, 10.0, 10.3 and 10.6 (the blue trace).

Figure 7

Crystal structures of the radical propagation pathway in NO₂Y-α2 mutants. The crystals were grown at pH 6.0-6.5. Oxygens are colored in red, nitrogens in blue, and sulfurs in gold. Pathway residues 731, 730 and 439 are shown with sticks. The structure of (a) [NO₂Y₇₃₀]-α2 and (b) [NO₂Y₇₃₁]-α2; the dotted lines indicate the distances between the phenolic oxygens, and the phenolic oxygen of the 730 residue and the sulfur of C₄₃₉ with distance variations associated with the three subunits in the asymmetric unit. The surrounding residues are shown as sheres. (c) An overlay of the structures of wt-α2 (green), [NO₂Y₇₃₀]-α2 (orange), and [NO₂Y₇₃₁]-α2 (yellow), generated using PyMOL 1.1 (DeLano Scientific LLC) software. Other residues shown are P₆₂₁, E₄₄₁, L₄₃₈, Y₄₁₃ and R₄₁₁. (d) An overlay of the structures of [NO₂Y₇₃₀]-α2 and the double mutants C₄₃₉S, C₄₃₉A, Y₇₃₁A and Y₇₃₁F. Residues shown are (from the left) 441, 439, 730 and 731.