Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation

Abstract

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their β-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.

Figure 1. Postulated mechanisms for the reaction of oxygen with the reduced Cu_B-heme a₃ binuclear center in CcO and for the reaction of H₂O₂ with the oxidized enzyme

In this scheme the reaction with O₂ with the reduced binuclear center starts from the lower left corner and progresses to the P_M intermediate shown in the center. P_M is associated with a radical on Y244, which donates an electron to the binuclear center following the O-O bond cleavage reaction. The reaction sequence progresses to the P_R and F intermediates and ultimately to the oxidized state on the top left. The reaction with H₂O₂ is initiated by its binding to the heme a₃ of the oxidized enzyme. The oxidized bCcO preparation used for the present work contains a peroxide bridged between Cu and Fe.^, Thus, the O species in the scheme are likely to be formed by the H₂O₂ reduction of the bridged peroxide. The O-O bond cleavage of the resulting hydroperoxy intermediate leads to a Compound I species with a porphyrin π-cation, which can accept an electron from Y244 to form P_H, leaving a radical on Y244 (Pathway A). The Y244 radical can be re-reduced by Y129 to form F^•. Alternatively, the porphyrin π-cation can accept an electron directly from Y129 to form F^• without passing through Y244 (Pathway B). The P_H intermediate formed via Pathway A is the same as that (P_M) generated in the O₂ reaction and depending on the pH can progress to form P_R and F.

Figure 2. Optical absorption difference spectra of the intermediates formed in the reaction bCcO with H₂O₂

Difference spectra were taken relative to the oxidized resting bCcO at either pH 6 (a) or pH 8 (b). Optical spectra were recorded at ~1 min following mixing. The bCcO/H₂O₂ concentrations for (a) and (b) were 220/280 µM and 390/500 µM, respectively.

Figure 3. X-band and D-band EPR spectra of the intermediates formed in the reaction of bCcO with H₂O₂ at pH 8

Panel (a) and (b) show the X-band and D-band spectra (solid lines), respectively, obtained following the reaction of bCcO with 36-fold excess of H₂O₂. Panel (c) shows the D-band spectrum (solid line) of bCcO obtained following its reaction with a stoichiometric amount of H₂O₂revealing contributions from both the 12 G and 46 G radicals. The experimental and simulation data are given by the solid and dotted lines, respectively. The parameters for the simulations are given in Table 1. In the X-band spectrum (a), the contribution from Cu_A has been removed by subtracting the spectrum of resting oxidized bCcO.

Figure 4. Crystal structures showing the orientation of Y244 and Y129

(Left) Crystal structure showing the orientation of Y244 and His240. The C_α—C_β bond is 31.3° to the plane of the phenol ring. Assuming sp³ geometry, the β-methylene protons are calculated to be 61.3° and 58.7° to the perpendicular axis to the phenol plane. (Right) Crystal structure showing the orientation of Y129. The C_α—C_β bond is 0.3° to the plane of the phenol ring, and the β-methylene protons are 30.3° and 29.7° to the perpendicular axis of the phenol plane. The inset defines the positions on the tyrosine.

Figure 5. X-band and D-band EPR spectra of the intermediates formed in the reaction of CcO with H₂O₂ at pH 6

The H₂O₂ (900 µM) used was ~2.5-fold excess of the enzyme (350 µM). Panels (b) and (d) are the observed spectra obtained at X-band and D-band, respectively while (a) and (c) are the corresponding spectra after subtraction of the 12 G radical. (f) is an expansion of the spectrum in (a) compared to that reported for PdCcO (e). The dashed lines in (e) and (f) are to help guide the eye. The dotted lines in (a–d) are simulated spectra with parameters given in Table 1. In the X-band spectra, the contributions from Cu_A have been removed by subtracting the spectrum of resting oxidized CcO.

Figure 6

Spin density for the optimized ferryl species without deprotonation of Y244 (main panel) and after a proton transfer from Tyr244 (lower right inset). The total unpaired spin density is shown with a green mesh.

Figure 7. The structure of the heme a₃-Cu_B binuclear center, Y244 and Y129 with respect to the positive and negative sides of the inner mitochondrial membrane

The red spheres are water molecules that are part of the water cluster near the heme a₃ propionates. These three water molecules directly link the propionates of heme a₃ to Tyr-129 by an H-bonding network (black dashed lines). The radical migration between Tyr-244 and Tyr-129 may occur through the linked Y244, H240, W236, Y129 network. The H-bond distances (in Å) between water molecules a, b and c and that between c and the oxygen of Y129 are indicated. The figure was made from PDB ID: 3AG3 with PyMol Molecular Graphics Software (Delano Scientific, LLC).