Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

Abstract

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.

Figure 1

H₂O₂-induced changes in the absolute and difference spectrum of cytochrome c oxidase. Main panel: time-dependent difference spectra of H₂O₂-reacted CcO minus oxidized CcO. Inset: the absolute spectra of oxidized CcO (thick line) and CcO after reaction with 1 mM H₂O₂ for 30 min. (thin line). Experimental conditions were 6 μM CcO in 20 mM Tris-SO₄ buffer at pH 7.4 containing 2 mM dodecylmaltoside.

Figure 2

Total sequence coverage of CcO subunits I, II and III by HPLC-ESI/MS/MS analysis of proteolytic peptides following in-gel digestion of the SDS-PAGE resolved subunits. Chymotryptic peptides detected by MS covered 65%, 46% and 28% of the subunits I, II and III sequences, respectively (sequences are underlined with a simple line). Elastase peptides detected by MS covered 52%, 44% and 38% of the subunit I, II and III sequences, respectively (sequences are underlined with bar-ended line, |——| ). Total sequence coverage by both experimental approaches was 84% of Subunit I, 66% of Subunit II and 54% of Subunit III (sequences are shaded blue). The underlined amino acids indicate the sequence coverage, not proteolytic peptides. For example, chymotryptic coverage of the Subunit I sequence 322–348 was accomplished by MS analysis of chymotryptic peptides 322–327, 328–334, 335–340, 341–344 and 345–348. After exposure of CcO to H₂O₂, the only oxidized amino acid that could be detected was W_334(I) (red box).

Figure 3

Electrospray ionization MS/MS data obtained for chymotryptic peptide 328–334 of Subunit I before (upper panel) and after (lower panel) exposure of CcO to 1 mM H₂O₂ for 30 min.

Figure 4

Location of the three tryptophans, which are oxidized by hydrogen peroxide, within the 3-dimensional structure of the CcO dimer. The color scheme is: oxidized tryptophans (red), Subunit I (cyan), all other subunits (blue) and the binuclear center (heme a₃, gold; Cu_B, magenta). Image was generated from the 3-dimensional coordinates of CcO (1v54) [25] using PyMol™ software, DeLano Scientific, LLC.

Figure 5

Stereo image of the aromatic amino acid networks within CcO that connect the binuclear center to W_334(I), W_19(VIIc) and W_48(IV). Aromatic amino acids that lead from the binuclear center toward W_19(VIIc) are, from left to right: H_376(I), H_429(I), F_430(I), F_426(I), F_346(I), W_323(I), F_348(I), Y_379(I), F_251(I), F_344(I), F_418(I), F_387(I), F_414(I), H_256(I), W_340(I), W_396(I), H_413(I), H_395(I), F_78(I), F_393(I), F_397(I), W_409(I), F_400(I), F_470(I), F_476(I), W_473(I) and Y_403(I). Aromatic amino acids that connect this first network to W_334(I) are: Y_260(I) and Y_261(I). Aromatic amino acids that lead from the binuclear center toward W_48(IV) are, from top to bottom: Y_372(I), H_376(I), H_429(I), F_430(I), F_426(I), F_32(II), F_321(I), W_65(II), F_268(I), Y_40(II), H_328(I), H_52(II), Y_39(Va), W_15(Va), Y_18(Va), F_68(IV), F_19(Va), F_64(IV), Y_60(IV) and F_61(Va). Image was generated from the 3-dimensional coordinates of CcO (1v54) [25] using PyMol™ software, DeLano Scientific, LLC.