The protonation state of the cross-linked tyrosine during the catalytic cycle of cytochrome c oxidase.

Cytochrome c oxidase is the terminal complex of the respiratory chain in mitochondria and some aerobic bacteria and is responsible for most of the O(2) consumption in biology. The key reaction in the catalysis of O(2) reduction is O-O bond scission that requires four electrons and a proton. In our recent work (Gorbikova, E. A., Belevich, I., Wikstrom, M., and Verkhovsky, M. I. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 10733-10737), it was shown that the cross-linked Tyr-280 (Paracoccus denitrificans numbering) provides the proton for O-O bond cleavage. The deprotonated Tyr-280 must be reprotonated later on in the catalytic cycle to serve as a proton donor for the next oxygen reduction event. To find the reaction step at which the cross-linked Tyr-280 becomes reprotonated, all further steps of the catalytic cycle after O-O bond cleavage were followed by infrared spectroscopy. We found that complete reprotonation of the tyrosine is linked to the formation of the one-electron reduced state coupled to reduction of the Cu(B) site.

The proton donor for O-O bond scission by cytochrome c oxidase.

Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate P(R) of the binuclear active site composed of heme a(3) and Cu(B). The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu(B). To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the P(R) intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximately 60 micros) appearance of the P(R) species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the P(R) species suggests transfer of a proton over a distance of approximately 4 A perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O(2) reduction in conditions where heme a is oxidized.

Time-resolved ATR-FTIR spectroscopy of the oxygen reaction in the D124N mutant of cytochrome c oxidase from Paracoccus denitrificans.

Real-time measurements of the cytochrome c oxidase reaction with oxygen were performed by ATR-FTIR spectroscopy, using a mutant with a blocked D-pathway of proton transfer (D124N, Paracoccus denitrificans numbering). The complex spectrum of the ferryl-->oxidized transition together with other bands showed protonation of Glu 278 with a peak position at 1743 cm-1. Since our time resolution was not sufficient to follow the earlier reaction steps, the FTIR spectrum of the CO-inhibited fully reduced-->ferryl transition was obtained as a difference between the spectrum before the laser flash and the first spectrum after it. A trough at 1735 cm-1 due to deprotonation of Glu 278 was detected in this spectrum. These observations confirm the proposal [Smirnova I.A., et al. (1999) Biochemistry 38, 6826-6833] that the proton required for chemistry at the binuclear site is taken from Glu 278 in the perroxy-->ferryl step, and that the rate of the next step (ferryl-->oxidized) is limited by reprotonation of Glu 278 from the N-side of the membrane in the D124N mutant enzyme. The blockage of the D-pathway in this mutant for the first time allowed direct detection of deprotonation of Glu 278 and its reprotonation during oxidation of cytochrome oxidase by O2.

Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy.

Reduction of cytochrome c oxidase is coupled to proton uptake, and the reduced-minus-oxidized FTIR spectrum should include signatures of protonation of protolytic centers. The major part of the spectrum shows only small differences between acidic and alkaline conditions, which is consistent with the rather weak pH dependence of the proton uptake stoichiometry. Here we aim at revealing redox state-dependent protonatable sites and present a comprehensive investigation over a wide pH range. The reduced-minus-oxidized transition of cytochrome c oxidase from Paracoccus denitrificans was studied by means of Fourier transform infrared spectroscopy in the pH range 5.2-9.5. Effects of pH were analyzed as the difference between reduced-minus-oxidized FTIR spectra at different pH values. Two pH-dependent processes with apparent pKa values of 6.6 and 8.4 and Hill coefficients 0.9 and 0.1, respectively, were found by this methodology. A sharp OH band appears in the IR "water region" on reduction of the enzyme, independent of pH in the range 6.5-9.0, and downshifted by approximately 940 cm-1 on changing the solvent to D2O and by 10 cm-1 on H216O/H218O isotope exchange. This feature of an asymmetric water molecule may belong to water that is produced in the binuclear center upon reduction or to a structured water molecule that loses a hydrogen bond.

Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy.

Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.