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.

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 OOO bond and formation of an intermediate PR of the binuclear active site composed of heme a3 and CuB. 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 CuB. To test this idea we used the Glu-278 -Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the PR intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (Ϸ60 s) appearance of the PR 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 PR species suggests transfer of a proton over a distance of Ϸ4 Å 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 OOO bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O2 reduction in conditions where heme a is oxidized.His/Tyr dimer ͉ cytochrome aa3 ͉ cell respiration ͉ proton transfer ͉ FTIR spectroscopy T he electrons required for oxygen reduction by cytochrome c oxidase (CcO) take a specific route from the water-soluble electron donor, cytochrome c, via Cu A and heme a to the binuclear heme a 3 /Cu B center where O 2 is bound. The energy released during the overall reaction is used for proton translocation across the mitochondrial or bacterial membrane (1). The catalytic cycle of CcO starts with binding of dioxygen to heme a 3 [formation of a ferrous O 2 adduct, compound A (2)]. This step is followed by scission of the OOO bond, which requires delivery of four electrons and a proton to dioxygen and leaves the binuclear site in the highly oxidized P state (3-5). Three of the four required electrons are donated by heme a 3 (two electrons) and Cu B (one electron). The source of the fourth electron depends on the initial reduction level of the enzyme. When catalysis starts from the fully reduced enzyme, the fourth electron is provided by heme a and the so-called P R state is formed at the binuclear site (6-9). When the reaction with oxygen starts from the mixed-valence (two-electron reduced) enzyme where both heme a and Cu A are oxidized, a P state is also formed (called P M ), and the fourth required electron is in this case thought to be donated by a nearby amino acid residue. The optical spectrum of P M is indistinguishable from P R (9), which indicates that the binuclear heme a 3 /Cu B site has a similar structure in these intermediates. Scission of the OOO bond also requires delivery of a proton; in both the ''fully r...

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.

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 crosslinked 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.Cytochrome c oxidase (CcO) 3 contains four metallic redox centers: Cu A , heme a, heme a 3 , and Cu B . heme a 3 and Cu B form a binuclear active site of O 2 binding and processing. The free energy of the reduction of O 2 is used by CcO for proton translocation across the mitochondrial or bacterial membrane, thus generating a transmembrane electrochemical proton gradient (1). During one catalytic turnover, CcO pumps four protons and takes up four more protons for the oxygen reduction chemistry. The proton uptake for both pumping and chemistry is accomplished via two proton-conducting channels: the D-and K-channels (see for example Ref. 2).Catalysis by CcO is initiated by binding of an oxygen molecule to heme a 3 when both redox centers of the binuclear active site are reduced. Oxygen binding leads to the formation of the first compound of the catalytic cycle, the ferrous-oxygen adduct, or compound A (3-6). For the intermediates of the catalytic cycle, their sequence, and corresponding structures, see Fig. 7.The next reaction in catalysis is scission of the O-O bond, which requires delivery of four electrons and a proton to dioxygen. Thus, the so-called peroxy intermediate (P) is formed, in which heme a 3 is in the ferryl state (7,8) and Cu B is cupric (9). Two electrons are provided by heme a 3 and one by Cu B . The source of the fourth electron depends on the initial reduction state of CcO. In the case of the fully reduced enzyme, where all four redox centers are reduced, the fourth electron is extracted from heme a (6, 10); in this case, the so-called P R species is formed (9). In the case of the mixed valence enzyme, where only heme a 3 and Cu B are initially reduced but Cu A and heme a are oxidized, the fourth electron is extracted from a nearby residue, most likely 12) that is located in close proximity to the binuclear center, at a distance of ϳ6 Å. This state of the binuclear center has been called P M . The source of the proton for O-O bond scission is in both cases (fully reduced and mixed valence enzyme) likely to be the same residue, viz. Tyr-280. Tyr-280 has a covalent cross-link to one of the His ligands of Cu B , namely 14). The cross-link should lower the pK a of the tyrosine (15), thus facilitating proton donation. The role of Tyr-280 in O-O bond rupture was first predicted from the crystal structure (13, 16) and further supported by radioactive labeling experiments (11), as more recently verified by Fourier transform infrared (FTIR) spectroscopy (12) alone and in combination with electrometry (17).The aim of this work was to...

Cytochrome c oxidase is the terminal enzyme of the respiratory chain that is responsible for biological energy conversion in mitochondria and aerobic bacteria. The membrane-bound enzyme converts free energy from oxygen reduction to an electrochemical proton gradient by functioning as a redox-coupled proton pump. Although the 3D structure and functional studies have revealed proton conducting pathways in the enzyme interior, the location of proton donor and acceptor groups are not fully identified. We show here by time-resolved optical and FTIR spectroscopy combined with time-resolved electrometry that some mutant enzymes incapable of proton pumping nevertheless initiate catalysis by proton transfer to a proton-loading site. A conserved tyrosine in the so-called D-channel is identified as a potential proton donor that determines the efficiency of this reaction.cell respiration | cytochrome aa3 | proton transfer | time-resolved electrometry T he electrochemical proton gradient across phospholipid membranes is the primary product of biological energy transduction during oxidation of foodstuffs by oxygen, followed by the synthesis of ATP by use of this gradient. The reduction of oxygen catalyzed by cytochrome c oxidase (CcO) may be summarized as follows. Electrons from cytochrome c, located on the positively charged P-side of the membrane, are transferred via the Cu A center at the membrane surface, via heme a, to a binuclear heme/ copper (a 3 ∕Cu B ) center (BNC) at a dielectric distance d about ⅓ into the membrane ( Fig. 1A; for reviews, see refs. 1-4). O 2 binding to the reduced BNC yields the oxygen adduct ferrousoxy intermediate (A) (Fig. 1B), and scission of the O-O bond in A yields the P ("peroxy") intermediate. If the reaction with O 2 is started with fully reduced enzyme (R), as in this study, the P state is denoted as P R . The scission of the O-O bond requires simultaneous transfer of four electrons to O 2 . Three of these electrons are taken from the BNC itself, while the fourth is supplied by heme a (5, 6). Protonation of the BNC in the P R state converts it into intermediate F (ferryl), and uptake of another electron and another proton yields the oxidized state O (see Fig. 1B) (7,8). Two more electron and proton transfer steps convert the O state back to the reduced form, which can react with the next O 2 molecule.Each electron transfer into the BNC is thus associated with uptake of a proton into this site from the negatively charged N-side of the membrane and is in addition coupled to translocation (pumping) of one more proton across the entire membrane (9). Proton uptake for formation of the equivalent of water at the BNC takes place via two different pathways, the so-called D-and K-channels, whereas proton uptake for pumping is restricted to the D-channel (Fig. 1A) (2, 3, 10).The dual role of the D-channel has raised much experimental and computational interest (see ref. 11), but its intriguing properties are not yet fully understood. It starts with a conserved aspartate (D124) at the entrance and e...