The Protonation State of the Cross-linked Tyrosine during the Catalytic Cycle of Cytochrome c Oxidase

Abstract

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...

Protonation of the binuclear active site in cytochrome c oxidase decreases the reduction potential of CuB

“…However, even if there may be an error in the calculations with regard to the order of the O B and the O H states, the O B state will not be significantly lower than the O H state, and the copper reduction potential would still be high in the O B state. It can also be noted that the small energy difference between the O H and O B states, is in line with the observation that the tyrosine residue becomes partially reprotonated in the oxidized intermediate at acidic conditions [24].…”

Kinetic gating of the proton pump in cytochrome c oxidase

“…The remaining two chemical protons are transferred via the K pathway. A likely factor for this duality is the special role of Tyr-244 at the end of the K pathway, a residue implicated in the chemistry of dioxygen bond-breaking as a proton and electron donor (30,31).…”

Computational study of the activated O H state in the catalytic mechanism of cytochrome c oxidase

“…FTIR data (21) suggested that the cross-linked tyrosine is a deprotonated tyrosinate in both the F and O states. The proton taken up into the BNC upon reduction of state F is thus likely to form a ferric heme a 3 hydroxide (Fig.…”

“…The crosslinked tyrosine is unprotonated (21) so that the cupric Cu B will have either a water molecule as the fourth ligand or no fourth ligand at all. The reason for this is that the oxidation of the fully reduced enzyme is coupled to net uptake of no more than two protons, which occurs in the steps P→F and F→O (Fig.…”