Initiation of the proton pump of cytochrome c oxidase

Abstract

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...

Analyzing the electrogenicity of cytochrome c oxidase

“…3) up to the point where the proton has been transferred to the binuclear center (BNC). The estimated observed voltage changes for the WT were 0.94 (the sum of the voltage changes originating from A→P and P→F) and 1.06 (the sum of the voltage changes of the two F→O components), respectively (43). In considering the normalization for this system, we note that it includes several transitions; the three transitions in the WT (A→P/P→F/F→O) (3.7 charges; i.e., a full oxidative cycle) (table 2 in ref.…”

“…Consequently, we normalized the calculated and observed results to the corresponding ratio between the given voltage and the voltage generated by a transfer of a full charge across the membrane (see ref. 43 for a related treatment). The resulting quantity is called here "fractional voltage," which is related to the so-called "dielectric distance" (ref.…”

Conformational coupling between the active site and residues within the K C -channel of the Vibrio cholerae cbb 3 -type (C-family) oxygen reductase

“…In the A-family HCOs, the P R →F reaction can occur without proton uptake from solution, but in the absence of a mutation blocking the entrance of the D-channel, a proton is taken up during this step to reprotonate the internal proton source. Two residues within the D-channel of the A-family of HCOs are involved in providing internal protons to the active site (28,29). Further reduction of the active site beyond the F intermediate requires proton uptake from bulk in both A-family and C-family HCOs (15), so that in the absence of the E49 IIIVc , this reaction is severely delayed, as evidenced by the slow oxidation of heme c (Fig.…”

Characterizing the proton loading site in cytochromecoxidase

“…The basic cycle assumes: (i) a new active site redox state is initiated with the electron transfer from heme a to the BNC; (ii) this increases the BNC proton affinity so a proton is bound via the D-or K-channel that will be used for chemistry. Other residues can substitute even when proton input into the D-channel is blocked by mutation (37,38); (iii) BNC protonation leads to proton release from the nearby PLS to the P-side; (iv) heme a is reduced by cytochrome c via Cu A ; (v) which leads to loading the nearby PLS by proton transfer from E286. The second electron transfer to the BNC moves the system to the next redox state restarting the sequence.…”