Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential

Abstract

The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

Fig. 1

Experimental system and model. a CytcO and ATP-synthase orientation in SMPs and mitochondria, respectively. b The reaction studied in this work. Upon initiation of the reaction by a light flash CO dissociates to form state R. Then, O₂ binds to the reduced CytcO after which the reaction proceeds as shown and described in the text. The data from this work suggest that the F_R → O step is slowed/blocked by the electrochemical proton gradient (red arrow). The release of H₂O is indicated in the O → R step, but one of the H₂O molecules could be released earlier in the cycle. One of the protons taken up in this reaction is bound by a Tyr residue that is not explicitly drawn in the figure (the Tyr also donates a proton upon forming P_R)

Fig. 2

Generation of an ATP-induced transmembrane electrochemical potential in SMPs. a The membrane electrical potential (ΔΨ) was monitored using the dye oxonol VI. The absorbance changes at 623 nm were measured as a function of time. ATP was added to the SMPs solution, containing oxonol VI, at t = 0 (black trace). The signal increased, consistent with proton pumping into the SMPs. Addition of nigericin after ~60 s (red trace) resulted in an increase in absorbance as the proton gradient (ΔpH) was converted into an electrical potential. In the presence of valinomycin, (val., added after ~120 s) the absorbance decreased to the same level as that before addition of ATP (red trace). Addition of FCCP (at ~540 s) removes both ΔΨ and ΔpH (black trace). Mixing artifacts caused by the additions were removed for clarity. The absorbance changes were also resolved on a shorter time scale using a stopped-flow device (see inset in a). The arrow indicates the time at which the reaction was started in the experiments shown in Fig. 3a, b. Experimental conditions: 0.1 mg SMPs in 1 ml buffer, 2 µM oxonol VI, 1 µM valinomycin. The final ATP concentration was 160 µM. In the stopped-flow experiment it was 1 mM. b Proton pumping to the interior of the SMPs (initiated by addition of ATP at t = 0) was monitored using the fluorescent dye ACMA. As the inside of the SMPs became acidified the emission of the fluorescent dye was quenched. Experimental conditions: 0.1 mg SMPs in 1 ml buffer, 200 nM ACMA, the final ATP concentration was 160 µM. The fluorophore was excited at 410 nm and the emission was recorded at 480 nm. (a.u.) is arbitrary units

Fig. 3

Absorbance changes associated with the reaction of CytcO with O₂ in sub-mitochondrial particles. The reduced CytcO was mixed with an O₂-saturated solution containing ATP. After 0.8 s the reaction of the CytcO with O₂ was initiated by a laser flash (at t = 0 in the graph). It was monitored at 445 nm (a), which reflects the redox states of hemes a and a₃, and at 605 nm (b), which reflects the redox state mainly of heme a. Experimental conditions after mixing: 250 mM sucrose, 50 mM KCl, 10 mM phosphate buffer at pH 7.4, 5 mM MgCl₂, 0.1 mM EDTA, 25 mM ATP, 1.9 mg ml⁻¹ SMPs with or without 10 µM valinomycin and 200 nM FCCP. The mixing ratio was 1:1. The grey graph is the 280-s time point from c. Typically, 10 or 20 traces were averaged at 445 nm and 605 nm, respectively. In b a laser artifact has been truncated. c Amplitude of the 5-ms component as a function of time monitored at 445 nm (filled symbols) and 605 nm (open symbols). ATP was added to the anaerobic SMP solution before the sample was transferred to the stopped-flow apparatus. The first time point in this graph was obtained before addition of ATP (set to 100%). The errors, estimated from the noise level of the traces, were typically smaller than the marks. Experimental conditions before mixing: 250 mM sucrose, 50 mM NaCl, 20 mM Hepes pH 7.4, 5 mM MgCl, 1 mM ATP and 5.5 mg ml⁻¹ SMP. The SMP:O₂ solution mixing ratio was 1:5. The time delay between mixing and laser flash was 0.2 s