Redox-coupled proton translocation in biological systems: proton shuttling in cytochrome c oxidase

Abstract

In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 --> Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O2). However, in this mutant CcO, the rate by which O2 is reduced to H2O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 A from N(I-139) and is an internal proton donor to the catalytic site, located in the membrane-spanning part of CcO. The mutation results in raising the effective pKa of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons.

Fig. 1.

(A) The structure of CcO from R. sphaeroides (1m56 in the Protein Data Bank, www.rcsb.org/pdb; ref. 7). (B) The redox-active groups (except Cu_A) and the D proton-transfer pathway in the R. sphaeroides CcO. The red spheres are water molecules in the D pathway. The location of residues discussed in this work is indicated. The D pathway is used for the transfer of both substrate and pumped protons (see arrows). At E(I-286), there is a possible branching point from which substrate protons are transferred to the binuclear center and pumped protons are transferred to an acceptor (dashed arrow) in contact with the proton output side. In the wild-type and ND(I-139) mutant CcO, the apparent pK_a values of E(I-286) are 9.4 and ≈11, respectively. The pK_a of the acceptor at the binuclear center is suggested to be ≥12 (see text), whereas that at the hemes a and a₃ propionate–Arg(I-481/482) cluster (acceptor A) is only slightly higher than that of E(I-286) in the wild-type CcO. In the ND(I-139) mutant CcO proton transfer from E(I-286) to A is impaired due to the increased pK_a of E(I-286). (C) A schematic representation of the reaction of CcO having a reduced binuclear center (state R) with dioxygen. Reduction of the oxidized (state O) binuclear center (with two electrons) is associated with net proton uptake from the bulk solution. Oxygen binds to the reduced heme a₃ (state A) after which the O[ONK]O bond is broken, forming state P_M. The transfer of the third and fourth electron (from an external electron donor) to the binuclear center are each associated with the uptake of one proton from solution through the D pathway, forming states F and O, respectively. If the CcO is initially fully reduced (blue pathway), after binding of O₂ an electron is transferred rapidly from heme a to the binuclear center with a time constant of 50 μs, followed by proton uptake from solution, forming state F with a time constant of ≈100 μs. The fourth electron is transferred from Cu_A through heme a. The images in A and B were prepared by using VISUAL MOLECULAR DYNAMIC software (8).

Fig. 2.

Proton pumping by CcO reconstituted in phospholipid vesicles in H₂O and D₂O as indicated. With valinomycin (K⁺ ionophore), only the net proton release to the outside of the vesicles is monitored. With both valinomycin and CCCP (H⁺ ionophore) the net proton consumption by CcO is monitored (an average of one H⁺ per electron). The slow drift is due to proton leakage across the vesicles. The experimental conditions are described in Materials and Methods.

Fig. 3.

(A) Absorbance changes at 580 nm associated with the P_R → F (increase in absorbance, τ ≅ 100 μs at pH 7.5) and F → O (decrease in absorbance, τ ≅ 1 ms at pH 7.5) transitions in the wild-type (WT, two lower traces) and ND(I-139) mutant (two upper traces) CcOs at pH 7.5 and 10, as indicated in the graph. The traces measured with the wild-type CcO have been moved down for clarity (compare absorbance levels at t < 0). Conditions: 2.5 μMCcO solubilized in 0.1% dodecyl-β-d-maltoside, 100 mM KCl, 5 mM sodium ascorbate, 1 μM PMS, 1 mM CO. The buffers Hepes and CAPS at 100 mM were used at pH 7.5 and 10, respectively. (B) The rate of the P_R → F transition (see A) in the wild-type (squares) and ND(I-139) (circles) CcOs as a function of pH. The solid lines are fits of the data with standard titration curves with pK_a values of 9.4 and 11, respectively.