Structure and function of the S. pombe III-IV-cyt c supercomplex.

The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII2), via a mobile electron carrier, cytochrome c. In the present study, we isolated the CIII2CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn2+, coordinated in the CIII subunit Cor1, which is encoded by the same gene (qcr1) as the mitochondrial-processing peptidase subunit ß. Our data show that the isolated CIII2CIV supercomplex displays proteolytic activity suggesting a dual role of CIII2 in S. pombe. As in the supercomplex from S. cerevisiae, subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII2 respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII2-CIV supercomplex.

Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers.

Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was ∼100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of ∼60 nm(2). Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm(2), but not when below 30 nm(2). We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is ∼100 times slower than that observed over submillimeter distances by proton-pulse experiments (Ds ∼ 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

Intermediates generated during the reaction of reduced Rhodobacter sphaeroides cytochrome c oxidase with dioxygen.

Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O2 and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.

The membrane modulates internal proton transfer in cytochrome c oxidase.

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.

Functional interactions between membrane-bound transporters and membranes.

One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated protein-protein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the two-dimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.