Mårten Wikström

Safranine as a probe of the mitochondrial membrane potential

One of the most central postulates of the chemiosmotic hypothesis of oxidative phosphorylation is the existence of a large electrochemical proton gradient (protonmotive force) across the mitochondrial membrane [ 1,2] . This gradient, which is composed of a membrane potential and a pH differential, is postulated to be the obligatory link between respiration and phosphorylation in the process of oxidative phosphorylation. The membrane potential has been suggested to be the major component of the electrochemical proton gradient under most conditions, and measurements of this potential are therefore essential for the understanding of the mechanism of oxidative phosphorylation and mitochondrial ion transport. The mitochondrial membrane potential has mainly been estimated from the distribution of K’ or Rb’ across the mitochondrial membrane in the presence of the ionophore valinomycin [3-g]. This method has given fairly consistent results and membrane potentials in the range 130190 mV (positive polarity extramitochondrially) have generally been reported in coupled mitochondria supplemented with either substrate or ATP. The differences in the values reported by different workers may for the most part be attributed to the use of different values for the matrix volume. The use of a method of this kind, which requires partially artificial experimental conditions, makes confirmation by other independent methods most desirable. This has prompted several workers to search for suitable spectroscopic membrane potential probes. The fluorescent probe MC V has been used by Chance and collaborators [9,10] as a membrane potential indicator in mitochondria, and Laris et al. [ 111 have employed a cyanine dye

Proton pump coupled to cytochrome c oxidase in mitochondria

CYTOCHROME c oxidase is an important enzyme of aerobic metabolism, catalysing electron transport between cytochrome c and molecular oxygen. Apart from this essential redox reaction the enzyme must be involved in conservation of the released redox energy for utilisation in ATP synthesis at the socalled third site of oxidative phosphorylation. I report here that the redox activity of mitochondrial cytochrome c oxidase is coupled to the translocation of hydrogen ions across the inner mitochondrial membrane. This finding may be a clue to the role of cytochrome oxidase in energy conservation.

A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.

Proton-Coupled Electron Transfer in Cytochrome Oxidase

3.3. Electron Tunneling between the Heme Groups 7068 4. Catalytic Cycle and States of the Binuclear Center 7069 4.1. O2 Binding and Bond Splitting 7069 4.2. Intermediate States of the Binuclear Center 7071 4.2.1. States PM, PR, and F 7071 4.2.2. Metastable States of the Binuclear Center 7072 4.2.3. Bridging Ligand in the Binuclear Site 7072 5. Proton Pumping 7073 5.1. Proton Transfer Pathways 7073 5.2. PCET and Proton Pumping 7073 5.3. Proton Pumping via the PR State 7076 5.4. Is the Proton-Loading Site “Loaded” in the Fully Reduced Enzyme? 7076

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site.

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

Oxidoreduction of cytochrome b in the presence of antimycin

Abstract 1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators. 2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site. 3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558. 4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant. 5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14. 6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c1 (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH2/QH• couple and reduction of cytochrome b.

Water-gated mechanism of proton translocation by cytochrome c oxidase.

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O(2) reduction at the haem a(3)-Cu(B) centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 ). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 ). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Delta-propionate of haem a(3) (pumping), or to haem a(3)-Cu(B) (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a(3)-Cu(B), which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O(2) reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, CuA and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.

Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone

The spectrophotometric indicators neutral red and safranine were used to determine the relative H+/e− ratios of proton uptake from the mitochondrial matrix, and the q+/e− ratios of electrical charge translocation, during oxidation of β‐OH‐butyrate and succinate by ferricyanide in rat liver mitochondria. With β‐OH‐butyrate both ratios were higher than with succinate by a factor close to 3.0. Since there is full agreement that H+/e− of proton uptake and q+/e− of charge translocation are both equal to unity for oxidation of succinate (or ubiquinol) by ferricytochrome c, the corresponding ratios for oxidation of NADH by ubiquinone and cytochrome c are near 2.0 and 3.0, respectively.

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild‐type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc 1 complex by myxothiazol. Coupled to this activity, which involves only b‐type cytochromes, there is translocation of 1.5–1.9 H+/e− across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa 3 to which it is structurally related.