Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.

Protonation changes accompanying conversion of oxidised (O state) cytochrome c oxidase to the 2-electron-reduced P state, and 3-electron-reduced F state at pH 8.0 have been measured. It was found that 2 and 3 protons, respectively, were taken up. The fourth proton required for the reduction of O2 to H2O must therefore be consumed in the remaining F----O portion of the catalytic cycle.

The spectral characteristics of the ‘655 nm’ band of cytochrome oxidase were found to be affected by ligands of the binuclear centre, including formate and chloride, and by the resting/pulsed transition. The band titrated with near n =1 characteristics at a midpoint of about 400 mV, in contrast to haem a 3 , which exhibits strong redox interaction and a titration range at significantly lower potential. Thus, although the total reduced‐oxidised difference spectrum of haem a 3 , shows a trough at about 655 nm, this characteristic is absent in the low potential region. The 655 nm feature may arise from a charge transfer band of ferric high‐spin haem a 3 , which is modulated by the redox state of Cu B , as suggested by Beinert et al. [(1976) Biochim. Biophys. Acta 423, 339–355].

The main aim of this brief contribution is to suggest that our understanding of the general principles of osmochemistry may provide useful insights into the type of mechanism by which solute-translocating catalysts work. In particular, I would like to encourage a more widespread and explicit recognition of the special merits of the mobile barrier type of mechanism (Mitchell, 1957, 1987), not as a panacea, but to explain the translocation of the characteristically hydrophilic and somewhat bulky solutes that are the main substrates of solute porters and of some osmoenzymes in bacterial membranes.

Conference Article| October 01 1989 The redox midpoint potential of haem a in cytochrome-c oxidase is sensitive to extramitochondrial pH J. ROY MITCHELL; J. ROY MITCHELL 1Glynn Research Institute, Bodmin, Cornwall PL30 4AU, U.K. Search for other works by this author on: This Site PubMed Google Scholar PETER MITCHELL PETER MITCHELL 1Glynn Research Institute, Bodmin, Cornwall PL30 4AU, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1989) 17 (5): 892–893. https://doi.org/10.1042/bst0170892 Article history Received: March 28 1989 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation J. ROY MITCHELL, PETER MITCHELL; The redox midpoint potential of haem a in cytochrome-c oxidase is sensitive to extramitochondrial pH. Biochem Soc Trans 1 October 1989; 17 (5): 892–893. doi: https://doi.org/10.1042/bst0170892 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1989 Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.

Imposition of a protonmotive force across the inner membrane of coupled cyanide-inhibited, beef heart mitochondria by addition of ATP causes reduction of cytochrome c and CuA with concomitant oxidation of haem aA. The data are consistent with previous demonstrations of an intramembrane location of haem aA but further indicate that CuA is very close to the cytosolic surface of the membrane. The implications of this finding for electron transfer route and the site of the proton pumping chemistry are discussed.

In recent papers on protonmotive redox mechanisms in cytochrome oxidase in [(1987) FEBS Lett. 222, 235-245] and [Glynn Biological Research Reports (1987) 3, 1-7], I have suggested that a copper centre may enable the H2O/OH or H2O/O couple to act as the hydrogen-carrying arm of a redox loop by means of a (CuOH2)+/(CuOH)+ or (CuOH2)+/(CuO)+ system at the centre. I here explain that critical comments by Malmström [(1988) FEBS Lett. 231, 268-269] on the first of these papers, which might also be levelled at the second, depend on a misunderstanding. I also respond to Malmström's comment about testing conformationally coupled proton-pump mechanisms.

The stoichoimetry of vectorial H + ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the ← H o + 2 e − ratio was found to be constant during the time-course of oxygen reduction, and thus independent of ΔpH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.

A new hypothetical type of redox loop is described, which translocates hydroxide ions instead of protons. Conventional protonmotive redox loops use carriers of protons with electrons (e.g. QH2/Q systems) to couple electron transfer to the translocation of protons. The putative hydroxidemotive redox loop uses carriers of hydroxide ions against electrons (e.g. transition‐metal centres) to couple electron transfer to the translocation of hydroxide ions. This simple idea leads to the proposal of a hydroxidemotive Cu loop mechanism that may possibly be applicable to the CuA or CuB centre of cytochrome oxidase, and might thus account for the coupling of electron transfer to net proton translocation in that osmoenzyme.

The observation in this laboratory that respiration and Sr2+ import were stimulated by the addition of 3-hydroxybutyrate to suspensions of N-ethylmaleimide-treated mitochondria respiring in state 6, after the addition of Sr2+, in a sucrose medium containing choline as substrate, led to the proposal by Moyle and Mitchell [(1977) FEBS Lett. 84, 135-140] that there is a Ca2+(Sr2+)-3-hydroxybutyrate symporter in rat liver mitochondria. However, experiments described in the present paper support a different interpretation. Under the conditions of the experiments by Moyle and Mitchell, the rate of respiration and the poise of Sr2+ accumulation are mainly limited, not by delta mu H+, but by lack of respiratory substrate. Even though N-ethylmaleimide is a potent inhibitor of 3-hydroxybutyrate dehydrogenase, we have found that, somewhat surprisingly, under the special conditions of these experiments, sufficient 3-hydroxybutyrate dehydrogenase activity remains available to account for the 3-hydroxybutyrate-dependent respiratory stimulation and Sr2+ import.

When O2 was injected into an anaerobic suspension of valinomycin-treated rat liver mitochondria inhibited with rotenone, antimycin, and myxothiazol, a small amount of O2 (0.23-0.33 ng-atom of O/mg of protein) was reduced extremely rapidly (within the 2 s time-resolution of the oxygen electrode). The subsequent steady-state rate of flow of electrons to oxygen was very low [less than 3 nequiv. X s-1 X (g of mitochondrial protein)-1]. In the presence of valinomycin there was a rapid ejection of protons synchronous with the rapid phase of O2 consumption corresponding to 0.38-0.61 nequiv. of H+ X (mg of mitochondrial protein)-1. When valinomycin was replaced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) there was a rapid alkalification of the medium corresponding to 0.20-0.42 nequiv. of H+ X (mg of mitochondrial protein)-1. When 2 mM-Fe(CN)6(4-) was present to re-reduce endogenous cytochrome c, O2 consumption was still biphasic but the second phase of O2 consumption was very much more rapid [600 nequiv. X s-1 X (g of protein)-1], and resulted in the virtually complete consumption of the O2 in the pulse within 4 s. With 60 microM-Ru(NH3)6(2+) as reductant, O2 consumption was even faster [1200 nequiv. X s-1 X (g of protein)-1]. In a medium containing 150 mM-choline chloride with Ru(NH3)6(2+) as reductant, the proton per reducing equivalent stoichiometry (delta H+O/e-) was +0.95 in the presence of valinomycin and -0.94 in the presence of FCCP. In choline chloride medium containing Ru(NH3)6(2+) and valinomycin, there was an uptake of K+ ions corresponding to 1.86 K+/e-. It is concluded that nearly 1 proton is translocated outwards through cytochrome oxidase per oxidizing equivalent injected in this medium. In low ionic strength sucrose-based medium, with Ru(NH3)6(2+) as reductant, delta H+O/e- was 1.05 in the presence of valinomycin, and -0.71 in the presence of FCCP. It is concluded that the translocation of protons is accompanied by net acid production in this medium.

Using the principle of specific vectorial ligand conduction, we outline directly coupled protonmotive O loop and O cycle mechanisms of cytochrome oxidase action that are analogous to protonmotive Q loop and Q cycle mechanisms of QH2 dehydrogenase action. We discuss these directly coupled mechanisms in the light of available experimental knowledge, and suggest that they may stimulate useful new research initiatives designed to elucidate the osmochemistry of protonmotive oxygen reduction in cytochrome oxidase.

The reversible protonmotive F0F1 ATPases perform the uniquely important function of balancing the forces, and interconverting the potential energies, of phosphoryl transfer and proton translocation. The molecular mechanics of the processes of ligand conduction catalysed by the F0F1 ATPases is therefore especially interesting. This paper summarises the main structural and functional knowledge of the F0F1 ATPases in the light of current mechanistic hypotheses, and suggests a new type of rotating subunit hypothesis, which is related to that recently developed for bacterial flagellar motors.

Journal Article The Correlation of Chemical and Osmotic Forces in Biochemistry Get access Peter MITCHELL Peter MITCHELL Glynn Research InstituteBodmin, Cornwall PL30 4AU, England Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 97, Issue 1, January 1985, Pages 1–18, https://doi.org/10.1093/oxfordjournals.jbchem.a135033 Published: 01 January 1985

Thiol modulation of the chloroplast protonmotive ATPase (CF0-CF1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall P2e ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in P2e ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in P2e ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and P2e ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF0-CF1 increased the observed Michaelis constant for ADP (Km(ADP)) and the apparent maximum rate (Vapp of photophosphorylation by the same factor, so that ratio VappKm was not altered. VappKm was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF0-CF1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF0-CF1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.

Bacterial ion‐driven flagellar motors are the smallest known rotatory mechanical devices, natural or artificial, their overall diameter being only about 25 nm or one millionth of an inch. They are unique in the fields of biology and engineering. This paper develops a possible osmoelectric or local electrokinetic mechanism of molecular rotatory motion in bilayer membranes, which may help to explain how bacterial flagellar motors work, and may incidentally encourage new developments in the bioenergetics and biomechanics of enzyme, osmoenzyme and porter action.

The electrochemical aspects of oxygen evolution in photosynthesis are considered. A four-electron mechanism is proposed for the photooxidation of water in the reaction center of photosystem II of chloroplasts. It is supposed that water can be oxidized by a reaction center consisting of four chloropyll molecules, a Mn cluster, two pheophytin molecules and several plastoquinone molecules. The catalytic complex responsible for the oxidation of water is assumed to include four manganese ions. Two cations are necessary for the catalytic formation of the hydrated chlorophyll dimer; these can be substituted by other cations of multivalent metals. The partition of protons between the inner and outer side of thylakoid membranes can be controlled by the pool of plastoquinones, lipids, polar groups of proteins and ATP-synthetase. The probability of the four-electron is analyzed thermodynamically and kinetically. The role of manganese in photosynthesis is discussed.