Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler.

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa3 in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230). 2. The Km for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 micrometer in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation. 3. The apparent Km for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts "competitively" towards oxygen. 4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H+ gradients modify the oxidase activity in reconstituted vesicles.     

Orientation and reactivity of cytochrome aa3 heme groups in proteoliposomes

Reduction of cytochrome aa₃ in proteoliposomes with ascorbate plus cytochrome c confirms that not more than 55% of the molecules are externally accessible and that the remainder are reduced only on the addition of membrane-permeable N,N,N′,N′tetramethyl-p-henylenediamine. Reduction in the presence of terminal inhibitors such as cyanide, azide, and carbon monoxide shows that likewise 50% of the cytochrome a is accessible and 50% inaccessible. Dithionite reduces part of the cytochrome a₃ in the presence of azide, and none in the presence of cyanide. Methyl viologen, which is somewhat membrane permeable, can reduce part of the cyanide-complexed cytochrome a₃ at low concentrations and all of it at high concentrations. Cytochrome a₃ is therefore also distributed randomly inside and outside the vesicles. Cytochrome c oxidase with externally facing cytochrome a is stimulated to high activity by its membrane association. Its turnover is dependent on the external pH and it is inhibited by external azide; trapping of azide cannot be used to demonstrate the orientation of the cytochrome a₃ hemes associated with externally facing cytochrome a. Cytochrome c oxidase with internally facing cytochrome a is rather sluggishly reactive. Its low activity accounts for the apparent failure of detergents to release extra activity on lysing proteoliposomes. Double reciprocal plots of the reaction of added cytochrome c with proteoliposomes indicate apparent biphasic binding in the energized state, which is abolished upon the addition of uncouplers and valinomycin. But no transmembraneous effect upon the oxidase reaction other than energization has been identified.     

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. I. The reaction with carbon monoxide

The kinetics of CO binding by the cytochrome c oxidase of pigeon heart mitochondria were studied as a function of membrane energization at temperatures from 180 to 280°K in an ethylene glycol/water medium. Samples energized by ATP showed acceleration of CO binding compared to those untreated or uncoupled by carbonylcyanide p-trifluoromethoxyphenylhydrazone but only at relatively low temperatures and high CO concentrations. Experiments using samples in a “mixed valency” (partially oxidized) state showed that the acceleration of ligand binding is not due to the formation of a partially oxidized state via reverse electron transport.It is concluded that in the deenergized state one CO molecule can be closely associated with the cytochrome a₃ heme site without actually being bound to the heme iron; in the energized state, two or more ligand molecules can occupy this intermediate position.The change in the apparent ligand capacity of a region near the heme iron in response to energization is evidence for an interaction between cytochrome oxidase and the ATPase system. Furthermore, these results suggest a control mechanism for O₂ binding.     

Energized transport of potassium ions in the absence of valinomycin by cytochrome c oxidase-reconstituted vesicles

Valinomycin-independent energized uptake of K⁺ was observed in cytochrome c oxidase reconstituted proteoliposome. The rate of K⁺ influx was proportoinal to the magnitude of electron flux. The energized uptake of K⁺ was abolished by p-trifluoromethoxycarbonylcyanide phenylhydrazone or by nigericin. Using the safranine fluorescence technique, it was demonstrated that even in the absence of valinomycin, liposomes and proteoliposomes reconstituted with cytochrome c oxidase are able to discriminate between Na⁺ and K⁺ and show a preference for K⁺ in the presence of excess Na⁺.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

1. Beef heart mitochondria have a cytochrome c₁ : c : aa₃ ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s^?1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s^?1, at 28° – 30°C, pH 7.4.2. Ascorbate plus N,N,N′,N′-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c₁ and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c₁ is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+N,N,N′,N′-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c₁ and c in the aerobic steady state, with a rate constant for the c₁ → c reduction step greater than 10³ s^?1.4. The greater apparent response of the $\text{c}\text{aa}{}_3$ electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the $\text{c}{}_1\text{c}$ step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa₃ units in situ. Cytochrome c₁ can either reduce the cytochrome c-cytochrome aa₃ complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Studies on the red oxidase (cytochrome o) of Azotobacter vinelandii

In an attempt to isolate and to study the electron transport system of $\text{Azotobacter vinelandii}$, we have isolated and purified a membrane-bound cytochrome $\text{o}$. The cytochrome $\text{o}$, purified as a detergent (Triton X-100) and hemoprotein complex, contained 1.6 nmoles heme per mg of protein. Cold-temperature spectrum showed that no other cytochrome was associated with the purified preparation, and electrophoresis revealed that only one type of hemoprotein was obtained. The purified cytochrome $\text{o}$ reacted with both carbon monoxide and cyanide readily. Only in the reduced form did it combine with carbon monoxide, whereas the oxidized form reacted with cyanide. An “oxygenated” form of the cytochrome $\text{o}$ was demonstrated to be spectrally distinguishable from both the oxidized and the reduced forms.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Active K+ transport in Mycoplasma mycoides var. Capri. Relationships between K+ distribution,electrical potential and ATPase activity

The addition of 5 · 10^?5 M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K⁺ influx rather than efflux and reduces by 30–40% the activity of the membrane-bound Mg²⁺-ATPase. Adding valinomycin to metabolizing cells does not markedly affect K⁺ distribution but induces a rapid and complete loss of intracellular K⁺ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$, dissipate the K⁺ concentration gradient only when combined with valinomycin.Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (Δψ) is generated on cell energization. This Δψ, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms.These experiments suggest that the membrane-bound Mg-ATPase activity controls K⁺ distribution in these organisms through the generation of a transmembrane electrical potential difference.     

A periplasmic location for methanol dehydrogenase from Paracoccus denitrificans: implications for proton pumping by cytochrome aa3

Evidence is presented that methanol dehydrogenase from $\text{Paracoccus}\text{denitrificans}$ has a periplasmic location. The implications for the mechanism of proton translocation during electron flow from methanol to oxygen via cytochromes c and aa₃, or to nitrite via cytochrome c and nitrite reductase, are discussed.     

Comparison of cerebral NADH and cytochrome aa3 redox shifts during anoxia or hemorrhagic hypotension.

The reduction-oxidation reactions of NADH and cytochrome $\text{aa}{}_3$ to incipient oxygen insufficiency caused by nitrogen ventilation or hemorrhagic hypotension were examined in the exposed cerebral cortex of the cat. A comparison of the onset of redox changes with each procedure shows that cytochrome $\text{aa}{}_3$ reduction precedes the reduction of mitochondrial NAD. This constitutes evidence that, in the living brain, NADH maintains its resting oxidation state at lower cellular oxygen tensions than cytochrome $\text{aa}{}_3$ does, consistent with the differences in oxygen affinity these respiratory chain components exhibit during oxygen titration $\text{in vitro}$.     

Changes in accessibility of the membrane bound transport enzyme glucose phosphotransferase of E. coli to protein group reagents in presence of substrate or absence of energy source

The inactivation of α-methyl-D-glucoside transport in $\text{E. coli}$ by N-ethyl-maleimide and 1-fluoro-2,4-dinitrobenzene is strongly enhanced by the presence of substrate or of an inhibitor of phosphoenol pyruvate synthesis. It is demonstrated in the case of N-ethylmaleimide that the target of the inhibition is the membrane bound component of the phosphoenol pyruvate glucose phospho-transferase system: enzyme II. Enzyme II could exist, in course of the transport, in two alternate conformational states: an energized and a deenergized state, the energized one being protected against inactivation by N-ethylmaleimide.     

The invisible copper of cytochrome c oxidase. pH and ATP dependence of its midpoint potential and its role in the oxygen reaction.

Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a₃ and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a₃ or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a₃ or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations.A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing $\text{sol}\text{[ATP]}\text{[ADP]}\text{× [}\text{P}{}_1\text{]}$ ratio.By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a₃²⁺-O₂-Cu¹⁺ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.     

Energy-dependence of nitrate reductase induction in Candidautilis

The requirement of a suitable energy source during the induced synthesis of nitrate reductase in $\text{Candida}\text{utilis}$ was investigated. The levels of nitrate reductase induced were shown to be energy-dependent, and to vary in response to the type of carbon source provided. Glycerol, fructose, ethanol, glucose, and sucrose served as efficient energy sources. Growth rate of the yeast and the induced level of nitrate reductase were dependent on the ratio of carbon to nitrogen in the induction medium, and ratio of 2 being optimal. Induction of nitrate reductase was inhibited by uncouplers, 2,4-dinitrophenol (DNP), dicumarol and carbonyl cyanide $\text{p}$-trifluoromethoxy phenyl hydrazone (CCCP), and by cyanide and azide, indicating an absolute energy-dependency. The facilitation of induction of a high level of nitrate reductase by exogenously added ATP as sole source of energy confirmed the obligate requirement of ATP for the synthesis of nitrate reductase in $\text{Candida}$$\text{utilis}$.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

d-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute

d-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5 μM}$). Uptake of d-gluconate was not mediated by a phosphoenolpyruvate: hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, ΔpH, of ?63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, $\text{ΔpH = 0}$. Addition of 8 μM d-gluconate significantly reduced the ΔpH. The transmembrane electrical potential, Δψ, which was ?87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. d-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the Δψ did not energize transport. Rather, the rate of d-gluconate uptake correlated with and appeared to be determined by the rate of d-gluconate metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both d-gluconate uptake and the metabolism of pre-accumulated d-gluconate; (b) the effects of cyanide and azide on d-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on d-gluconate metabolism; (c) extraction and chromatography of intracellular label from d-gluconate uptake revealed that accumulation of unaltered d-gluconate was negligible; (d) a series of mutant strains with lower d-gluconate kinase activities also exhibited low rates of d-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both d-gluconate kinase activity and wild type levels of uptake.