Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

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.     

Cytochrome c reactivity in its complexes with mammalian cytochrome c oxidase and yeast peroxidase

1.

1. The ascorbate reducibility of cytochrome c (beef or horse heart) in its complexes with cytochrome c oxidase (beef heart) and cytochrome c peroxidase (yeast) has been studied.     

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase to inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II–IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site in subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Structure of the cytochrome c oxidase dimer electron microscopy of two-dimensional crystals

We have studied the structure of beef heart mitochondrial cytochrome c oxidase dimers by image-processing of electron micrographs of the vesicle crystal form. Specimens were prepared by different procedures, which contrast different features of the crystals. Heavy-atom shadowing of freeze-dried crystals contrasts the exterior or M-side surface (mitochondrial matrix-side) and reveals a 100 Å long ellipsoidal dimer oriented with its long axis in the (?1, 1) direction of the 95 Å × 125 Å rectangular unit cell. The M-side surface structure correlates well with the intra-bilayer structure revealed by contrast matching extra-bilayer protein with glucose. Frozen suspensions of vesicle crystals fracture predominantly along hydrophilic surfaces revealing the interior C-side (mitochondrial cytoplasm-facing surface) of vesicle crystals. The C-side surface revealed in shadowed replicas of fracture surfaces shows the ends of the dimers furthest from the bilayer surface; they consist of two structural domains separated by 70 to 80 Å. We present a new interpretation of the structure of the cytochrome oxidase dimer based on these data and on the y-shaped monomer structure described by Fuller et al. (1979). A cytochrome oxidase dimer is formed from two y-shaped monomers joined along one set of identical M-domain arms with the other arms approximately 70 Å apart along a unit cell diagonal in the (?1, 1) direction. The arms of the monomers lie within and perpendicular to the phospholipid bilayer, and they protrude approximately 25 Å beyond the bilayer surface on the M-side. The y tails represent the C-side domains, which are closely apposed across the dimer 2-fold axis near the C-side bilayer surface. Further away from the bilayer surface, C-side domains split away from one another forming a large cleft.     

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N′,N′-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827–5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(l-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.     

Structural studies on the cytochrome c oxidase proton pump using a spin-label probe

We report studies in which we have used N-(2,2,6,6-tetramethylpiperidyl-1-oxyl)-N′-cyclohexylcarbodiimide, a spinlabel analogue of N,N′-dicyclohexylcarbodiimide, to investigate the structural aspects of the cytochrome c oxidase proton pump. We establish that the spin label binds to the reconstituted enzyme at the same site as does N,N′-dicyclohexylcarbodiimide, i.e., within subunit III. ESR studies of the bound spin label indicate that its binding site is situated in an apolar region of the enzyme, though close to its surface. The binding of the spin label to the free oxidase is different from that with the reconsituted enzyme, leading to spin-spin exchange between the bound probe molecules. From this and the fact that N,N′-dicyclohexylcarbodiimide binds to subunits III and IV in the free oxidase, we conclude that these two subunits are at the most 20 Å apart.     

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.     

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (?Ψ_m) in living cells and tissues at low values (100-140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ?Ψ_m values (180-220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ?Ψ_m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ?Ψ_m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ?Ψ_m was found. From the data we conclude that high matrix ATP/ADP ratios keep ?Ψ_m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ?Ψ_m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ?Ψ_m according to the Mitchell Theory.     

Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.     

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues

We re-determined the near infrared (NIR) spectral signatures (650–980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric Cu_A, which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a₃/Cu_B binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting ‘perturbed’ binuclear centre is also characterised by a previously unreported broad 715–920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the Cu_A centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a₃/Cu_B binuclear centre.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P_M, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P_M is produced at high pH with CO/O₂, no EPR-detectable radicals are formed. Conversion of the CO/O₂-generated P_M into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in P_M which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.     

Studies on the mechanism of electron transport in the bc1-segment of the respiratory chain in yeast. II. The binding of antimycin to mitochondrial particles and the function of two different binding sites

1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>1</mtext>}}\text{≦ 4 · 10}{}^{\mathrm{?13}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext><mtext>2</mtext>}}\text{= 3 · 10}{}^{\mathrm{?9}}\text{M}$, respectively.2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 – 150 pmol per mg of mitochondrial protein.3. Antimycin bound to the stronger site mainly inhibits NADH- and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN.4. Under certain conditions cytochrome b and c₁ are dispensible components for antimycin-sensitive electron transport.5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G. and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71–85).