Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.     

Intramitochondrial positions of cytochrome haem groups determined by dipolar interactions with paramagnetic cations.

E.p.r.(electron-paramagnetic-resonance) spectra of the ferricytochromes were studied in normal and 'nickel-plated' pigeon heart mitochondria and pigeon heart submitochondrial particles. NiCL2 added to either mitochondria or particles was bound completely to the membranes, but none was transported across the vesicles. Hence, any perturbations of the haem e.p.r. spectra by Ni(II) should occur only for those cytochromes in close proximity to the exterior surface. Whenever Ni(II) can approach to within 1 nm of cytochrome haem. the consequent acceleration of the haem e.p.r. relaxation kinetics should elicit dipolar line broadening. Relaxation acceleration should also increase the incident power level required to saturate the haem e.p.r. signal. In pigeon heart mitochondria, at least three e.p.r. resonances, attributable in part to cytochromes c1, bK and br, are observed at gz=3.3 resonance. In these submitochondrial particles, the peak at gz=3.5 is missing, and the resonance at gz=3.6 resolves into two components, neither of which is sensitive to added Ni(ii). Addition of free haemin (ferric, a paramagnetic anion) to intact mitochondria elicits the same e.p.r. signal changes as does a preparation of submitochondrial particles. Saturation curves for cytochrome oxidase obtained for e.p.r. spectra of the high-spin form (g = 6) and the low-spin form (gz=3.1) also reveal no effect of Ni(II) on the haem e.p.r. relaxation in either mitochondria or inverted submitochondrial particles. Further, Ni(II) fails to alter the spectra or saturation properties of cytochrome c in either mitochondria or submitochondrial particles therefrom. Only with a 50-fold molar excess of Ni(II) can one accelerate the e.p.r. relaxation of cytochrome c in aqueous solution, although other more subtle types of magnetic interactions may occur between the cytochrome and either Ni(II) or ferricyanide. Addition of haemin to mitochondria likewise failed to alter the e.p.r. characteristics of either cytochrome c or cytochrome oxidase. The present observations strongly suggest that cytochromes bK, br and c1 reside on the exterior surface of the inner mitochondrial membrane. On the other hand, we find no positive evidence for the location of cytochrome c or cytochrome oxidase haem groups within 1 nm of either membrane surface. Because of possible shielding effects from the protein moieties, however, we cannot unequivocally assign the location of the haem groups to the membrane interior. The present results are not inconsistent with the observations of other investigators who used different techniques. However, it is clear that any model of energy coupling in mitochondrial oxidative phosphorylation must account for the positioning of all the b-c cytochrome haem groups on the outside.     

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.     

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.     

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.     

Effect of adenosine 5'-triphosphate and adenosine 5'-diphosphate on the oxidation of cytochrome c by cytochrome c oxidase

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and inorganic pyrophosphate partially inhibit the oxidation of exogenous cytochrome c by cytochrome c oxidase of submitochondrial particles (with or without detergent treatment) or by a purified preparation when it is assayed polarographically in buffers of nonbinding ions at pH 7.8. ATP is somewhat more inhibitory than ADP. The inhibition is never greater than 50%, and it is always less than an equal concentration of Mg2+ ions is present or when the assays are run at pH 6. In contrast, the effect of ATP, ADP, and pyrophosphate on oxidase assays run spectrophotometrically is a similar slight stimulation of the oxidase of submitochondrial particles treated with deoxycholate and little or no effect on purified oxidase. The reaction of the oxidase of submitochondrial particles with the endogenous cytochrome c is stimulated by the nucleotides, as is the reduced nicotinamide adenine dinucleotide (NADH) oxidase activity. The observations can be explained by binding of ATP, ADP, or pyrophosphate to cytochrome c so that the formation of an especially reactive combination of cytochrome c and cytochrome oxidase previously postulated [Smith, L., Davies, H. C., & Nava, M. E. (1979) Biochemistry 18, 3140] is prevented. The data give no evidence that respiration via cytochrome c oxidase is regulated physiologically by direct effects of ATP or ADP on its activity.     

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 ahve 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 degrees-30 degrees 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 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 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 c1/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.     

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.     

Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.

The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.     

Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation

A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles.     

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > MP > IMP. The apparent K(m) values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 degrees C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.     

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.     

Orientation of cytochrome c oxidase molecules in the two populations of reconstituted vesicles resolved by column chromatography on DEAE-Sephacryl

Vesicles reconstituted with bovine heart cytochrome c oxidase and dioleoylphosphatidylcholine can be resolved into two populations by column chromatography in DEAE-Sephacryl (Madden, T.D. and Cullis, P.R. (1984) J. Biol. Chem. 259, 7655-7658). These two fractions (I and II) were treated with two proteases. These are trypsin, which has been found to cleave subunit IV in the M domain of the cytochrome c oxidase molecule, and chymotrypsin, which has been found to cleave subunit III in the C domain. These studies show that fraction I vesicles contain cytochrome c oxidase orientation with the M domain outside, i.e., in the same topology as in submitochondrial particles, while fraction II vesicles contain enzyme molecules with their C domain outside, and thus in the same orientation as in mitochondria.     

A peptide-sensitive channel of large conductance is localized on mitochondrial outer membrane.

Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.     

Mitochondrial localization of cytochrome c1 with specific antibodies]

A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Reaction of oxidized cytochrome oxidase with cyanide. Effects of pH, cytochrome c and membrane environment

Binding of HCN with ferric beef heart cytochrome oxidase has been studied in submitochondrial particles, as with the enzyme solubilized in detergent or reconstituted into proteoliposomes. Under all conditions, the reaction proceeds via an intermediate and its kinetics can be described by formal parameters Km and kmax in keeping with the Michaelis-type equation. Km of the reaction strongly depends on the enzyme environment; thus it increases 100-1000 fold upon solubilization of cytochrome oxidase but can be subsequently decreased by incorporation of the enzyme in liposomes and by addition of cytochrome c. pH-dependence of the reaction rate shows that, in submitochondrial particles and proteoliposomes as well as in the case of solubilized enzyme supplement with cytochrome c, HCN specifically binds the form of cytochrome oxidase in which a heme-linked ionizable group with pKa 6,5-6,9 is protonated.     

Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis.

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef-heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial-mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.     

Immunochemical evidence for an inactive form of cytochrome oxidase in mitochondrial membranes of ethanol-fed rats

Previous studies have established that rats fed ethanol chronically exhibit a 50% decrease in hepatic mitochondrial cytochrome oxidase compared to pair-fed controls, based on both heme aa3 content and specific activity. To determine whether the 'missing' 50% of cytochrome oxidase is present in the membrane but catalytically inactive, or entirely absent, we used immunochemical techniques to determine the content of cytochrome oxidase protein in hepatic submitochondrial particles. Rabbit antiserum against purified rat liver cytochrome oxidase precipitated cytochrome oxidase from detergent-solubilized submitochondrial particles. Immunoinhibition titrations of a fixed amount of anti-oxidase serum with increasing amounts of submitochondrial particle protein showed that similar percentages of added oxidase activity were recovered in supernatants after immunoprecipitation with preparations from both alcoholic and control rats. Similarly, titrations of a fixed amount of submitochondrial particle protein with increasing amounts of antiserum showed comparable decreases in oxidase activity. Equivalent amounts of protein were obtained in immunoprecipitates from both preparations. Immunoprecipitates demonstrated comparable oxidase subunit profiles by electrophoresis, except that one additional band, migrating in the region of oxidase subunit IV, was present in samples from alcoholic rats. The data indicate that cytochrome oxidase immunologic reactivity is quantitatively similar in both types of membranes. The results suggest that the 'missing' cytochrome oxidase is actually present within the membranes of alcoholic animals in an inactive form, apparently devoid of heme aa3.