Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates

Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Br?nsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.     

Structure and function of cytochrome-c oxidase

Recent works on the structure and the function of cytochrome-c oxidase are reviewed. The subunit composition of the mitochondrial enzyme depends on the species and is comprised of between 5 and 13 subunits. It is reduced to 1 to 3 subunits in prokaryotes. The complete amino acid composition has been derived from protein sequencing. Gene sequences are partially known in several eukaryote species. Metal centers are only located in subunits I and II. The mitochondrial cytochrome-c oxidase is Y-shaped; the arms of the Y cross the inner membrane, the stalk protrudes into the intermembrane space. The bacterial enzyme has a simpler, elongated shape. A number of data have been accumulated on the subunit topology and on their location within the protein. All available spectrometric techniques have been used to investigate the environment of the metal centers as well as their interactions. From the literature, attention must be paid to what may be considered or not as an active form. The steady improvement of the instrumentation has yielded evidence for different kinds of heterogeneities which could reflect the in vivo situation. The 'pulsed' and 'resting' conformers have been well characterized. The 'oxygenated' form has been identified as a peroxide derivative of the fully oxidized cytochrome-c oxidase. The mammalian enzyme has been isolated in fully active monomeric form which does not preclude the initially suggested dimeric behavior in situ. The role of the lipids is still largely investigated, mainly through reconstitution experiments. Kinetic studies of electron transfer between cytochrome c and cytochrome-c oxidase lead to a single catalytic site model to account for the multiphasic kinetics. Results related to the low temperature investigation of the intermediate steps in the reaction between oxygen and cytochrome-c oxidase received a sound confirmation by the resolution of compound A at room temperature. It is also pointed out that the so-called mixed valence state might not be a transient state in the catalytic reduction of oxygen. The functioning of cytochrome-c oxidase as a proton pump has been supported by a number of experimental results. Subunit III would be involved in this process. The redox link to the proton pump has been suggested to be at the Fea-CuA site. The molecular mechanism responsible for the proton pumping is still unknown.     

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.     

Purification of cytochrome-c oxidase retaining its pulsed form

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.     

Inactivation of cytochrome-c with glucose oxidase

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10 nM enzyme.     

The pathway of the quinol/quinone transhydrogenation reaction in ubiquinol: cytochrome-c reductase of Neurospora mitochondria

Ubiquinol: cytochrome-c reductase, isolated from Neurospora mitochondria as a protein/Triton X-100 preparation, and reconstituted into phospholipid membranes, catalyses the electron transfer from duroquinol to 2.3-dimethoxy-5-decyl-6-methyl-benzoquinone (decQ) on a myxothiazol-insensitive, but antimycin-sensitive, ping-pong pathway. Duroquinol reacts first to form the altered, reduced enzyme E'. This reaction is followed by dissociation of duroquinone making way for E' to bind decQ and convert it into decQH2.     

Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.

The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Characterization of cytochrome-c oxidase mutants in human fibroblasts

Skin fibroblasts were selected as having cytochrome-c oxidase deficiency by activity measurements in whole cells. Each cell line was cultured in sufficient amount to isolate mitochondria for biochemical characterization. Cytochrome-c oxidase was then examined by activity measurements, by heme determination and by polypeptide analysis using antibodies specific to the enzyme subunits. The cytochrome-c oxidase activity in the different cell lines ranged from 9% to 54% of that of normal fibroblasts. Heme determinations and polypeptide analysis established that the lowered cytochrome-c oxidase activity was due to reduced amounts of the complex in the mitochondrial inner membrane. In all cases, there was defective assembly of the enzyme, with the amounts of mitochondrially coded and nuclear coded subunits being reduced proportionally. These studies show that fibroblasts can be used for prenatal diagnosis of mitochondrial diseases and are a useful system in which to study mitochondrial biogenesis.     

Kinetic alterations of cytochrome-c oxidase in cystic fibrosis

We compared the kinetics of cytochrome-c oxidase (cytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) in fibroblasts derived from normal and cystic fibrosis individuals. The Km of the enzyme for reduced cytochrome c was significantly increased in CF cells; the change, however, was observed only at temperatures above 25 degrees C. The Vmax values were comparable in both types of individuals.     

Effects of subunit V antibodies on the topology of the subunit and the activity of beef heart cytochrome-c oxidase

Redox-sensitive epitopes on subunit V of beef heart cytochrome-c oxidase were demonstrated previously using polyclonal subunit-specific antibodies raised in rabbits. The antibodies only slightly inhibited electron transfer, and the accessibility of their epitopes depended on the presence of a membrane and on the redox state of the oxidase. The present paper describes additional preparations of antibodies raised against subunit V. These antibodies have an even higher subunit specificity, they are more than three times as inhibitory against electron transfer, and their binding does not require a membrane. Moreover, the redox-sensitive nature of their binding to detergent-dispersed oxidase is sensitive to the method of its isolation. We discuss inferences that can be drawn from a detailed quantitative comparison of the interactions of the two antibody preparations with the antigen in different environments. The techniques used in the comparison can be used to examine other perturbants of the oxidase as to their effects on specific segments of the enzyme.     

Electron paramagnetic resonance and spectrophotometric studies of the peroxide compounds of manganese-substituted horseradish peroxidase, cytochrome-c peroxidase and manganese-porphyrin model complexes

Peroxide compounds of manganese protoporphyrin IX and its complexes with apo-horseradish peroxidase and apocytochrome-c peroxidase were characterized by electronic absorption and electron paramagnetic resonance spectroscopies. An intermediate formed upon titration of Mn(III)-horseradish peroxidase with hydrogen peroxide exhibited a new electron paramagnetic resonance absorption at g = 5.23 with a definite six-lined 55Mn hyperfine (AMn = 8.2 mT). Neither a porphyrin pi-cation radical nor any other radical in the apoprotein moiety could be observed. The reduced form of Mn-horseradish peroxidase, Mn(II)-horseradish peroxidase, reacted with a stoichiometric amount of hydrogen peroxide to form a peroxide compound whose electronic absorption spectrum was identical with that formed from Mn(III)-horseradish peroxidase. The electronic state of the peroxide compound of manganese horseradish peroxidase was thus concluded to be Mn(IV), S = 3/2. Mn(III)-cytochrome-c peroxidase reacted with stoichiometry quantities of hydrogen peroxide to form a catalytically active intermediate. The electronic absorption spectrum was very similar to that of a higher oxidation state of manganese porphyrin, Mn(V). Since the peroxide compound of manganese cytochrome-c peroxidase retained two oxidizing equivalents per mol of the enzyme (Yonetani, T. and Asakura, T. (1969) J. Biol. Chem. 244, 4580-4588), this peroxide compound might contain an Mn(V) center.     

Gene mapping and gene enrichment by the avidin-biotin interaction: use of cytochrome-c as a polyamine bridge.

A modification of previously described methods of electron microscopic gene mapping and of gene enrichment based on the avidin-biotin interaction is presented. The modification consists of coupling cytochrome-c instead of pentane diamine to the oxidized 2', 3' terminus of an RNA by Schiff base formation and BH-4 reduction. The RNA-cytochrome-c conjugate is purified by a simple chromatographic procedure; several biotins are attached to the cytochrome moiety by acylation. The extended arm between biotin and RNA gives efficient electron microscopic gene mapping of DNA:RNA-biotin hybrids with avidin-ferritin and avidin-polymethacylate sphere labels and efficient gene enrichment by buoyant banding of DNA:RNA-biotin:avidin-spheres in CsCl. A 1400 fold enrichment (thus, 25% pure) and a 90% yield of long Drosophila DNA strands with 5S RNA genes is achieved.     

Influence of non-esterified fatty acids on respiratory control of reconstituted cytochrome-c oxidase

Bovine heart cytochrome-c oxidase was reconstituted in liposomes (asolectin) and the activity measured in the presence and absence of uncoupler at increasing concentrations of non-esterified fatty acids. Palmitic and stearic acids resulted in a decrease of about 40% in the respiratory control ratio at a concentration of 1 microM, when measured using a spectrophotometric procedure but not with a polarographic assay method. At higher fatty acid concentrations no further change was found. A 50% decrease in respiratory control was determined when the enzyme was reconstituted in pure phosphatidylcholine containing 2% cardiolipin. The respiratory control of reconstituted cytochrome-c oxidase from bovine liver was not influenced by fatty acids.     

Nitric oxide, cytochrome-c oxidase and myoglobin

Myoglobin, the monomeric haemoprotein expressed in red muscle, is reported in biochemistry and physiology textbooks to function as an intracellular oxygen carrier and oxygen reservoir. Here, Maurizio Brunori argues that myoglobin can also play the role of intracellular scavenger of nitric oxide, an inhibitor of mitochondrial cytochrome-c oxidase, thereby protecting respiration in the skeletal muscle and the heart.     

The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme.

The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB-F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus. The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103] is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium.     

Binding and oxidation of mutant cytochromes c by cytochrome-c oxidase

Mutation of conserved Phe-82 of yeast iso-1 cytochrome c to Tyr, Gly, Ser, Leu, or Ile affects binding to and reaction with cytochrome-c oxidase from beef heart. The observed changes of binding and kinetic constants reflect mutation-induced rearrangements in the heme vicinity brought about by the replacement of Phe-82. Such conformational rearrangements are also revealed by altered circular dichroism spectra of the oxidase-bound mutant cytochromes c. Variations in Km for cytochrome c oxidation do not parallel variations in Kd, the dissociation constant for binding of cytochrome c to the oxidase. This observation does not support an enzymatic mechanism in which the rate of cytochrome c oxidation is governed by product dissociation.     

Identification of different quaternary structures of beef heart cytochrome-c oxidase by two-dimensional polyacrylamide gel electrophoresis

A two-dimensional gel electrophoresis is described to identify different quaternary structures of the heart cytochrome-c oxidase. Bovine enzyme was purified and separated by discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the 1st dimension into several discrete complexes and thereupon shown to be heterodisperse in Triton X-100 and dodecyl maltoside. A discontinuous SDS-polyacrylamide gel electrophoresis in the 2nd dimension was used to determine the subunit composition of the isolated complexes. One of these represents the intact enzyme with 12 different polypeptides while the others have an incomplete subunit composition.     

Specific effects of ATP on the kinetics of reconstituted bovine heart cytochrome-c oxidase

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured by the polarographic and photometric method under uncoupled conditions in the presence of various polyvalent anions. In order to distinguish between specific and unspecific ionic effects of ATP, the photolabelling reagent 8-azido-ATP was applied. Covalently bound ATP at the enzyme complex caused the same increase of Km for cytochrome c as free ATP, if measured by the photometric assay. The increase of Km by photolabelling with 8-azido-ATP was completely prevented by ATP, but not by ADP. The data indicate the occurrence of a specific binding site for ATP at the cytosolic side of cytochrome-c oxidase, which, after binding of ATP, changes the kinetics of cytochrome c oxidation.     

Apoptosis induced by gamma irradiation in peripheral blood mononuclear cells is not mediated by cytochrome-c release and only partially involves caspase-3-like proteases

Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after gamma-irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after gamma irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (delta psi m) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5-Gy gamma irradiation, is characterized by a decrease in delta psi m, but surprisingly no release of cytochrome-c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in delta psi m with cytochrome-c release and a clear caspase 3 activation. We were unable to block the decrease in delta psi m with the caspase-inhibitors zVAD-fmk or zDEVD-fmk after gamma irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in gamma irradiated mature PBL, caspase-dependent and -independent pathways, but not cytochrome c, seem to be involved in the apoptotic process.