Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.     

ATP-induced spectral changes in cytochrome c oxidase. A kinetic investigation.

Mixing ATP with soluble oxidized cytochrome c oxidase induces a spectral perturbation in the Soret region of the enzyme. This spectral perturbation is observed at ATP concentrations similar to those found to modulate the catalytic activity of cytochrome c oxidase [Malatesta, Antonini, Sarti & Brunori (1987) Biochem. J. 248, 161-165]. The process is reversible and corresponds to a simple binding with Kd = 0.2 mM at 25 degrees C. The absorbance change follows a first-order time course, and analysis of the ATP-concentration-dependence indicates the presence of a rate-limiting monomolecular step that governs the process. From the temperature-dependence of this process, studied at saturating concentrations of ATP, an activation energy of 44 kJ/mol (10.6 kcal/mol) was measured. The spectral perturbation also occurs when cytochrome c oxidase is reconstituted into artificial phospholipid vesicles, with equilibria and kinetics similar to those observed with the soluble enzyme. Mixing ATP with soluble oxidized cyanide-bound cytochrome c oxidase induces a different spectral perturbation, and the apparent affinity of ATP for the enzyme is substantially increased. There is no absolute specificity for ATP, because EGTA, inositol hexakisphosphate, sulphate and phosphate are all able to induce an identical spectral perturbation with the same kinetics, although the value of the apparent Kd is different for the various anions. The presence of Mg2+ ions decreases, in a saturation-dependent fashion, the apparent affinity of cytochrome c oxidase for ATP. The absorbance change can be correlated to the displacement of the Ca2+ bound to cytochrome c oxidase.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.     

Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles.

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.     

Demonstration of short-lived complexes of cytochrome c with cytochrome bc1 by EPR spectroscopy: implications for the mechanism of interprotein electron transfer

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc(1). To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc(1), we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths. Our data show a successive decrease of the bound cytochrome c fraction as the ionic strength increases, with a limit of approximately 120 mm NaCl above which essentially no bound cytochrome c can be detected by EPR. This limit does not apply to all of the interactions of cytochrome c with cytochrome bc(1) because the cytochrome bc(1) enzymatic activity remained high over a much wider range of ionic strengths. We concluded that EPR monitors just the tightly bound state of the association and that an averaged lifetime of this state decreases from over 100 mus at low ionic strength to less than 400 ns at an ionic strength above 120 mm. This suggests that at physiological ionic strength, the tightly bound complex on average lasts less than the time needed for a single electron exchange between hemes c and c(1), indicating that productive electron transfer requires several collisions of the two molecules. This is consistent with an early idea of diffusion-coupled reactions that link the soluble electron carriers with the membranous complexes, which, we believe, provides a robust means of regulating electron flow through these complexes.     

Reconstitution of escherichia coli succinoxidase from soluble components.

1. The membrane-bound succinoxidase of Escherichia coli was fractionated with deoxycholate into three soluble components, viz. succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase complex, and a factor identified as a phospholipid-containing component. 2. The dehydrogenase and cytochrome oxidase complexes were partially purified by filtration on Amicon membranes, Sepharose 4B chromatography, and sucrose gradient centrifugation. 3. Reconstitution of membranous succinoxidase, which catalyzes the oxidation of succinate by molecular oxygen by an integrated CN(-)-sensitive pathway, was achieved by mixing the soluble succinate dehydrogenase.cytochrome b1 complex with the soluble cytochrome oxidase complex in the presence of deoxycholate and then removing the detergent by gel filtration on Sephadex G-75. The phospholipid-containing factor stimulated the formation of succinoxidase by about 100% over that observed with the two complexes. 4. Isopycnic sucrose gradient centrifugation of succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase, and the reconstituted succinoxidase gave buoyant densities (p value) as 1.167, 1.229, and 1.194, respectively. 5. Electron microscopic evidence is presented for the vesicular nature of the reconstituted succinoxidase.     

Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Kinetics of the interaction of the cytochrome c oxidase of Paracoccus denitrificans with its own and bovine cytochrome c

We have devised a relatively simple method for the purification of cytochrome aa3 of Paracoccus denitrificans with three major subunits similar to those of the larger subunits of the mitochondrial cytochrome oxidase. This preparation has no c-type cytochrome. Studies were made of the oxidation of soluble cytochromes c from bovine heart and Paracoccus. The cytochrome-c oxidase activity was stimulated by low concentrations of either cytochrome c, providing an explanation for the multiphasic nature of plots of v/S versus v. Kinetics of the oxidation of bovine cytochrome c by the Paracoccus oxidase resembled those of bovine oxidase with bovine cytochrome c in every way; the Paracoccus oxidase with bovine cytochrome c can serve as an appropriate model for the mitochondrial system. The kinetics of the oxidation of the soluble Paracoccus cytochrome c by the Paracoccus oxidase were different from those seen with bovine cytochrome c, but resembled the latter if poly(L-lysine) was added to the assays. The important difference between the two species of cytochrome c is the more highly negative hemisphere on the side of the molecule way from the heme crevice in the Paracoccus cytochrome. Thus, the data emphasize the importance of all of the charged groups on cytochrome c in influencing the binding or electron transfer reactions of this oxidation-reduction system. The data also permit some interesting connotations about the possible evolution from the bacterial to the mitochondrial electron transport system.     

A salinity-induced C3-CAM transition increases energy conservation in the halophyte Mesembryanthemum crystallinum L

A strongly increased ATP/ADP ratio was found during the nocturnal phase I in crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants. Conversely, during the daytime phase III in CAM-performing plants the ATP/ADP ratio dropped to a similar level to that of C3 plants, cytochrome c oxidase activity was stimulated and mitochondrial Mn-superoxide dismutase activity was strongly increased. The findings suggest that a salinity-induced C3-CAM transition might be an efficient energy-conserving strategy for M. crystallinum plants, in which the strong nocturnal ATP production seems to be, at least partially, independent from the coupled mitochondrial electron transport.     

Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside

Cytochrome oxidase is purified from rat liver and beef heart by affinity chromatography on a matrix of horse cytochrome c-Sepharose 4B. The success of this procedure, which employs a matrix previously found ineffective with beef or yeast oxidase, is attributed to thorough dispersion of the enzyme with nonionic detergent and a low density of cross-linking between the lysine residues of cytochrome c and the cyanogen bromide activated Sepharose. Beef heart oxidase is purified in one step from mitochondrial membranes solubilized with lauryl maltoside, yielding an enzyme of purity comparable to that obtained on a yeast cytochrome c matrix [Azzi, A., Bill, K., & Broger, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2447-2450]. Rat liver oxidase is prepared by hydroxyapatite and horse cytochrome c affinity chromatography in lauryl maltoside, yielding enzyme of high purity (12.5-13.5 nmol of heme a/mg of protein), high activity (TN = 270-400 s-1), and very low lipid content (1 mol of DPG and 1 mol of PI per mol of aa3). The activity of the enzyme is characterized by two kinetic phases, and electron transfer can be stimulated to maximal rates as high as 650 s-1 when supplemented with asolectin vesicles. The rat liver oxidase purified by this method does not contain the polypeptide designated as subunit III. Comparisons of the kinetic behavior of the enzyme in intact membranes, solubilized membranes, and the purified delipidated form reveal complex changes in kinetic parameters accompanying the changes in state and assay conditions, but do not support previous suggestions that subunit III is a critical factor in the binding of cytochrome c at the high-affinity site on oxidase or that cardiolipin is essential for the low-affinity interaction of cytochrome c. The purified rat liver oxidase retains the ability to exhibit respiratory control when reconstituted into phospholipid vesicles, providing definitive evidence that subunit III is not solely responsible for the ability of cytochrome oxidase to produce or respond to a membrane potential or proton gradient.     

Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.     

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.     

Interaction of cytochrome c with mitochondrial proteins and cybacrone-dextran

Interaction of cytochrome c with electron carriers in intact and damaged (with destroyed outer membrane) rat liver mitochondria was studied. It was shown that the increase in ionic strength causes changes in the respiration rate of damaged mitochondria due to the reduction of the cytochrome c affinity for its binding sites in the organelles. This suggests that cytochrome c concentration in the intermembrane space of intact mitochondria is increased by salts, whereas the increase in ionic strength has a slight influence on the rates of succinate oxidase and external rotenone-insensitive NADH-oxidase of intact mitochondria. At low ionic strength values, the Michaelis constant (KM) value of external NADH-oxidase for cytochrome c exceeds by one order of magnitude that for succinate oxidase, while the maximal activity of these two systems is nearly the same. The increase in ionic strength causes an increase in the KM value for both oxidases. Interaction of cytochrome c with mitochondrial proteins was modelled by cytochrome c interaction with cibacron-dextran anions. It was concluded that the ionic strength-sensitive electrostatic interactions play a decisive role in cytochrome c binding to electron carriers in mitochondrial membranes. However, cytochrome c content and its binding parameters in intact-mitochondrial membranes prevent the latent activity of external NADH oxidase to be revealed in intact mitochondria after the increase in the ionic strength of the surrounding medium.     

Cross reactivity of monoclonal antibodies and cDNA hybridization suggest evolutionary relationships between cytochrome c oxidase subunits VIa and VIc and between VIIa and VIIb.

Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody-producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC-purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa-h (heart-type), after amplification by the polymerase chain reaction, with a probe of VIa-l (liver-type).     

Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM.

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.