The effects of ADP on reverse electron flow and the oxygen exchange reactions catalyzed by bovine heart muscle submitochondrial particles

1,N⁶-Ethenoadenosine diphosphate (ϵ-ADP) inhibits reverse electron flow (succinate → NAD⁺ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non- or uncompetitive inhibition, the former occuring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP. ADP, which stimulates ATP ⇌ H₂O and P_i ⇌ H₂O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP-supported P_i ⇌ H₂O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg²⁺ complexes of ADP and P_i. Thus only exchanges involving medium ADP and P_i would be expected to show arsenate sensitivity.     

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.     

Low-level chemiluminescence of bovine heart submitochondrial particles

Submitochondrial particles from bovine heart mitochondria showed low-level chemiluminescence when supplemented with organic hydroperoxides. Chemiluminescence seems to measure integratively radical reactions involved in lipid peroxidation and related processes. Maximal light-emission was about 1500 counts/s and was reached 2-10min after addition of hydroperoxides. Ethyl hydroperoxide, cumene hydroperoxide and t-butyl hydroperoxide were effective in that order. Antimycin and rotenone increased chemiluminescence by 50-60%; addition of substrates, NADH and succinate did not produce marked changes in the observed chemiluminescence. Cyanide inhibited chemiluminescence; half-maximal inhibitory effect was obtained with 0.03mm-cyanide and the inhibition was competitive with respect to t-butyl hydroperoxide. Externally added cytochrome c (10-20mum) had a marked stimulatory effect on chemiluminescence, namely a 12-fold increase in light-emission of antimycin-inhibited submitochondrial particles. Stimulation of hydroperoxide-induced chemiluminescence of submitochondrial particles by cytochrome c was matched by a burst of O(2) consumption. O(2) is believed to participate in the chain radical reactions that lead to lipid peroxidation. Superoxide anion seems to be involved in the chemiluminescence reactions as long as light-emission was 50-60% inhibitible by superoxide dismutase. Singlet-oxygen quenchers, e.g. beta-carotene and 1,4-diazabicyclo[2,2,2]-octane, affected light-emission. beta-Carotene was effective either when incorporated into the membranes or added to the cuvette. The present paper suggests that singlet molecular oxygen is mainly responsible for the light-emission in the hydroperoxide-supplemented submitochondrial particles.     

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c₁ complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K_m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b-c₁ complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K_m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V_max and the K_m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.     

Thermodynamics of the electrochemical proton gradient in bovine heart submitochondrial particles.

The electrical and chemical components of the electrochemical proton gradient of submitochondrial particles can be monitored simultaneously by continuously recording optical signals from the probes oxonol-VI and 9-aminoacridine. Either respiration or ATP hydrolysis causes a red shift in the absorption spectrum of oxonol-VI indicative of a membrane potential and a decrease of the fluorescence of 9-aminoacridine indicative of a pH gradient. The magnitude of the membrane potential and pH gradient formed by respiring submitochondrial particles can be modulated by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi] where deltaG'o is the standard free energy of ATP hydrolysis. Increasing values of deltaGp cause an increase in the steady state magnitudes of both the membrane potential and pH gradient. Thermodynamic phosphorylation potential titration experiments indicate that the electrochemical proton gradient normally maintained by respiring submitochondrial particles has an energy equivalent to 10.5 to 10.9 kcal/mol.     

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.     

The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions.

1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.     

H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells.

Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO. These data suggest that (i) the H+/e- stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.     

Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron(III)-adriamycin complex.

The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.     

The cytochrome chain of mitochondria exhibits variable H+/e- stoichiometry.

A study is presented of the ----H+/e- stoichiometry for H+ pumping by the cytochrome chain in isolated rat liver mitochondria under level-flow and steady-state conditions. It is shown that the ----H+/e- stoichiometry for the cytochrome chain varies under the influence of the flow rate and transmembrane delta microH+. The rate-dependence is shown to be associated with cytochrome c oxidase, whose ----H+/e- ratio varies from 0 to 1, whilst the ----H+/e- ratio for the span covered by cytochrome c reductase is invariably 2.     

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.     

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.     

Occurrence of an uncoupler-resistant intermediate type of phosphate-water oxygen exchange reaction catalyzed by heart submitochondrial particles

The hydrolysis of ATP catalyzed by phosphorylating vesicles prepared from bovine heart mitochondria by ultrasonic disruption was studied in H218O. Provided that an ATP-generating system was included to prevent accumulation of ADP due to hydrolysis, the addition of 20 mM arsenate or 0.5 mM 2,4-dinitrophenol to the incubation mixture either singly or together, had little or no effect on the number of oxygen atoms from H2O incorporated (on the average) into each molecule of Pi formed by hydrolysis (the O:P ratio). As the ATP concentration was reduced from 2.0 to 0.05 mM, the O:P ratio increased from about 1.4 to over 2.0 and, although dinitrophenol significantly increased the ATPase activity, it did not significantly alter the O:P ratio for a given ATP level. This implies that the uncoupler does not act directly on the terminal transphosphorylation step. Companion experiments were performed in which 18O label was placed either initially in H2O or Pi. Under conditions where extensive exchange from H218O into Pi occurred, no 18O was lost from medium Pi under identical circumstances, thus showing that the exchange was intermediate and did not involve medium Pi. Kinetic plots of v vs. v/S were nonlinear with respect to ATPase activity. The kinetic data, as well as the Pi = H218O exchange data, are consistent with enzyme models having multiple forms of catalytic sites. Several models are evaluated and attempts are made to distinguish between some of the simpler cases of these models.     

Reticulocyte lipoxygenase changes the passive electrical properties of bovine heart submitochondrial particles

Purified reticulocyte lipoxygenase oxygenates the polyunsaturated phospholipids of sonified submitochondrial particles from bovine heart as measured by a burst of oxygen uptake. Over the frequency range of 0.5 to 100 MHz, the complex impedance of the submitochondrial particles as a function of the frequency before and after lipoxygenase attack was measured. From these data, the membrane capacity, the conductivity of the membrane and the conductivity inside the particles were calculated. Lipoxygenase action causes a 4-fold increase in the membrane capacity and a 2-fold increase in the membrane conductivity. Using the method of deformation of electric pulses, kinetic measurements were performed. In parallel to the changes of the passive electric properties, a partial inhibition of NADH oxidase and succinate oxidase was caused by the lipoxygenase attack. Oxygen uptake, changes of the passive electric properties and the inhibition of respiratory enzymes were prevented by lipoxygenase inhibitors. Owing to the high oxygen consumption produced by the lipoxygenase reaction, anaerobiosis was reached within the first 30 s in the closed chamber. Therefore, it must be concluded that the changes in passive electric properties and the inhibition of the respiratory enzymes are due to secondary anaerobic processes such as the hydroperoxidase reaction catalyzed by the lipoxygenase or a slow redistribution of peroxidized membrane lipids. The results are discussed in relation to the breakdown of mitochondria during the maturation process of red cells.     

Pentachlorophenol inhibition of succinate oxidation by the respiratory chain in submitochondrial particles from the bovine heart

Study on the effect of pentachlorophenol on the succinate oxidase activity of submitochondrial particles and on the reduction level of cytochromes b revealed that the Ki value for PCP is equal to 2-4 microM. The succinate-DCPIP-reductase activity is noncompetitively inhibited with PCP (by 75-85%) (Ki = 3.6 microM). In the case of the succinate-PMS-reductase activity PCP at micromolar concentrations decreases the value of V only by 40% (C50 = 2 microM) with a simultaneous increase of the Km value for PMS. The identity of Ki values for PCP under these conditions suggests that the effect of PCP is due to the inhibitor interaction with the same component of the succinate dehydrogenase complex. The type of action of PCP on the succinate-acceptor-reductase activities indicates that the inhibiting effect of PCP on succinate oxidations is similar to that exerted by traditional inhibitors of succinate dehydrogenase--tenoyltrifluoroacetone and carboxins. Since PCP inhibits succinate dehydrogenase at low concentrations, it seems likely that the biological (pesticidal) effect of PCP is provided for not only by its uncoupling action but also by the inhibition of succinate oxidation in the respiratory chain.