ATP synthase. Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP.

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.

Glucose catabolism in African trypanosomes. Evidence that the terminal step is catalyzed by a pyruvate transporter capable of facilitating uptake of toxic analogs.

The protozoan parasite Trypanosoma brucei derives its metabolic energy exclusively from a unique type of glycolysis in which pyruvate derived from glucose catabolism is released into the host bloodstream. In this study, this terminal metabolic step has been examined in detail. Pyruvate release from trypanosomal cells supplied with glucose is very rapid, proceeding with an apparent Vmax of 214 nmol x min-1 x mg-1. Counterflow experiments with [14C]pyruvate demonstrate that this metabolic end product can be taken up by actively metabolizing cells consistent with the presence of a plasma membrane transporter. The findings that [14C] acetate exhibits a much lower capacity for cell entry and that the structural analog alpha-cyano-3-hydroxycinnamic acid inhibits pyruvate release provide additional support for the presence of a pyruvate transporter. The substrate analog and alkylating agent 3-bromopyruvate inhibits completely both cell motility and pyruvate release. Surprisingly, however, it is a poor inhibitor of pyruvate transport per se. Rather, its preferential site of action and that of iodoacetic acid were identified by radiolabeling studies and microsequence analysis as glyceraldehyde-3-phosphate dehydrogenase. In extending these studies, 3-bromopyruvate was found to be over 20 times less effective in inhibiting glyceraldehyde-3-phosphate dehydrogenase in intact erythrocytes than in trypanosomal cells. However, in sonicated preparations from both cell types, the enzyme exhibits nearly identical sensitivities to inhibition by 3-bromopyruvate. Experiments reported here provide the first direct evidence that pyruvate release in African trypanosomes is catalyzed by a specific transport system and implicate this transporter as a vehicle for delivering toxic alkylating agents into trypanosomal cells.

Comparison of energy-transducing capabilities of the two- and three-subunit cytochromes aa3 from Paracoccus denitrificans and the 13-subunit beef heart enzyme.

In the accompanying paper, we have shown that the two-subunit cytochrome aa3 isolated from Paracoccus denitrificans displays the same kind of complex and interactive redox behavior as the 13-subunit cytochrome aa3 from beef heart. Therefore, the redox characteristics are not dependent on the additional 11 subunits. In the current work, we have examined the energy-transducing capabilities of both the two- and three-subunit enzymes obtained from Paracoccus denitrificans in relation to that of the 13-unit mammalian enzyme. We have found that in all of the tested functions, which included the development of delta psi and delta pH, and the pumping of protons, that the two-subunit enzyme is at least as efficient as the structurally more complex mammalian enzyme. There is thus a correlation between the complex redox behavior and energy transducing capabilities of the two enzymes. There was also no difference in energy-transducing capabilities between the two- and three-subunit forms of the bacterial enzyme. It seems that only 2 subunits are required for an efficient energy-transducing cytochrome aa3. The most likely role of the additional subunits in the mammalian enzyme, therefore, seems to be in regulation.

The polyphasic reduction of oxygen to water by purified cytochrome c oxidase.

The time course of oxygen consumption by purified cytochrome oxidase has been studied in reactions where the fully reduced enzyme was rapidly mixed with molecular oxygen. Similar to intact mitochondria (Reynafarje & Davies, Am. J. Physiol. 258, 1990), the enzyme reduces oxygen to water in a kinetically and well defined polyphasic event. The initial rates of O2 consumption depended hyperbolically on O2 concentration, with a bimolecular rate constant of near 10(7) M-1 s-1. The Vmax of O2 uptake was, however, a complex function of the concentrations of both enzyme and cytochrome c. It is concluded that the reduction of oxygen to water takes place in a cyclic process in which the oxidase undergoes redox changes at rates depending on the relative concentration of the enzyme and its 3 substrates: O2, electrons and protons. No evidence was found for impairments in the intramolecular flow of electrons per se.

The polyphasic nature of the respiratory process at the mitochondrial level.

The kinetics of oxygen consumption by rat liver mitochondria, respiring under a variety of metabolic conditions, have been studied. Respiration was initiated by injecting oxygen into anaerobic suspensions of mitochondria. It was found that, irrespective of the metabolic state of the mitochondria and the nature of the respiratory substrate, the rates of electron flow and oxygen consumption follow the pattern of a polyphasic reaction. The rates of oxygen uptake during the first phase are extremely fast and depend on oxygen concentration. The second phase represents a transition in which net oxidation of cytochrome-c oxidase stops and the rates of oxygen consumption suddenly decrease. The third phase is characterized by its changeability. Depending on initial conditions the rates may increase, decrease, or remain constant, although the reaction is not one of zero order. During the last phase, the rates decrease and the oxidase becomes increasingly reduced. It is postulated that the mitochondrial respiratory process is basically a cyclic event in which the redox state of the membrane and the rates of oxygen consumption oscillate with amplitudes and frequencies conditioned by the energy demand and energy-yielding capacity of the cell.

Proton stoichiometry of electron transport in rodent tumor mitoplasts.

The mechanistic vectorial H+/O translocation ratios characteristic of energy-conserving sites 2 + 3 and site 3 of the respiratory chain of two tumor cell lines were determined using succinate and ferrocytochrome c, respectively, as electron donors. The measurements were carried out on mitoplasts in order to allow ferrocytochrome c free access to its binding site on the inner mitochondrial membrane. The tumor cell lines used were Ehrlich ascites tumor and the AS30-D ascites tumor. K+ was used as charge-compensating cation in the presence of valinomycin. The O2 uptake rate measurements were made with a fast-responding membrane-less electrode whose response time was closely matched with that of a pH electrode. The rates of O2 uptake and H+ ejection during the apparent zero-order rate phase of respiration, analyzed by computer, were extrapolated to zero time. The observed H+/O ratios for succinate oxidation in both tumors exceeded 7 and approached 8 and the H+/O ratios for the cytochrome oxidase reaction closely approached 4.0, in agreement with data or normal mitochondria. However, the rates of H+ back decay in the tumor mitochondria are relatively high and may influence the net efficiency of oxidative phosphorylation under intracellular conditions.

Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria.

The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO-cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed.

Upper and lower limits of the proton stoichiometry of cytochrome c oxidation in rat liver mitoplasts.

The stoichiometry of vectorial H+ translocation coupled to oxidation of added ferrocytochrome c by O2 via cytochrome-c oxidase of rat liver mitoplasts was determined employing a fast-responding O2 electrode. Electron flow was initiated by addition of either ferrocytochrome c or O2. When the rates were extrapolated to level flow, the H+/O ratios in both cases were less than but closely approached 4; the directly observed H+/O ratios significantly exceeded 3.0. The mechanistic H+/O ratio was then more closely fixed by a kinetic approach that eliminates the necessity for measuring energy leaks and is independent of any particular model of the mechanism of energy transduction. From two sets of kinetic measurements, an overestimate and an underestimate and thus the upper and lower limits of the mechanistic H+/O ratio could be obtained. In the first set, the utilization of respiratory energy was systematically varied through changes in the concentrations of valinomycin or K+. From the slope of a plot of the initial rates of H+ ejection (JH) and O2 uptake (JO) obtained in such experiments, the upper limit of the H+/O ratio was in the range 4.12-4.19. In the second set of measurements, the rate of respiratory energy production was varied by inhibiting electron transport. From the slope of a plot of JH versus JO, the lower limit of the H+/O ratio, equivalent to that at level flow, was in the range 3.83-3.96. These data fix the mechanistic H+/O ratio for the cytochrome oxidase reaction of mitoplasts at 4.0, thus confirming our earlier measurements (Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7218-7222). Possible reasons for discrepancies in published reports on the H+/O ratio of cytochrome oxidase in various mitochondrial and reconstituted systems are discussed.

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria. Reply to a commentary.

Costa, L.E., Reynafarje, B. and Lehninger, A.L. [(1984) J. Biol. Chem. 259, 4802-4811] have reported 'second-generation' measurements of the H+/O ratio approaching 8.0 for vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria. In a Commentary in this Journal [Krab, K., Soos, J. and Wikström, M. (1984) FEBS Lett. 178, 187-192] it was concluded that the measurements of Costa et al. significantly overestimated the true H+/O stoichiometry. It is shown here that the mathematical simulation on which Krab et al. based this claim is faulty and that data reported by Costa et al. had already excluded the criticism advanced by Krab et al. Also reported are new data, obtained under conditions in which the arguments of Krab et al. are irrelevant, which confirm that the H+/O ratio for succinate oxidation extrapolated to level flow is close to 8.

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase.

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

O2 solubility in aqueous media determined by a kinetic method.

A kinetic method for the determination of O2 solubility in air-saturated aqueous solutions of widely varying composition and temperature is described. It is based on the precise molar stoichiometry between the rates of uptake of H+ and O2, measured with response-matched electrodes, in the reaction NADH + H+ + 1/2O2----NAD+ + H2O, catalyzed by an NADH oxidase preparation. To the initially anaerobic test system, which contains an excess of NADH and NADH oxidase in a buffered medium, an aliquot of the O2-containing solution to be tested is added and the rates of both O2 uptake and H+ uptake are recorded; the H+ electrode is calibrated against standard HCl. From these data the amount of O2 in the aliquot is calculated. Some representative values for O2 solubility at 25 degrees C and 760 mm in air-saturated systems are (i) distilled H2O, 516 nmol O/ml, (ii) 0.15 M KCl, 480 nmol O/ml, and (iii) 0.25 M sucrose, 458 nmol O/ml. Data and equations are also given for the solubility of O2 at 760 mm in air-saturated and lightly buffered 0.15 M KCl and 0.25 M sucrose over the range 5 to 40 degrees C. In the method described the rates of O2 and H+ uptake are precisely linear and stoichiometric when NADH is present in large excess over O2. However, when O2 is in excess and small additions of 340-nm-standardized NADH are made, as in earlier methods based on NADH oxidation, the endpoint is approached very gradually and tends to overestimate O2 solubility, owing to (i) the higher Km for NADH than for O2, (ii) the relatively slow response of the Clark O2 electrode, and (iii) the incomplete oxidation of NADH in the presence of 340-nm-absorbing inhibitory substances.

Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain.

Stoichiometry of mitochondrial H+ translocation coupled to succinate oxidation at level flow.

The mechanistic stoichiometry of vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria in the presence of a permeant cation has been determined under level flow conditions with a membraneless fast responding O2 electrode kinetically matched with a glass pH electrode. The reactions were initiated by rapid injection of O2 into the anaerobically preincubated test system under conditions in which interfering H+ backflow was minimized. The rates of O2 uptake and H+ ejection, obtained from computer-fitted regression lines, were monotonic and first order over 75% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero delta mu H+ and thus level flow prevails, yielded vectorial H+/O flow ratios above 7 and closely approaching 8. The mitochondria undergo no irreversible change and give identical H+/O ratios on repeated tests. In a further refinement, the lower and upper limits of the mechanistic H+/O ratio were determined to be 7.55 and 8.56, respectively, from plots of the rates of O2 uptake versus H+ ejection at increasing malonate and increasing valinomycin concentrations, respectively. It is therefore concluded that the mechanistic H+/O ratio for energy-conserving sites 2 + 3 is 8, in confirmation of earlier measurements. KCl concentration is critical for maximal observed H+/O ratios. Optimum conditions and possible errors in determination of mechanistic H+/O translocation ratios are discussed.

Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant derivative.

Obligately alkalophilic Bacillus firmus RAB had a higher molar growth yield on L-malate (Ymal = 38 mg, dry weight/mmol of L-malate) than its non-alkalophilic mutant derivative, strain RABN (Ymal = 12 mg, dry weight/mmol of L-malate). Measurements of respiration dependent proton translocation by the two strains in the presence of K+ and valinomycin showed that the alkalophile also has much higher H+/O stoichiometries (at pH 9.0) than does the mutant (at pH 7.0). H+/O ratios for B. firmus RAB at pH 9.0 were as high as 13, with a frequently observed value of 9. These high values were observed in the first phase of a set of biphasic curves for both oxygen consumption and proton ejection. At pH 7.0, both the wild type and the mutant exhibited H+/O ratios near 4 in a single phase of oxygen consumption and proton ejection. The results are consistent with suggestions that the alkalophilic respiratory chain is especially well adapted for effective energy transduction at alkaline but not neutral pH.

Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.

The 1,2,3-benzothiadiazoles. A new type of compound acting on coupling site I, in rat liver mitochondria.

1. In rat liver mitochondria, 6-chloro-1,2,3-benzothiadiazole inhibited ADP phosphorylation and Ca2+-transport when the energy required for these processes came from the oxidation of NAD-linked substrates. The inhibition was characterized by substantial reduction in oxygen consumption, H+-movement and disappearance of acceptor control ratio. 2. When the substrate oxidized was succinate, depending on the 6-chloro-1,2,3-benzothiadiazole concn., little or no effect was observed on ADP phosphorylation and Ca2+-transport. 3. The results suggest that 6-chloro-1,2,3-benzothiadiazole can block site I at low concn., but at higher concn. can affect site I and site II, although site I is always more affected.