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The effect of pH on the kinetics of the following transhydrogenation reactions catalyzed by energized and nonenergized submitochondrial particles has been studied: NADH leads to 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP), NADH leads to thionicotinamide adenine dinucleotide phosphate (thioNADP), NADPH leads to AcPyADP, and NADPH leads to thioNADP. The effect of membrane energization on reaction rates can be approximated in the case of NADH leads to AcPyADP and thioNADP transhydrogenations, or equaled in the case of NADPH leads to AcPyADP and thioNADP transhydrogenations by lowering the assay pH to less than or equal to 6.0. For the reactions NADH leads to AcPyADP and thioNADP under energy-linked conditions, substrate Km values are lowest and Vmax values are highest at pH 7 to 7.5, the optimum pH for mitochondrial energy transduction processes. Under non-energy-linked conditions, however, Km values were lowest and Vmax values were highest at the most acid conditions (pH = 5.5) examined. Plots of ln (Vmax/Km) (an index of enzyme-substrate affinity to form a complex) versus pH showed the highest affinity at pH 7 to 7.5 for energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions. Similar plots for non-energy-linked conditions showed a sharp and linear increase in the value of ln (Vmax/Km) as the assay pH was lowered from 8.5 to 6 to 6.5. This was followed by a less steep line down to pH 5.5, with a clear break at pH 6 to 6.5. These results suggested the involvement of ionizable group(s) with pK value(s) at pH 6 to 6.5 affecting enzyme-substrate binding under non-energy-linked conditions. An analogous mechanism, possibly by way of proton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.roton-induced conformation change of the transhydrogenase enzyme (EC 1.6.1.1), might be involved in increasing enzyme-substrate affinity and consequently the rate of transhydrogenation under energy-linked conditions.

The mitochondrial energy-linked transhydrogenase enzyme catalyzes hydride ion transfer between NAD and HADP, of which the reaction NADH leads to NADP is slow in the absence of energy and is accelerated 10-fold or more when the mitochondrial membrane is energized by ATP hydrolysis or respiration. The enzyme is a proton pump and effects proton translocation coupled to hydride ion transfer from NADPH to NAD (Earle, S.R., and Fisher, R.R. (1980) J. Biol Chem. 255, 827-830). The present studies have shown that submitochondrial particles also catalyze transhydrogenation from NADPH to two NADP analogs, namely 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP) and thionicotinamide adenine dinucleotide phosphate (thioNADP). Both reaction rates are greatly accelerated when the system is energized by ATP hydrolysis (inhibitable by uncouplers or rutamycin) or succinate oxidation (inhibitable by uncouplers or antimycin A). As in the case of NAD(H) in equilibrium with NADP(H) reactions, the transhydrogenations from NADPH to AcPyADP and thioNADP are inhibited by treatment of submitochondrial particles with trypsin or the arginyl residue modifier, butanedione. The Km values of the above substrates and the Vmax values under energy-linked conditions have been determined. The finding that the mitochondrial energy-linked transhydrogenase enzyme catalyzes transhydrogenation from NADPH to NADP analogs has revealed features regarding substrate site specificities and the effect of substrates on the directionality of proton translocation by the enzyme.

Submitochondrial particles catalyze transhydrogenation from NADPH to [14C]NADP. This transhydrogenation is energy-linked, since its rate increases several-fold when the system is energized by succinate oxidation in the presence of rotenone (inhibitable by antimycin A or uncouplers), or by ATP hydrolysis (inhibitable by rutamycin or uncouplers). As in the case of transhydrogenation reactions from NAD(P)H to 3-ace-tylpyridine adenine dinucleotide phosphate and to thionicotinamide adenine dinucleotide phosphate, transhydrogenation from NADPH to [14C]NADP is also sensitive to treatment of the particles with trypsin or the arginyl residue modifier, butanedione. However, unlike the former reactions, transhydrogenation from NADPH to [14C]NADP cannot accumulate energy in the concentrations of the products, because, except for radioactivity, the nature and concentrations of the reactants and products remain unchanged throughout the course of the reaction. Therefore, the unrecoverable energy utilization by this region could be ascribed to an entropic component of the process, very likely an enzyme conformation change necessary for facilitation of hydride ion transfer from NADPH to [14C]NADP. This interpretation is in agreement with our previous kinetic evidence for enzyme conformation change associated with energy-linked transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate and thionicotinamide adenine dinucleotide phosphate, and with our conclusions regarding the mechanism of action of the transhydrogenase enzyme (Galante, Y.M., Lee, Y., and Hatefi, Y. (1980) J. Biol. Chem. 255, 9641-9646).

Two N-1 type iron-sulfur clusters in NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) were potentiometrically resolved: one was titrated as a component with a midpoint oxidation-reduction potential of -335 mV at pH 8.0, and with an n-value equal to one; the other as an extremely low midpoint potential component (Em 8.0 less than -500 mV). These two clusters are tentatively assigned to N-1b and N-1a, respectively. Cluster N-1b is completely reducible with NADH and has a spin concentration of about 0.8/FMN. Its EPR spectrum can be simulated as a single rhombic component with principal g values of 2.019, 1.937, and 1.922, which correspond to the Center 1 reported earlier by Orme-Johnson, N. R., Hansen, R. E., and Beinert, H. (1974) J. Biol. Chem. 249, 1922-1927. At extremely low oxidation-reduction potentials (less than -450 mV), additional EPR signals emerge with apparent g values of gz = 2.03, gy = 1.95, and gx = 1.91, which we assign to cluster N-1a. It is difficult, however, to simulate the detailed spectral line shape of this component as a single rhombic component, suggesting some degree of protein modification or interaction with a neighboring oxidation-reduction component. EPR spectra of soluble NADH dehydrogenase, containing 5-6 g atoms of non-heme iron and 5-6 mol of acid-labile sulfide/mol of FMN, were examined. Signals from at least two iron-sulfur species could be distinguished in the NADH-reduced form: one of an N-1b type spectrum; the other of a spectrum with g values of 2.045, 1.95, and 1.87 (total of about 0.5 spin equivalents/FMN). This is the first example of an N-1 type signal detected in isolated soluble NADH dehydrogenase.