Oxidative phosphorylation and respiratory control phenomenon in Paracoccus denitrificans plasma membrane.

Changes in respiratory activity, transmembrane electric potential, and ATP synthesis as induced by additions of limited amounts of ADP and P(i) to tightly coupled inverted (inside-out) Paracoccus denitrificans plasma membrane vesicles were traced. The pattern of the changes was qualitatively the same as those observed for coupled mitochondria during the classical State 4-State 3-State 4 transition. Bacterial vesicles devoid of energy-dependent permeability barriers for the substrates of oxidation and phosphorylation were used as a simple experimental model to investigate two possible mechanisms of respiratory control: (i) in State 4 phosphoryl transfer potential (ATP/ADP × P(i)) is equilibrated with proton-motive force by reversibly operating F(1)·F(o)-ATPase (thermodynamic control); (ii) in State 4 apparent "equilibrium" is reached by unidirectional operation of proton motive force-activated F(1)·F(o)-ATP synthase. The data support the kinetic mechanism of the respiratory control phenomenon.

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I).

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.

Proton-translocating ATP-synthase of Paracoccus denitrificans: ATP-hydrolytic activity.

Tightly coupled membranes of Paracoccus denitrificans catalyze oxidative phosphorylation but are incapable of ATP hydrolysis. The conditions for observation and registration of the venturicidin-sensitive ATPase activity of subbacterial particles derived from this organism are described. The ATP hydrolytic activity does not appear after prolonged incubation in the presence of pyruvate kinase and phosphoenol pyruvate (to remove ADP), EDTA (to remove Mg2+) and/or inorganic phosphate, whereas the activity dramatically increases after energization of the membranes. ATP hydrolysis by -activated ATPase is coupled with electric potential formation. Inorganic phosphate prevents and azide promotes a decline of the enzyme activity during ATP hydrolysis. The addition of uncouplers results in rapid and complete inactivation of ATPase. The -dependent ATPase activity increases upon dilution of the membranes. The results are discussed as evidence for the presence of distinct ATP-synthase and ATP-hydrolase states of F(o)F(1) complex in the coupling membranes (Vinogradov, A. D. (1999) Biochemistry (Moscow), 64, 1219-1229). The proposal is made that part of the free energy released from oxidoreduction in the respiratory chain is used to maintain active conformation of the energy-transducing proteins.

Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase.

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax. There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.