No abstract

Considerable quantitative variations in the competitive inhibition of NADH oxidase activity of bovine heart submitochondrial particles (SMP) by different samples of NAD- were observed. ADP-ribose (ADPR) was identified as the inhibitory contaminating substance responsible for variations in the inhibition observed. ADPR competitively inhibits NADH oxidation with Ki values (25 degrees C, pH 8.0) of 26 microM, 30 microM, and 180 microM for SMP, purified Complex I and three-subunit NADH dehydrogenase (FP), respectively. ADPR decreases NADH-induced flavin reduction and prolongs the cyclic bleaching of FP during aerobic oxidation of NADH. Ki for inhibition of the rotenone-sensitive NADH oxidase in SMP by ADPR does not depend on delta mu H+. The initial rate of the energy-dependent NAD+ reduction by succinate is insensitive to ADPR. The inhibitor increases the steady-state level of NAD+ reduction reached during aerobic succinate-supported reverse electron transfer catalyzed by tightly coupled SMP. The results obtained are consistent with the proposal on different nucleotide-binding sites operating in the direct and reverse reactions catalyzed by the mitochondrial NADH-ubiquinone reductase.

F 0 ⅐F 1 -ATPases (ATP synthases) are the oligomeric molecular machines that couple ATP hydrolysis (synthesis) with proton translocation across the energy-transducing membranes in mitochondria, chloroplasts, and bacteria. The structural arrangement of the subunits within F 0 ⅐F 1 complexes of various organisms is assumed to be very similar (1-3). The hydrophilic F 1 is composed of a trimer of tightly packed ␣⅐␤-subunit pairs and one copy each of the ␦-, ⑀-, and ␥-subunits (Escherichia coli nomenclature for the subunits is used). Long rod-like ␥-subunit is asymmetrically positioned in the central cavity of the almost spherical globular ␣⅐␤ trimer. F 0 component is the hydrophobic complex composed of 10 -14 transmembraneously positioned c-, one a-, and two b-subunits. The hydrophilic parts of b-subunits are bound to F 1 (to one pair of ␣⅐␤-and one ␦-subunit), thus forming the peripheral stalk. The other central stalk is formed by ␥⅐⑀ complex, which interacts with c-subunit(s) arranged in a ring. F 1 bears three "catalytic" nucleotide-binding sites located on ␤-subunits, and F 0 serves as a proton-conducting path. It is generally believed that the coupling between the ATP hydrolysis (synthesis) and flow of protons across the membrane results from the consequence of the long distance conformational change: ␣⅐␤-pair-associated chemical catalysis 3 ␥⅐⑀ 3 ab 2 c n leading to rotation of the rotor (␥⅐⑀ bound to c-ring) within the stator (␣⅐␤ trimer fixed by two b-and one ␦-subunits) (4).The kinetics of ATP hydrolysis catalyzed by the soluble F 1 or membrane-bound F 0 ⅐F 1 preparations of the enzyme (coupled or uncoupled) are very complex (5). It has been documented that the key factor for such a complexity is a formation of so-called ADP(Mg 2ϩ )-inhibited form of the enzyme originally described (6) and kinetically characterized in a number of reports published by our (7-11) and other groups (12-16). Several phenomena such as hysteresis in onset of the catalytic activity (17), slow inhibition of ATPase by Mg 2ϩ (18), activation of ATP hydrolysis by sulfite and other anions (9,19,20), and the inhibitory effect of azide (9) can be consistently explained by the kinetic scheme in which the slowly reversible interconversion between catalytically competent enzyme-ADP intermediate and its inactive "isomer" plays the central role (5). Perhaps the most intriguing property of the ADP(Mg 2ϩ )-deactivated ATPase is that being inactive in ATP hydrolysis, it is fully competent in the ATP synthase activity (21,22).The ADP(Mg 2ϩ )/ATP-and possibly ⌬ H ϩ 1 -induced rearrangement within F 0 ⅐F 1 complex that trigger its ATP hydrolase and ATP synthase activities remains unclear. The ⑀-subunit, an endogenous inhibitor of the ATP hydrolase activity of F 1 and F 0 ⅐F 1 (23, 24), seems to be the most likely candidate for the triggering function. It has been shown that two distinct domains of the ⑀-subunit can exist in different conformations interacting differently with ␥-subunit (25-27). Most recently, it has been reported that the isolated ⑀...

The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD + ) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex Iassociated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern : the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (K i = 40 W WM) than with APAD + (K i = 150 W WM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.z 1999 Federation of European Biochemical Societies.

The presence of medium Pi (half-maximal concentration of 20 microM at pH 8.0) was found to be required for the prevention of the rapid decline in the rate of proton-motive force (pmf)-induced ATP hydrolysis by Fo.F1 ATP synthase in coupled vesicles derived from Paracoccus denitrificans. The initial rate of the reaction was independent of Pi. The apparent affinity of Pi for its "ATPase-protecting" site was strongly decreased with partial uncoupling of the vesicles. Pi did not reactivate ATPase when added after complete time-dependent deactivation during the enzyme turnover. Arsenate and sulfate, which was shown to compete with Pi when Fo.F1 catalyzed oxidative phosphorylation, substituted for Pi as the protectors of ATPase against the turnover-dependent deactivation. Under conditions where the enzyme turnover was not permitted (no ATP was present), Pi was not required for the pmf-induced activation of ATPase, whereas the presence of medium Pi (or sulfate) delayed the spontaneous deactivation of the enzyme which was induced by the membrane de-energization. The data are interpreted to suggest that coupled and uncoupled ATP hydrolysis catalyzed by Fo.F1 ATP synthases proceeds via different intermediates. Pi dissociates after ADP if the coupling membrane is energized (no E.ADP intermediate exists). Pi dissociates before ADP during uncoupled ATP hydrolysis, leaving the E.ADP intermediate which is transformed into the inactive ADP(Mg2+)-inhibited form of the enzyme (latent ATPase).