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A D Vinogradov

Publications and source records attributed to A D Vinogradov.

At least 19 recordsLinked to original sources
PubMed2005

Generation of superoxide-radical by the NADH:ubiquinone oxidoreductase of heart mitochondria.

Besides major NADH-, succinate-, and other substrate oxidase reactions resulting in four-electron reduction of oxygen to water, the mitochondrial respiratory chain catalyzes one-electron reduction of oxygen to superoxide radical O(2)(-.) followed by formation of hydrogen peroxide. In this paper the superoxide generation by Complex I in tightly coupled bovine heart submitochondrial particles is quantitatively characterized. The rate of superoxide formation during Deltamu(H(+))-controlled respiration with succinate depends linearly on oxygen concentration and contributes approximately 0.4% of the overall oxidase activity at saturating (0.25 mM) oxygen. The major part of one-electron oxygen reduction during succinate oxidation (approximately 80%) proceeds via Complex I at the expense of its Deltamu(H(+))-dependent reduction (reverse electron transfer). At saturating NADH the rate of O(2)(-.) formation is substantially smaller than that with succinate as the substrate. In contrast to NADH oxidase, the rate-substrate concentration dependence for the superoxide production shows a maximum at low (approximately 50 microM) concentrations of NADH. NAD+ and NADH inhibit the succinate-supported superoxide generation. Deactivation of Complex I results in almost complete loss of its NADH-ubiquinone reductase activity and in increase in NADH-dependent superoxide generation. A model is proposed according to which complex I has two redox active nucleotide binding sites. One site (F) serves as an entry for the NADH oxidation and the other one (R) serves as an exit during either the succinate-supported NAD+ reduction or superoxide generation or NADH-ferricyanide reductase reaction.

Animals↗
PubMed2003

Unidirectional effect of lauryl sulfate on the reversible NADH:ubiquinone oxidoreductase (Complex I).

Lauryl sulfate inhibits the Deltamu;(H)(+)-dependent reverse electron transfer reactions catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in coupled bovine heart submitochondrial particles and in vesicles derived from Paracoccus denitrificans. The inhibitor affects neither NADH oxidase (coupled or uncoupled) nor NADH:ferricyanide reductase and succinate oxidase activities at the concentrations that selectively prevent the succinate-supported, rotenone-sensitive NAD(+) or ferricyanide reduction. Possible uncoupling effects of the inhibitor are ruled out: in contrast to oligomycin and gramicidin, which increases and decreases the rate of the reverse electron transfer, respectively, in parallel with their coupling and uncoupling effects, lauryl sulfate does not affect the respiratory control ratio. A mechanistic model for the unidirectional effect of lauryl sulfate on the Complex I catalyzed oxidoreduction is proposed.

Animals↗
PubMed2003

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.

Adenosine Triphosphatases↗
PubMed2001

H+/2e- stoichiometry of the nadh:ubiquinone reductase reaction catalyzed by submitochondrial particles.

Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio H+/2e- (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q1 gives the value of n = 4. Thermally induced deactivation of Complex I [1, 2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q1-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q1-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.

Animals↗
PubMed2001

Respiratory complex I: structure, redox components, and possible mechanisms of energy transduction.

Structural arrangements and properties of redox components of the mitochondrial and bacterial proton-translocating NADH:quinone oxidoreductases are briefly described. A model for the mechanism of proton translocation at first coupling site, which emphasizes participation of specifically Complex I-associated ubisemiquinones, is discussed. An alternative mechanism is proposed where all redox reactions take place in a hydrophilic part of the enzyme and the free energy accumulated as conformational constraint drives the proton pump associated with the hydrophobic polypeptides.

Animals↗
PubMed2000

Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition.

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.

Alamethicin↗
PubMed2000

Steady-state and pre-steady-state kinetics of the mitochondrial F(1)F(o) ATPase: is ATP synthase a reversible molecular machine?

H(+)-ATP synthase (F(1)F(o) ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F(1), which is ideally suited for the alternating binding change mechanism, with a rotating gamma-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (P(i)) and Mg(2+) participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F(1) and F(1)F(o) demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, P(i), Mg(2+) and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F(1)F(o) ATPase is proposed.

Adenosine Diphosphate↗
PubMed1999

Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling.

The sensitivities of NADH oxidase and/or NADH-ubiquinone reductase activities of submitochondrial particles and purified complex I towards N-ethylmaleimide (NEM) and other SH-reagents were studied. Only thermally de-activated preparations IA.D. Vinogradov (1998) Biochim. Biophys. Acta 1364, 169-185] were inhibited by SH-reagents whereas the redox-pulsed, activated enzyme was resistant to the inhibitors. The pH profile of the pseudo-first order inhibition rate suggested a pKa of about 10 for the de-activation-dependent, NEM-reactive sulfhydryl group. NADH-ubiquinone reductase of activated particles treated with an excess of NEM followed by removal of the inhibitor was still capable of slow reversible active/de-active transition. When active, NEM-treated particles were de-activated and further inhibited by N-fluorescein maleimide, specific incorporation of the fluorescence label into low molecular mass polypeptide was evident. Comparison of the specific fluorescence labeling of submitochondrial particles, crude and purified complex I showed that the active/de-active state-dependent SH-group is located in a 15 kDa polypeptide (most likely in the 15 kDa IP subunit of the iron-sulfur protein-containing fraction of complex I).

Animals↗
PubMed1999

-->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q1. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t1/2 < or = 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H+ decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting -->H+/2e- stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q1 reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same -->H+/2e- stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q1-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+.

Adenosine Diphosphate Ribose↗
PubMed1999

Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase.

The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.

Catalysis↗
PubMed1999

Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.

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 I-associated 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 (Ki = 40 microM) than with APAD+ (Ki = 150 microM) 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.

Animals↗
PubMed1999

Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).

Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1 reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Ki of 1x10-5 M (pH 8.0, 25 degrees C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5x10-5 M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+ reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1 reductase activity of SMP is inhibited non-competitively by added Q1 whereas a simple competition between Q1 and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.

Animals↗
PubMed1999

Catalytic properties of mitochondrial NADH-ubiquinone reductase (Complex I).

Qualitative and quantitative characteristics of the reactions catalyzed by the most complex and least understood proton translocating unit of the mammalian respiratory chain (NADH-ubiquinone oxidoreductase, Complex I) are described for enzyme preparations differing in degree of resolution--from intact mitochondria to homogeneous small enzyme fragments. Special attention is given to the problems and pitfalls of reliable interpretation of the kinetic analysis of the enzyme activities. Detailed analysis of the problems concerning the slow active/inactive reversible enzyme transition is provided.

Catalysis↗
PubMed1999

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis.

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.

Adenosine Triphosphate↗
PubMed1999

Mitochondrial ATP synthase: fifteen years later.

Mitochondrial Fo.F1-H+-ATP synthase is the main enzyme responsible for the formation of ATP in aerobic cells. An alternating binding change mechanism is now generally accepted for the operation of the enzyme. This mechanism apparently leaves no room for the participation of nucleotides and Pi other than sequential binding to (release from) the catalytic sites. However, the kinetics of ATP hydrolysis by mitochondrial ATPase is very complex, and it is difficult to explain it in terms of the alternating binding change mechanism only. Fo.F1 catalyzes both delta muH+-dependent ATP synthesis and ATP-dependent delta muH+ generation. It is generally believed that this enzyme operates as the smallest molecular electromechanochemical reversible machine. This essay summarizes data which contradict this simple reversible mechanism and discusses a hypothesis in which different pathways are followed for ATP hydrolysis and ATP synthesis. A model for a reversible switch mechanism between ATP hydrolase and ATP synthase states of Fo. F1 is proposed.

Adenosine Triphosphate↗
PubMed1998

Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain.

Our recent experimental data on iron-sulfur clusters and semiquinones in the complex I segment of the respiratory chain is presented, focusing on the Paracoccus (P.) denitrificans and bovine heart studies. The iron-sulfur cluster N2 has attracted the attention of investigators in the research field of complex I, since the mid-point redox potential of this cluster is the highest among all clusters in complex I, and is pH dependent (60 mV/pH). It is known that this cluster is located either in the NQO6 (NuoB in E. coli/PSST in bovine heart nomenclature) or in the NQO9 (NuoI/TYKY) subunit in the amphipathic domain of complex I. Our preliminary data indicate that the cluster N2 is located in the NuoB rather than the long-advocated NuoI subunit, and may have a unique ligand structure. We previously reported spin-spin interactions between cluster N2 and two distinct species of semiquinone (designated SQNf and SQNs) associated with complex I. A parallel intensity change was observed between the SQNf (g = 2.00) signal and the N2 split g parallel signal, further supporting our proposed interaction between SQNf and N2 spins.

Animals↗