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).

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.

Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition.

The interaction of rotenone with active ('pulsed') and thermally de-activated ('resting') membrane-bound Complex I (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) as revealed by inhibition of NADH-ubiquinone- and ubiquinol-NAD+ reductase activities was studied. Ki = 1 x 10(-9) M, k(on) = 5 x 10(7) M-1 min-1 and k(off) = 0.02 min-1 (inhibitory effect of rotenone on NADH oxidation) and Ki = 2 x 10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K approximately 100) after the slow strongly temperature-dependent de-activation process has completed. Rotenone partially prevents and reverses the enzyme de-activation. About two order of magnitude difference in affinity of rotenone to the active and de-activated forms of the enzyme was demonstrated. The strong difference in rotenone sensitivity of the direct and reverse reactions can not be accounted for delta mu H(+)-dependence of rotenone binding. We propose that two rotenone-specific inhibitory sites exist in Complex I: one is involved in NADH oxidation by ubiquinone and the other is operating in ubiquinol-NAD+ reductase reaction. The affinities of rotenone for both sites are strongly altered upon the slow enzyme active/inactive transition.

Kinetics of the mitochondrial three-subunit NADH dehydrogenase interaction with hexammineruthenium(III).

The steady-state kinetics of the NADH dehydrogenase activity of the three-subunit flavo-iron-sulfur protein (FP, Type II NADH dehydrogenase) in the presence of the one-electron acceptor hexammineruthenium(III) (HAR) were studied. The maximal catalytic activities of FP with HAR as electron acceptor calculated on the basis of FMN content were found to be approximately the same for the submitochondrial particles, Complex I and purified FP. This result shows that the protein structure responsible for the primary NADH oxidation by FP is not altered during the isolation procedure and the lower (compared with Complex I) catalytic capacity of the enzyme previously reported was due to the use of inefficient electron acceptors. Simple assay procedures for NADH dehydrogenase activity with HAR as the electron acceptor are described. The maximal activity at saturating concentrations of HAR was insensitive to added guanidine, whereas at fixed concentration of the electron acceptor, guanidine stimulated oxidation of low concentrations of NADH and inhibited the reaction at saturating NADH. The inhibitory effect of guanidine was competitive with HAR. The double-reciprocal plots 1/v vs. 1/[NADH] at various HAR concentrations gave a series of straight lines intercepting on the ordinate. The plots 1/v vs. 1/[HAR] at various NADH concentrations gave a series of straight lines intercepting in the fourth quadrant. The kinetics support the mechanism of the overall reaction where NADH is oxidized by the protein-Ru(NH3)3+(6) complex in which positively charged electron acceptor is bound at the specific site close to FMN, thus stabilizing the flavosemiquinone intermediate.

An increase in the energy coupling capacity of submitochondrial particles in the presence of lanthanides.

NADH and succinate oxidase activities of inside-out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Inhibition by both oligomycin and oligomycin plus lanthanides is completely relieved by an uncoupler. The respiratory control, measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin-treated particles, is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess of EDTA. La3+ increases the extent of the aerobic energy-linked succinate-supported NAD+ reduction catalyzed by the oligomycin-treated submitochondrial particles. Lanthanides seem to be a useful tool to increase the energy coupling capacity of the submitochondrial particles.

An increase of the energy coupling capacity of submitochondrial particles by lanthanides.

NADH and succinate oxidase activities of inside-out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Both inhibition by oligomycin and oligomycin plus lanthanides are completely relieved by an uncoupler. The respiratory control measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin-treated particles is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess EDTA. La3+ increases the extent of the aerobic energy-linked succinate-supported NAD+ reduction catalyzed by the oligomycin-treated submitochondrial particles. Lanthanides seem to be useful tools to increase the energy coupling capacity of the submitochondrial particles.

Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction.

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase.

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.

Pyridoxal phosphate-induced dissociation of the succinate: ubiquinone reductase.

Treatment of the soluble ubiquinone-deficient succinate: ubiquinone reductase with pyridoxal phosphate results in the inhibition of the carboxin-sensitive ubiquinone-reductase activity of the enzyme. The inactivation is prevented by the soluble homolog of ubiquinone (Q2) but is insensitive to the dicarboxylates interacting with the substrate binding site of succinate dehydrogenase. The reactivity of the pyridoxal phosphate-inhibited enzyme with different electron acceptors suggests that the observed inhibition is due to the dissociation of succinate dehydrogenase from the enzyme complex. The soluble succinate dehydrogenase was recovered in the supernatant after treatment of the insoluble succinate: ubiquinone reductase with pyridoxal phosphate. The data obtained strongly suggest the participation of amino groups in the interaction between succinate dehydrogenase and the ubiquinone reactivity conferring peptide within the complex.

Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components.

1. A protein fraction containing three polypeptides (the major one with Mr < 13 000) was isolated by means of Triton X-100 extraction of submitochondrial particles specifically treated to remove succinate dehydrogenase. 2. The mixing of the protein fraction with the soluble reconstitutively active succinate dehydrogenase results in formation of highly active succinate-DCIP reductase which is sensitive to thenoyltrifluoroacetone or carboxin. 3. The maximal turnover number of succinate dehydrogenase in the succinate-DCIP reductase reaction revealed in the presence of a saturating amount of the protein fraction is slightly higher than that measured with phenazine methosulfate as artificial electron acceptor. 4. The protein fraction greatly increases the stability of soluble succinate dehydrogenase under aerobic conditions. 5. The titration of soluble succinate dehydrogenase by the protein fraction shows that smaller amounts of the protein fraction are required to block the reduction of ferrycyanide by Hipip center than that required to reveal the maximal catalytic capacity of the enzyme. 6. The apparent Km of the reconstituted system for DCIP depends on the amount of protein fraction; the more protein fraction added to the enzyme, the lower the Km value obtained. 7. A comparison of different reconstituted succinate-ubiquinone reductases described in the literature is presented and the possible arrangement of the native and reconstituted succinate-ubiquinone region of the respiratory chain is discussed.

[Reconstitution of succinate-ubiquinone reductase of the respiratory chain of mitochondria]. / Rekonstruktsiia suktsinat-ubikhinon-reduktaznogo uchastka dykhatel'noi tsepi mitokhondrii.

A soluble protein fraction, which confers the reactivity of soluble succinate dehydrogenase towards ubiquinone, was isolated from beef heart mitochondria. This fraction contains three polypeptides as revealed by SDS-electrophoresis; the major peptide (about 80% of protein) has a molecular weight less than 13 000. Several properties of the reconstituted succinate-ubiquinone reductase, i. e. the turnover number of succinate dehydrogenase inhibitor sensitivity, stability and reactivity towards artificial electron acceptors were found to be identical to those of the native succinate-ubiquinone region of the respiratory chain. A model of the minimal functionally active structure capable of reduction of ubiquinone by succinate is proposed.

Studies on the succinate dehydrogenating system. I. Kinetics of the succinate dehydrogenase interaction with a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine.

1. The activities of the soluble reconstitutively active succinate dehydrogenase (EC 1.3.99.1) measured with three artificial electron acceptors, e.g. ferricyanide, phenazine methosulfate and free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (WB), have been compared. The values estimated by extrapolation to infinite acceptor concentration using double reciprocal plots 1/v versus 1/[acceptor] are nearly the same for ferricyanide and phenazine methosulfate and about twice as high for the WB. 2. The double reciprocal plots 1/v versus 1/[succinate] in the presence of malonate at various concentrations of WB give a series of straight lines intercepting in the third quadrant. The data support the mechanism of the overall reaction, in which the reduced enzyme is oxidized by WB before dissociation of the enzyme-product complex. 3. The dependence of the rate of the overall reaction on WB concentration shows that only one kinetically significant redox site of the soluble succinate dehydrogenase is involved in the reduction of WB. 4. Studies of the change of V and Km values during aerobic inactivation of the soluble enzyme suggest that only 'the low Km ferricyanide reactive site' (Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys, Res. Commun. 65, 1264--1269) is involved in reoxidation of the reduced enzyme by WB. 5. The pH dependence of V for the succinate-WB reductase reaction shows that the group of the enzyme with the pKa value of 6.7 at 22 degrees C is responsible for the reduction of dehydrogenase in the enzyme-substrate complex. 6. When WB interacts with the succinate-ubiquinone region of the respiratory chain, the double reciprocal plot 1/v versus 1/[WB] gives a straight line. The thenoyltrifluoroacetone inhibition of succinate-ubiquinone reductase or extraction of ubiquinone alter the 1/v versus 1/[WB] plots for the curves with a positive initial slope intercepting the ordinate at the same V as in the native particles. The data support the mechanism of succinate-ubiquinone reduction, in which no positive modulation of succinate dehydrogenase by ubiquinone exist in the membrane.

Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase.

Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by N-ethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation of alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a site-directed inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pKa value for the enzyme sulfhydryl group equal to 7.0 at 22 degrees C in 50 mM Tris-sufate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation, including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.

[Kinetic and structural characteristics of succinate dehydrogenase components reacting with natural and artificial electron acceptors]. / Kineticheskie i strukturnye kharakteristiki komponentov suktsinatdegidrogenazy, reagiruiushchikh s estestvennym i iskusstvennymi aktseptorami élektronov

A new catalitic activity of soluble succinate dehydrogenase, i.e. the reduction of low (20-200 muM) concentration of ferricyanide in the presence of succinate is described. The apparent Km value for the acceptor is about 200 muM. The turnover numbers of the enzyme measured in this reaction, with PMS as an electron acceptor and in the system reconstituted from soluble enzyme and alkali-treated submitochondrial particles (succinate oxidase) are found to be almost the same. The new succinate. ferricyanide reductase activity is very sensitive to oxygen, high (3 mM) ferricyanide concentration and mercaptide-forming agents. When the enzyme is stored under aerobic conditions the loss of this activity occurs according to the first-order kinetics with the same rate constants as the reconstitutive activity decreases. The rate constants both for ferricyanide reductase and reconstitution decay do not depend on pH within the range of 6,5--7,5 (k = 8.10(-2) min-1) and increase dramatically at pH 8,5 (K = 4.10(-1) MIN-1). When these two activities are lost after oxygen exposure the PMS-reductase fall down to about 50% of its original activity. The new ferricyanide reductase is found only in the soluble preparation of the enzyme succinate: cytochrome c reductase, succinate dehydrogenase of submitochondrial particles and reconstituted succinate oxidase do not interact with low concentrations of ferricyanide. The treatment of the enzyme after inactivation by oxygen exposure with sulfide ion--iron--mercaptoethanol mixture followed by Sephadex filtration completely restores the original reconstitutive, ferricyanide and PMS reductase activities. The hypothesis is suggested that succinate dehydrogenase contains at least two red-ox centers reacting with electron acceptors. The first one is located in hydrophylic environment (mitochondrial matrix) being accessible for high concentrations of ferricyanide. The second one (iron--sulfur complex, Hipip-type) is responsible for ferricyanide reductase activity described, being located intramembraneously and involved in the electron transfer between dehydrogenase and the rest of the respiratory chain.

[Reaction ability and alkylation kinetics of sulfhydride groups of soluble succinate dehydrogenase]. / Reaktsionnosposobnost' i kinetika alkilirovaniia sul'fgidril'nykh grupp rastvorimoi suktsinatdegidrogenazy

Inhibition kinetics of succinate--an acceptor of oxidoreductase activity of soluble succinate dehydrogenase by N-ethylmaleimide is studied. The alkylation reaction is described by the kinetic equation of the first order, its stechiometric coefficient being 1. The binding of enzyme sulphhydride groups by p-chloromercuriumbenzoate blocks the enzyme alkylation and its inhibition by oxaloacetate. Succinate protects succinate dehydrogenase from the inhibitory effect of N-ethylmaleimide. The reaction of the enzyme with an alkylating agent in the presence of different substrate concentrations corresponds kinetically to the model, according to which a sulphhydride group acts in the active site of the enzyme. pKa of this group is 7.0 at 20degreesC. The dependency of the maximal substrate oxidation reaction rate and that of the enzyme alkylation rate on pH coinside at the pH range 5.8--7.8. The presence of anions in the alkylation medium decreases the reaction ability of the active site with respect to N-ethylmaleimide. A mechanism of the initial stage of succinate oxidation with the cooperation of the sulphhydride group of the enzyme active site is postulated.

[Study of the fractional composition of protein substances in gluten by the gel-chromatographic method]. / Izuchenie fraktsionnogo sostava belkovykh veshchestv kleikoviny metodom gel'-khromatografii

The fractional composition of protein substances of the gluten was studied by gel chromatography. The gluten proteins from the mormal wheat flour and eurygaster damaged wheat flour were compared. The content of the high molicular weight fraction was measured for the gluten of weak and normal flour. The fractional composition of gliadin from the normal and weak flour was investigated. The molecular weight of gliadin from eurygaster damaged grain was shown to change during autolysis.