The effects of lipid phase transitions on the interaction of mitochondrial NADH--ubiquinone oxidoreductase with ubiquinol--cytochrome c oxidoreductase.

1. The endogenous phosphatidylcholine and phosphatidylethanolamine of Complexes I and III from bovine heart mitochondria may be completely replaced with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine with at least partial retention of activity. 2. The lipid-replaced enzymes associate in 1:1 molar ratio to give a Complex I--III unit catalysing NADH-cytochrome c oxidoreductase activity. 3. On increasing the concentration of ubiquinone-10 and the synthetic phospholipid, the lipid-replaced Complexes appear to operate independently of each other as in the natural membrane. Thus the lipid-replaced enzymes associate in exactly the same ways as the enzymes containing natural phospholipids. 4. Arrhenius plots of NADH--cytochrome c oxidoreductase activity reconstituted from lipid-replaced Complexes I and III exhibit changes in slope at 24 degrees C. When the concentrations of phospholipid and ubiquinone-10 are increased, the Arrhenius plots show discontinuities at 24 degrees C as well as changes in slope. 5. The kinetics of cytochrome b reduction by NADH were measured in mixtures containing 2 mol of Complex III/mol of Complex I. When the enzymes contained natural phospholipids. the reduction kinetics were biphasic. When the enzymes had been supplemented with further phospholipid and ubiquinone-10 the kinetics were monophasic. When lipid-replaced enzymes were supplemented with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine and ubiquinone-10, reduction of cytochrome b was monophasic above the phase-transition temperature of the lipid but biphasic below it. 6. These findings are interpreted in terms of the model for the interaction of Complexes in the natural membrane proposed by Heron, Ragan & Trum-power [(1978) Biochem. J. 174, 791--800].     

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association.

1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.     

The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase

NADH-ubiquinone oxidoreductase (Complex I) can be recombined with ubiquinol-cytochrome c oxidoreductase (Complex III) to reconstitute NADH-cytochrome c oxidoreductase. Two modes of interaction have been found. In one, the Complexes interact stoichiometrically in one to one molar ratios to give a binary Complex I-III unit. In the other, the kinetics of NADH-cytochrome c oxidoreductase are characteristic of 'Q-pool' behaviour seen in intact mitochondria and submitochondrial particles in which the Complexes need not interact directly but can do so via a pool of mobile ubiquinone. Stoichiometric behaviour is found when only boundary layer or annular lipid is present or the lipid is in the gel phase. The lipid is immobile on the ESR time scale and protein rotational diffusion, measured by saturation transfer ESR, is very slow. Q-pool behaviour is found when mobile extra-annular lipid phase is also present. Protein rotational diffusion is rapid and characteristic of a fully disaggregated state. We have also used freeze-fracture electron microscopy of reconstituted NADH-cytochrome c oxidoreductase to monitor protein aggregation and lateral phase separation of lipids and proteins under various conditions. We discuss our findings in relation to models for lateral interactions between respiratory chain enzymes.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour.

1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358--368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.     

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.     

Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide–adenine dinucleotide–ubiquinone oxidoreductase from bovine heart mitochondria

1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.     

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.     

A cytochrome b/c1 complex with ubiquinol--cytochrome c2 oxidoreductase activity from Rhodopseudomonas sphaeroides GA

A cytochrome b/c1 complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-10 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol--cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.     

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.     

Spatial organization of redox active centers in the bovine heart ubiquinol-cytochrome c oxidoreductase

We have examined the spatial organization of the redox active centers in the Site II segment of the bovine heart respiratory chain by using reconstituted proteoliposomes of ubiquinol-cytochrome c oxidoreductase (Complex III or cytochrome bc1 complex) and EPR techniques. 1) Mutual spin-spin interactions between intrinsic redox active centers were detected. The spin relaxation of the Rieske iron-sulfur cluster was enhanced by the paramagnetic cytochrome c1 and b566 hemes but not by cytochrome b562. 2) Relative distances of the individual redox active centers to the P-side and N-side surfaces of the reconstituted Complex III proteoliposome were measured by our paramagnetic probe method (Blum, H., Bowyer, J. R., Cusanovich, M. A., Waring, A. J., and Ohnishi, T. (1983) Biochim. Biophys. Acta 748, 418-428). The cytochrome b562 heme was shown to be close to the middle of the phospholipid bilayer, while the Rieske iron-sulfur cluster and cytochrome b566 heme were assigned to be near the P-side surface level of the membrane. This probe method is a low resolution technique from the structural viewpoint; however, it can provide direct and reliable assignment of the topographical locations of redox active centers within the membrane. This is the first direct demonstration of the transmembranous location of the two cytochrome b hemes, although electron transfer between these two hemes crosses only half of the membrane thickness. Our data support the assignment of transmembranous distribution of the redox active centers based on electrochromic measurements (Robertson, D.E., and Dutton, P.L. (1988) Biochim, Biophys. Acta 935, 273-291). The implication of these results on the mechanism of Site II energy coupling is discussed.     

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.     

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.     

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.     

The recognition of a special ubiquinone functionally central in the ubiquinone-cytochrome b-c2 oxidoreductase.

Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.     

Reactivity with ubiquinone of quinoprotein D-glucose dehydrogenase from Gluconobacter suboxydans

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent oxidoreductase linked to the respiratory chain of a wide variety of bacteria. There is a controversy as to whether the glucose dehydrogenase is linked to the respiratory chain via ubiquinone or cytochrome b. In this study, it was shown that the glucose dehydrogenase of Gluconobacter suboxydans has the ability to react directly with ubiquinone. The enzyme purified from the membranes of G. suboxydans was able to react with ubiquinone homologues such as ubiquinone-1, -2, or -6 in detergent solution. Furthermore, in order to demonstrate the reactivity of the enzyme with native ubiquinone, ubiquinone-10, in the native membranous environment, the dehydrogenase was reconstituted together with cytochrome o, the terminal oxidase of the respiratory chain, into a phospholipid bilayer containing ubiquinone-10. The proteoliposomes thus reconstituted exhibited a reasonable glucose oxidase activity, the electron transfer reaction of which was able to generate a membrane potential and a pH gradient. Thus, D-glucose dehydrogenase of G. suboxydans has been demonstrated to donate electrons directly to ubiquinone in the respiratory chain.     

Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.     

One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556

Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms. The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme. The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification. In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity. This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon. The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule. The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate.     

Interaction between cytochrome c and ubiquinone-cytochrome c oxidoreductase: A study with water-soluble carbodiimides

The role of carboxyl groups on the interaction between ubiquinone-cytochrome c oxidoreductase (Complex III) and cytochrome c has been probed using the two water-soluble carbodiimides EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and CMC (1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide metho-p-toluensulphonate). The results suggest that: 1) carboxyl groups present on both cytochrome c₁ and subunit VIII are modified. Some of these residues are shielded by cytochrome c. 2) The enzyme activity decreases during the carbodiimide treatment and the extent of inhibition is larger in the presence of cytochrome c. 3) Cytochrome c, equimolar with the enzyme, cross-links to cytochrome c₁ and subunit VIII via the carbodiimide-activated carboxyl groups. The two subunits appear to be in contact in the isolated enzyme.     

Molecular study of the rat liver NADH: Cytochrome c oxidoreductase complex during development and aging

The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidorectuctase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined.An increase in Complex I-III activity was detected during development (142 ± 10 vs. 447 ± 23 mol cyt. c/mg/min, p < 0.001) ang ageing (447 ± 23 vs. 713 ± 45 mol cyt. c/mg//min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity.