Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q₂) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q₂ (DQ) -Q₂H₂ (DQH₂)-NAD⁺ in contrast to the order determined using Q₁ (Q₁-NADH-NAD⁺-Q₁H₂) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11–19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q₂ and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q₁ (one isoprenoid unit) is too short to induce the conformational change.     

The second coenzyme Q1 binding site of bovine heart NADH: coenzyme Q oxidoreductase

The rotenone sensitivity of bovine heart NADH: coenzyme Q oxidoreductase (Complex I) depends significantly on coenzyme Q₁ concentration. The rotenone-insensitive Complex I reaction in Q₁ concentration range above 300 M indicates an ordered sequential mechanism with Q₁ and reduced Q₁ (Q₁H₂) as the initial substrate to bind to the enzyme and the last product to be released from the enzyme product complex, respectively. This is the case in the rotenone-sensitive reaction although both K _m and V _max values of the rotenone-insensitive reaction for Q₁ are significantly higher than those of the rotenone-sensitive reaction (Nakashima et al., 2002, J. Bioenerg. Biomemb. 34, 11–19). This rigorous control mechanism between the nucleotide and ubiquinone binding sites strongly suggests that the rotenone-insensitive reaction is also physiologically relevant.     

Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III₂-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.     

The kinetics of NADH oxidation by complex I of isolated plant mitochondria

The oxidation of matrix NADH in the presence and absence of rotenone was investigated in submitochondrial particles prepared from purified beetroot ( Beta vulgaris L.) mitochondria. The submitochondrial particles oxidised NADH using oxygen and artificial electron acceptors such as ferricyanide (FeCN) and short-chain analogues of ubiquinone(UQ)-10, although the NADH-FeCN reductase activity was not inhibited by rotenone. NADH-oxygen reductase activity in the presence and absence of rotenone displayed different affinities for NADH (145 ± 37 and 24 ± 9 μ M , respectively). However, in the presence of 0.15 m M UQ-1 where any contribution from non-specific sites of UQ-reduction was minimal, the rotenone-insensitive oxygen uptake was stimulated dramatically and the K_m(NADH) decreased from 167 ± 55 μ M to 11 ± 1 μ M ; a value close to that determined for the total oxygen uptake which itself was virtually unaffected by the addition of UO-1 [K_m(NADH) of 13 ± 3 μ M ).
The similar affinity of NADH-oxygen reductase for NADH when UQ-1 was present in both the presence and absence of rotenone, suggested that there may be only one NADH binding site involved in the two activities. A quantitative two-stage model for Complex I is postulated with one NADH binding site and two sites of UQ-reduction (one of which is insensitive to rotenone) with a common intermediate 'P' whose level of reduction can influence the NADH binding site. The poor affinity that rotenone-insensitive NADH-oxygen reductase activity displayed for NADH results from a limitation on the interaction of its UQ-reduction site with UQ-10 in the membrane; possibly from a low concentration of UQ-10 around this site or from steric hindrance restricting the access of UQ-10 to this reduction site.     

Essentiality of coenzyme Q for the oxidation of -glycerophosphate by pig brain mitochondria

Serial extraction of lyophilized pig brain mitochondria with cold pentane resulted in complete loss of α-glycerophosphate oxidase activity. On titration with coenzyme Q₁₀ the activity was fully recovered. On comparing the decline of α-glycerophosphate, NADH, and succinoxidase activities during serial extraction with pentane, α-glycerophosphate oxidation was always the first to be lost. Extraction of coenzyme Q₁₀ from lyophilized brain mitochondria with pentane does not affect the activities of α-glycerophosphate or NADH dehydrogenase, but succinate dehydrogenase is partially inactivated. Reversible inactivation of the α-glycerophosphate oxidase system on depletion of the coenzyme Q content is taken as evidence that coenzyme Q is an obligatory component of this system. In accord with the conclusion that coenzyme Q is probably the physiological oxidant of α-glycerophosphate dehydrogenase, in antimycin-treated brain mitochondria α-glycerophosphate causes full activation of endogenous succinate dehydrogenase, in analogy to the previously observed activation by NAD-linked substrates in liver and heart mitochondria and by NADH in submitochondrial particles.     

Deficit of coenzyme Q in heart and liver mitochondria of rats with streptozotocin-induced diabetes

Mitochondrial dysfunction and oxidative stress participate in the development of diabetic complications, however, the mechanisms of their origin are not entirely clear. Coenzyme Q has an important function in mitochondrial bioenergetics and is also a powerful antioxidant. Coenzyme Q (CoQ) regenerates alpha-tocopherol to its active form and prevents atherogenesis by protecting low-density lipoproteins against oxidation. The aim of this study was to ascertain whether the experimentally induced diabetes mellitus is associated with changes in the content of endogenous antioxidants (alpha-tocopherol, coenzymes Q9 and Q10) and in the intensity of lipoperoxidation. These biochemical parameters were investigated in the blood and in the isolated heart and liver mitochondria. Diabetes was induced in male Wistar rats by a single intravenous injection of streptozotocin (45 mg x kg(-1)), insulin was administered once a day for 8 weeks (6 U x kg(-1)). The concentrations of glucose, cholesterol, alpha-tocopherol and CoQ homologues in the blood of the diabetic rats were increased. The CoQ9/cholesterol ratio was reduced. In heart and liver mitochondria of the diabetic rats we found an increased concentration of alpha-tocopherol, however, the concentrations of CoQ9 and CoQ10 were decreased. The formation of malondialdehyde was enhanced in the plasma and heart mitochondria. The results have demonstrated that experimental diabetes is associated with increased lipoperoxidation, in spite of the increased blood concentrations of antioxidants alpha-tocopherol and CoQ. These changes may be associated with disturbances of lipid metabolism in diabetic rats. An important finding is that heart and liver mitochondria from the diabetic rats contain less CoQ9 and CoQ10 in comparison with the controls. We suppose that the deficit of coenzyme Q can participate in disturbances of mitochondrial energy metabolism of diabetic animals.     

Virgin olive oil and coenzyme Q10 protect heart mitochondria from peroxidative damage during aging.

The mitochondrial theory of aging suggests that this phenomenon is the consequence of random somatic mutations in mitochondrial DNA, induced by long-term exposure to free radical attack. There are two potential dietary means of delaying the effects of free radicals on cellular aging, i.e., enrichment of mitochondrial membranes with monounsaturated fatty acids and supplementation with antioxidants. We have performed a preliminary study on male rats, 6 or 12 month old, fed with diets differing in the nature of the fat (virgin olive oil or sunflower oil) and/or with antioxidant supplementation (coenzyme Q10), analysing hydroperoxide and coenzyme Q9 and Q10 in heart mitochondria. Preliminary results allow us to conclude that the CoQ10 dietetic supplementation as well as the enrichment of the cellular membranes with monounsaturated fatty acids, successfully protect mitochondrial membranes from aged rats against the free radical insult.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

The locus of inhibition of NADH oxidation by benzothiadiazoles in beef heart submitochondrial particles

6-Chloro-1,2,3-benzothiadiazole (6-Cl-BTD) is an effective inhibitor of NADH oxidase (site I) but not of succinate oxidase in beef heart submitochondrial particles. For NADH oxidase activity maximal inhibition (80-85%) was achieved at 0.75mM 6-Cl-BTD. A similar level of inhibition was also observed (half maximal inhibitory concentration 0.5mM) towards NADH-duroquinone reductase; NADH-juglone reductase was slightly inhibited (23%) at 0.5mM 6-Cl-BTD while NADH-ferricyanide reductase was unaffected. The data suggest that 6-Cl-BTD interacts with an electron transport site on the oxygen side of NADH dehydrogenase and inhibitory studies with 6-Cl-BTD and rotenone indicate that it might correspond with one of the two sites affected by rotenone. The substituted 1,2,3-benzothiadiazoles (BTDs) are perhaps best known for their activity as inhibitors of cytochrome P-450-mediated mixed-function oxidation (MFO). In vitro, the BTDs are potent inhibitors of MFO activities in microsomes from mammalian liver and insect tissues and they have been demonstrated to inhibit aminopyrine metabolism in perfused rat liver. In vivo, they reportedly prolong hexobarbital sleeping time in mice, inhibit the irreversible binding of labeled trichloro-ethylene to microsomal protein and effectively enhance the toxicity (synergize) of pyrethrin, organophosphorus-containing and carbamate insecticides to insects.     

Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity

1. Enzymatic reduction of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) by NADH can be used in an assay procedure for the NADH dehydrogenase. The reduction of this quinone occurs in the region of the electron transport system between the primary dehydrogenase and the cytochrome system as defined by the almost complete loss of reductase activity following piericidin A treatment.

2. Duroquinone reduction can be distinguished from ubiquinone 2 reduction by the marked inhibition of the former following phospholipase C, poly- -lysine, or chloroquine diphosphate treatment. In addition, duroquinone reduction requires the presence of endogenous ubiquinone 10 specifically whereas ubiquinone 2 reduction does not require the presence of endogenous quinone. These observations are consistent with the nonequivalency of the reduction sites of duroquinone and ubiquinone 2.

3. Duroquinol can be utilized as an electron donor for the energy-linked reduction, of NAD⁺. Duroquinol reduction of NAD⁺ is dependent upon the presence of ATP, is inhibited by oligomycin, carbonyl cyanide p-trifluoro methoxyphenylhydrazone and piericidin A, and is not inhibited by antimycin A at levels which inhibit electron transport.

4. Duroquinone reduction as well as ubiquinone 2 reduction are inhibited almost completely by phospholipase A, p-chloromercuribenzoate, o-phenanthroline, and Triton X100 treatments.     

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ($\text{w}\text{v}$) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.     

Properties of succinate oxidation in tomato fruit mitochondria

Mitochondria from tomato fruit (Lycopersicon esculentum Mill.) exhibited a respiratory control ratio of 2.5 and an ADP:O ratio of 1.3 for succinate oxidation for 24 hours after isolation. They also showed a delay in response to the first addition of ADP. The addition of ATP and ADP before succinate eliminated the delayed response as did chelation of endogenous cations with ethylenediaminetetraacetic acid. The addition of ATP after succinate resulted in a longer delay in response than that obtained with ADP. Exogenous oxaloacetate in low concentration inhibited respiration in states 3 and 4 with succinate and resulted in delayed response to ADP. The function of adenine nucleotide during the delay in response may be to promote the metabolism of oxaloacetate or to decrease the affinity of oxaloacetate to its site of inhibition.