Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state

The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.     

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c reductases

The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.     

Dicyclohexylcarbodiimide inhibition of succinate- and ubiquinol-cytochrome c reductase in beef heart mitochondria

We have found that dicyclohexylcarbodiimide (DCCD) inhibits both the succinate-cytochrome c and the ubiquinol-cytochrome c reductases in cytochrome c-depleted mitochondria. On the other hand the succinate-ubiquinone reductase is not decreased at the same levels of the inhibitor. The inhibition curve of DCCD results sigmoidal for succinate-cytochrome c reductase, whereas it is hyperbolic for the ubiquinol-1-cytochrome c reductase, with also a lower apparent KI. The inhibition appears dependent both on the time of preincubation and on the mitochondrial concentration. The apparent Km for ubiquinol-1 is increased and the maximal velocity of ubiquinol-cytochrome c reductase is decreased by DCCD. The effects do not appear to be caused by unspecific modification of the physicochemical state of the bc1 region of the respiratory chain. The results therefore suggest the presence of a DCCD-sensitive electron transfer step in the redox pathways from ubiquinol to cytochrome c.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

Restoration of pool function type behavior to succinate dehydrogenase having latent reconstitutive activity in ubiquinol-cytochrome c reductase complex

Mitochondrial ubiquinol-cytochrome $\text{c}$ reductase complex contains small amounts of succinate dehydrogenase. Estimates from electrophoresis indicate there is one dehydrogenase per eight complexes. This dehydrogenase transfers electrons to the $\text{b}$-$\text{c}{}_1$ complex poorly, as judged by low succinate-ubiquinone and succinate-cytochrome $\text{c}$ reductase activities. Electron transfer to the $\text{b}$-$\text{c}{}_1$ complex is restored by reconstitution of the complex with phospholipid. This phospholipid dependent restoration of electron transfer indicates that either reconstitutive activity of the dehydrogenase is preserved under conditions where electron transfer is absent, or that addition of phospholipid allows one dehydrogenase to transfer electrons to multiple $\text{b}$-$\text{c}{}_1$ complexes.     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

The existence of an alternative electron-transfer pathway to the periplasmic nitrite reductase (cytochrome cd 1) in Paracoccus denitrificans

Exposure of aerobically-grown wild-type cells of Paracoccus denitrificans to a decreased aeration caused parallel increases in both PMS/ascorbate and succinate-linked activities of nitrite reductase. By contrast, the expression of the succinate-linked activity was considerably delayed in an insertion mutant specifically lacking the periplasmic 15 kDa cytochrome c-550. In this case the observed activity followed very closely the content of a 40 kDa cytochrome c. A subcellular fraction enriched in a haemoprotein of a similar apparent molecular weight showed the activity of cytochrome c peroxidase and was able to restore the antimycin-sensitive electron transport from membrane vesicles to nitrite reductase. It is concluded that P. denitrificans possesses an alternative nitrite-reducing pathway involving the 40 kDa cytochrome c instead of cytochrome c-550. This pathway branches from the respiratory chain after the cytochrome bc ₁ segment.Abbreviations PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulphate     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N′-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c₁, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c₁ complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c₁ complex. The binding of [¹⁴C]DCCD to the isolated b-c₁ complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c₁ complex are discussed.     

Role of the photosynthetic electron transfer chain in electrogenic activity of cyanobacteria

Certain anaerobic bacteria, termed electrogens, produce an electric current when electrons from oxidized organic molecules are deposited to extracellular metal oxide acceptors. In these heterotrophic “metal breathers”, the respiratory electron transport chain (R-ETC) works in concert with membrane-bound cytochrome oxidases to transfer electrons to the extracellular acceptors. The diversity of bacteria able to generate an electric current appears more widespread than previously thought, and aerobic phototrophs, including cyanobacteria, possess electrogenic activity. However, unlike heterotrophs, cyanobacteria electrogenic activity is light dependent, which suggests that a novel pathway could exist. To elucidate the electrogenic mechanism of cyanobacteria, the current studies used site-specific inhibitors to target components of the photosynthetic electron transport chain (P-ETC) and cytochrome oxidases. Here, we show that (1) P-ETC and, particularly, water photolysed by photosystem II (PSII) is the source of electrons discharged to the environment by illuminated cyanobacteria, and (2) water-derived electrons are transmitted from PSII to extracellular electron acceptors via plastoquinone and cytochrome bd quinol oxidase. Two cyanobacterial genera (Lyngbya and Nostoc) displayed very similar electrogenic responses when treated with P-ETC site-specific inhibitors, suggesting a conserved electrogenic pathway. We propose that in cyanobacteria, electrogenic activity may represent a form of overflow metabolism to protect cells under high-intensity light. This study offers insight into electron transfer between phototrophic microorganisms and the environment and expands our knowledge into biologically based mechanisms for harnessing solar energy.     

Enzymatic activity mastered by altering metal coordination spheres

Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd ₁ nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Rapid Nonsynonymous Evolution of the Iron-Sulfur Protein in Anthropoid Primates

Cytochrome c (CYC) and 9 of the 13 subunits of cytochrome c oxidase (complex IV; COX) were previously shown to have accelerated rates of nonsynonymous substitution in anthropoid primates. Cytochrome b, the mtDNA encoded subunit of ubiquinol-cytochrome c reductase (complex III), also showed an accelerated nonsynonymous substitution rate in anthropoid primates but rate information about the nuclear encoded subunits of complex III has been lacking.We now report that phylogenetic and relative rates analysis of a nuclear encoded catalytically active subunit of complex III, the ironsulfur protein (ISP), shows an accelerated rate of amino acid replacement similar to cytochrome b. Because both ISP and subunit 9, whose function is not directly related to electron transport, are produced by cleavage into two subunits of the initial translation product of a single gene, it is probable that these two subunits of complex III have essentially identical underlying rates of mutation. Nevertheless, we find that the catalytically active ISP has an accelerated rate of amino acid replacement in anthropoid primates whereas the catalytically inactive subunit 9 does not.     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

Kinetic indication for multiple sites of ubiquinol-1 interaction in ubiquinol-cytochrome c reductase in bovine heart mitochondria

We have assayed the ubiquinol-cytochrome c reductase activity either in situ or in different mitochondrial fractions, including the isolated bc₁ complex, employing ubiquinol-1 and exogenous cytochrome c as substrates. A clear biphasic behavior of both the time courses and the initial rates of cytochrome c reduction have been observed. Two K_m values have been found, one of 1–7 × 10^?6m ubiquinol-1, and another varying from 0.6 to 4.6 × 10^?5m ubiquinol-1, depending on the cytochrome c concentration and the type of mitochondrial fraction used. Either the kinetic phase with the lower K_m or the kinetic phase with the higher K_m exhibits an almost identical antimycin sensitivity. We have also monitored the rapid reduction of endogenous b cytochromes in the presence of antimycin, and the initial rates are again biphasic as a function of ubiquinol-1 concentration. These findings indicate that the steps conferring the biphasic kinetics to the ubiquinol-cytochrome c reductase activity involve the redox equilibria between exogenous ubiquinol-1 and the b cytochromes, and suggest that two redox pathways may be present in the electron transfer from ubiquinol to cytochrome c through the bc₁ segment of the mammalian respiratory chain.     

Towards the mechanisms involved in the antioxidant action of Mn<Superscript>III</Superscript> [<Emphasis Type="Italic">meso</Emphasis>-tetrakis(4-<Emphasis Type="Italic">N</Emphasis>-methyl pyridinium) porphyrin] in mitochondria

Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, Mn^IIITMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of Mn^IIITMPyP. SOD and cytochrome c antioxidant activities were also investigated. Mn^IITMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, Mn^IIITMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for Mn^IIITMPyP and do not exclude cytochrome c-like activity. However, considering that para Mn^IIITMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the Mn^IITMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of Mn^IITMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of Mn^IIITMPyP, whose efficiency is dependent on the NADPH/GSH content in cells.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.