The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

The absence of coupling between the formation of membrane potential and electron transfer via the alternative pathway of cyanide-resistant mitochondria

The estimation of membrane potential of cyanide-resistant mitochondria of Candida lipolytica yeast was carried out using positively charged dye phenosafranine. The electron transfer via alternative pathway of cynide resistant mitochondria was shown not to be coupled with the formation of potential on membrane mitochondria.     

Cause of the activity loss of the alternative pathway of electron transport in cyanide-resistant mitochondria of the yeast Candida lipolytica

The cause of the activity loss of alternative pathway of electron transport in mitochondria of the yeast Candida lipolytica has been investigated. Incubation of cyanide-resistant mitochondria at 25 degrees was shown to cause the loss by mitochondria of their ability to oxidize substrates in the presence of 1 mM cyanide. This suggests that in the course of incubation the alternative pathway loses its activity. Repeated washing of mitochondria with a solution containing 2,5 mM EDTA inhibits, while Ca2+, Mn2+, Cu2+ and Zn2+ (but not Sr2+) enhance the process of the activity loss of the alternative pathway. The loss of the cyanide-resistant respiration is also observed during incubation of mitochondria in the presence of phospholipases A, C and D or lysolecithin. In all cases studied the reactivation of the cyanide-resistant respiration of mitochondria is attained by addition of azolectin. The loss of cyanide-resistant respiration is accompanied by the activity reduction of the main respiratory chain, which is restored by addition of cytochrome c and Mg2+. These data indicate that the activity loss of the alternative pathway is not related to inactivation of any components in the alternative pathway itself or in the main respiratory chain. The most probable cause of the activity loss in the destruction of reducing equivalents in the alternative pathway of a donor as a result of a break of the structural entity of the internal membrane of mitochondria due to the detersive action of the phospholipid lysoforms produced either by endogenic or exogenic phospholipases.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.     

On energy conservation and transfer in mitochondria

Structural and functional features of energy conservation and transfer in mitochondria have been examined in the light of recent developments, and hypothetical schemes for energy coupling and transfer have been presented.Supported by USPHS grant AM-08126 to Y.H. and San Diego County Heart Association Grant-in-Aid 97-71 to W.G.H.Recipient of USPHS career Development Award 1-K4-GM38291.     

The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria

In addition to possessing multiple NAD(P)H dehydrogenases, most plant mitochondria contain a cyanide- and antimycin-insensitive alternative terminal oxidase. Although the general characteristics of this terminal oxidase have been known for a considerable number of years, the mechanism by which it is regulated is unclear and until recently there has been relatively little information on its exact nature. In the past 5 years, however, the application of molecular and novel voltametric techniques has advanced our understanding of this oxidase considerably. In this article, we review briefly current understanding on the structure and function of the multiple NADH dehydrogenases and consider, in detail, the nature and regulation of the alternative oxidase. We derive a kinetic model for electron transfer through the ubiquinone pool based on a proposed model for the reduction of the oxidase by quinol and show how this can account for deviations from Q-pool behaviour. We review information on the attempts to isolate and characterise the oxidase and finally consider the molecular aspects of the expression of the alternative oxidase.     

Discrimination between duroquinol oxidase activity and the terminal oxidation step of the cyanide-resistant electron transport pathway of plant mitochondria

Comparison of the cyanide-resistant duroquinol oxidase activity of sub-mitochondrial particles from Arum maculatum L. with their ability to carry out a cyanide-resistant oxidation of NADH and succinate shows that heat-inactivation of the duroquinol oxidase activity does not proportionally affect NADH and succinate oxidation. Moreover, 1 microM antimycin inhibits duroquinol oxidase activity by 50% while not decreasing the rates of NADH and succinate oxidation. Therefore, the cyanide-resistant electron transport does not appear to be mediated by a "duroquinol oxidase", and a convincing proof of the existence of a specific protein acting as a cyanide-resistant oxidase in plant mitochondria is still lacking.     

Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain

The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 m M of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate production. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells – presumably complex II – plays important role in the regeneration of ascorbate from its oxidized form, DHA.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.     

Activation by ATP of a proton-conducting pathway in yeast mitochondria.

The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H. Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed. It has been demonstrated already in S. cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels. In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H(+)-dependent swelling. We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane.     

Disulfiram inhibition of the alternative respiratory pathway in plant mitochondria

Disulfiram (tetraethylthiuram disulfide) was found to be a potent and selective inhibitor of the alternative respiratory path of plant mitochondria. The onset of inhibition by disulfiram takes several minutes and the inhibition is not readily reversed by washing, nor by metal ions. By contrast, thiols such as dithiothreitol not only reverse, but also prevent, disulfiram inhibition. Inhibition by disulfiram and by hydroxamic acids are not mutually exclusive. Structural analogs of disulfiram are far less potent inhibitors, with the exception of bisethyl xanthogen. Inhibition is due to disulfiram, per se, and not to its reduction product, diethyldithiocarbamate, a powerful chelator. Accordingly, the inhibitory effect of disulfiram is considered to involve the formation of mixed disulfides with one or more sulfhydryl groups in the alternative path. Disulfiram does not act as an electron sink diverting electron flow from oxygen.