David G. Whitehouse

The role(s) of adenylate kinase and the adenylate carrier in the regulation of plant mitochondrial respiratory activity

Abstract The effect of inhibitors of the ATP synthase, the adenylate carrier and adenylate kinae on the respiratory rate steady-state membrane potential and mitochondrial ATP levels have been investigated in potato and pea leaf mitochondria. Under ADP-limited conditions, it was found that the addition of oligomycin, aurovertin or efrapeptin increased the membrane potential and decreased the respiratory rate, implying that ATP synthesis was occurring prior to inhibition. Molybdate, NEM or vanadate had no effect on the ATP synthesised, suggesting that ADP-regeneration required for continued phosphorylation was not due to contaminating ATPases or phosphatases. ATP levels were significantly reduced by carboxyatractyloside (CAT) and increased by P1, P5-di(adenosine-5′) pentaphosphate (Ap5A). The respiratory rate could be stimulated by the addition of AMP and the stimulated rate was sensitive to oligomycin and aurovertin. Preincubation with CAT or Ap5A abolished AMP stimulation of NADH oxidation. It is suggested that respiration can sustain a limited but significant net formation of ATP, even in the absence of any added ADP. A model involving the combined activities of the adenylate carrier, adenylate kinase and the ATP synthase is proposed to account for the ATP synthesised under these conditions. Furthermore, it is suggested that the cycling of mitochondrial ADP and ATP via this model may represent a major regulatory influence on the activity of mitochondrial respiration under conditions of ADP-limitation — a condition likely to reflect the in vivo situation in plant cells.

The active site of the plant alternative oxidase: structural and mechanistic considerations

In the present review we seek to provide an up-to-date view on the molecular nature of the active site of the plant alternative oxidase which has been postulated to comprise of a binuclear iron centre. A three-dimensional model of the catalytic centre of the oxidase is presented which is based on the active site structure of the free radical component of ribonucleotide reductase and methane monooxygenase. The model indicates that a highly conserved carboxylate (Glu-270) occupies a central position within the proposed di-iron centre as it co-ordinates both iron atoms. The expression of an alternative oxidase protein in Schizosaccharomyces pombe in which Glu-270 was mutated to asparagine yields an inactive protein. The implications of this in relation to the structural model of the active site of the oxidase suggests that Glu-270 is essential for catalytic alternative oxidase activity. A kinetic mechanism is suggested which accounts for the full reduction of dioxygen to water catalysed by a single di-iron centre.

Maesaquinone: A novel inhibitor of plant mitochondrial respiratory enzymes that react with ubiquinone

The effect of maesaquinone, 2‐(14‐nonadecenyl)‐3,6‐dihydroxy5‐methyl‐1,4‐benzoquinone, on plant mitochondrial respiration has been investigated. In mitochondria isolated from thermogenic Arum maculatum spadices, this compound inhibits both cytochrome and alternative pathway activities. Kinetic analyses reveal that this inhibition is the result of potent effects of maesaquinone on the alternative oxidase (ID50 <0.3θM) and complex III (ID50 <5θM). Succinate dehydrogenase and external NADH dehydrogenase are also inhibited, albeit to a lesser extent (∼30% at 1 θM). These data suggest that maesaquinone specifically affects the interaction of the respective enzymes with ubiquinone.

Respiratory Chain and ATP Synthase

The respiratory chain and adenosine triphosphate (ATP) synthase are, in eukaryotic cells, located in the inner membrane of mitochondria. Mitochondria are oval-shaped organelles, typically 1–2 μm long and 0.5 μm in diameter, whose principal cellular functions are to generate energy, in the form of ATP and carbon skeletons for biosynthetic purposes. The shape and appearance of mitochondria vary considerably and these organelles appear to be most numerous in mammalian tissues with a high-energy demand (heart, liver, muscle, and brain and in rapidly dividing plants cells). The respiratory chain functions to oxidize NADH (+H + ) and FADH 2 and reduce molecular oxygen to water. These functions are electron transfer (electron motive) processes and result in a substantial release of energy, which generates a proton-motive force that is used to drive the ATP synthase in the forward direction thereby synthesizing ATP for use in cellular reactions.