A screen has been performed of possible inhibitors of the quinol oxidation sites of the two terminal oxidases of Escherichia coli, cytochromes bo and bd. Aurachin C and its analogues were found to be particularly effective inhibitors of both enzymes, whereas aurachin D and its analogues displayed a selectivity for inhibition of cytochrome bd. In addition, a tridecyl derivative of stigmatellin was found to inhibit cytochrome bo at concentrations which were without significant effect on cytochrome bd. Titration of membrane-bound cytochromes bo and bd with aurachin C gave an observed dissociation constant in the range of 10(-8) M. A similar observed dissociation constant was determined for aurachin D inhibition of cytochrome bd. For both enzymes, their kinetic behavior during a series of substrate pulses indicates that it is reduction of the enzyme by quinol, and not reaction with oxygen, which is inhibited. It is concluded that the aurachins are powerful inhibitors of the quinol oxidation sites of bacterial cytochromes bo and bd. The effects of aurachin C on cytochrome bo were investigated in more detail. The number of inhibitor binding sites on the purified enzyme was determined by titration to be 0.6 per enzyme. At an inhibitor/oxidase ratio of 1.0, electron donation into the enzyme from added quinol is extremely slow, making it very unlikely that there is more than one quinone-reactive site. Aurachin C caused a potent inhibition of electron donation from a pulse of quinol.(ABSTRACT TRUNCATED AT 250 WORDS)

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A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified.Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions.A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Q, site of the cytochrome be, complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase.Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondria1 fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 pg ml -I . A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 -C 0.15 nmol mg protein, or a ratio of ubiquinol oxidasekytochrome oxidase of about 15 2 7 : 1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 2 42 electrons . s '.Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (Z,,,) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I,,, values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in ratecontrolling steps with substrates of different activities.Keywords: ubiquinol oxidase; mitochondria; inhibitors ; higher plants ; quinone analogues.Specific inhibitors have been useful as probes of structural and mechanistic aspects of a wide range of enzymes, including those of respiration and photosynthesis. Most useful are those which are specific and which bind tightly to their target site so that only stoichiometric amounts are required. In the case of respiratory and photosynthetic electron transfer chains, a wide range of synthetic and natural com...

MOA-stilbene is known to be a specific inhibitor of the Qo site of mammalian cytochrome bc 1 complex. We show that it also binds to the chloroplast cytochrome bf complex. Binding to the reduced enzyme induces a red-shift of the Soret and visible absorption bands of the haems b. Steady state and single turnover experiments with thylakoid membranes show that MOA-stilbene promotes additional 'oxidant-induced reduction' of the b haems and slows their subsequent dark reoxidation. In single turnover experiments, the associated slow phase of the carotenoid bandshift at 518 nm is only partially decreased in apparent extent and rate. These and other effects are similar to those produced by NQNO, a Qi site effector, and by analogy indicate that MOA-stilbene should also be primarily a Qi-site effector of the cytochrome bf complex. MOA-stilbene has less effect on other parts of the photosynthetic chain. This confers an important advantage on MOA-stilbene in that its effects on the cytochrome bf complex can be studied by using Photosystem II to activate turnover. Myxothiazol displays effects on the cytochrome bf complex which are similar to, but much weaker than, those of MOA-stilbene.A Q cycle-based model of turnover of the cytochrome bf complex is presented, which can account for several unusual features of kinetic behaviour.