Mhn. Hoefnagel et al., NEW INHIBITORS OF THE UBIQUINOL OXIDASE OF HIGHER-PLANT MITOCHONDRIA, European journal of biochemistry, 233(2), 1995, pp. 531-537
A screen has been performed of possible inhibitors of the ubiquinol ox
idase of higher plant mitochondria by assaying their effects on cyanid
e-insensitive NADH oxidase of mitochondria of Arum maculatum. A number
of compounds which have powerful inhibitory effects have been identif
ied. Potent inhibition was found with compounds related to the previou
sly described il-propyl gallate, but with the n-propyl sidechain repla
ced with alkyl chains of greater hydrophobicity. Titration of a range
of partial reactions showed that the inhibitors act specifically on th
e ubiquinol oxidase. The concentrations of inhibitor required are depe
ndent on the respiratory substrate and on the amount of mitochondria u
sed in the assay. Octyl gallate also proved to be a potent inhibitor o
f the ubiquinol oxidase in tobacco cell suspensions. A second class of
compounds which strongly inhibit cyanide-insensitive NADH oxidation i
s aurachin C and its analogues. Compounds related to aurachin D are mu
ch 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(i) site
of the cytochrome bc(1) complex, as already known to be the case in ot
her systems, and so they are of more limited value for studies of the
ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquino
l oxidase in a purified mitochondrial fraction from the spadices of Ar
um maculatum with octyl gallate gave a half-maximal effect at a concen
tration of around 6 nM when the protein concentration was 14 mu g ml(-
1). 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 +/- 0.15 nmol mg(-1) p
rotein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15
+/- 7:1. The measurements also provide a minimal turnover number for
the ubiquinol oxidase of 186 +/- 42 electrons . s(-1). Titration of th
e ubiquinol oxidase in soybean cotyledon mitochondria with these compo
unds gave the concentration of inhibitor required to elicit 50% of the
maximum observed effect (I-50) values about one order of magnitude hi
gher than those found with Arum mitochondria, and again the values dep
ended on the respiratory substrate. An explanation for the variation i
n I-50 values may be found in terms of differences in oxidase concentr
ations in the different mitochondrial membranes and in the differences
in rate-controlling steps with substrates of different activities.