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EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

ABSTRACT: EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.

SUBMITTER: Shergill JK

PROVIDER: S-EPMC1136710 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Project description:In the midgut cytosol of Lepidoptera, ecdysteroids undergo inactivation by transformation via the 3-dehydro derivative to the corresponding 3-epiecdysteroid (3 alpha-hydroxy) and by phosphate conjugation. The oxygen-dependent oxidase catalyses formation of 3-dehydroecdysteroid, which can be reduced either irreversibly by 3-dehydroecdysone 3 alpha-reductase to 3-epiecdysteroid, or by 3-dehydroecdysone 3 beta-reductase back to the initial ecdysteroid. Furthermore, these ecdysteroids undergo further inactivation by phosphorylation. These ecdysteroid transformations have been investigated in last instar larvae of the cotton leafworm, Spodoptera littoralis. The products of the phosphorylation have been characterized as predominantly ecdysteroid 2-phosphate accompanied by smaller amounts of the corresponding 22-phosphate. The phosphotransferases require Mg2+ and ATP. Whereas the 3-dehydroecdysone 3 alpha-reductase has a clear preference for NADPH rather than NADH, the corresponding 3 beta-reductase markedly favours NADH. The physiological significance of the latter enzyme is unclear. The profiles of the various enzymic activities in dialysed midgut cytosol supplemented with appropriate cofactors were determined throughout the last larval instar. All activities were detectable throughout the instar, but the respective enzymes exhibited maxima at different times. Ecdysone oxidase showed a peak early in the instar, with 3-dehydroecdysone 3 alpha-reductase increasing to a peak as the former activity declined. The 3-dehydroecdysone 3 beta-reductase exhibited peak activity late in the instar, a profile similar to that observed for the corresponding haemolymph enzyme involved in reduction of the 3-dehydroecdysone product of the prothoracic glands to ecdysone. Thus, the significance of the midgut 3 beta-reductase may be related to production of active hormone. Both ecydsteroid 22- and 2-phosphotransferases showed high activities early in the instar and then declined. The physiological significance of the profiles for the ecdysone oxidase, the 3-dehydroecdysone 3 alpha-reductase and phosphotransferases is unclear.

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EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

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