EPR SPECTROSCOPIC CHARACTERIZATION OF THE IRON-SULFUR PROTEINS AND CYTOCHROME-P-450 IN MITOCHONDRIA FROM THE INSECT SPODOPTERA-LITTORALIS (COTTON LEAFWORM)

EPR spectroscopy was used to investigate the cytochrome P-450-dependen t steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafwor m (Spodoptera littoralis) and the redox centres associated with membra nes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been as signed to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bou nd) was tentatively assigned to a signal at g = 8.0, which was detecta ble from membranes as prepared. An EPR signal characteristic of a [2Fe -2S] cluster detected from the soluble mitochondrial matrix fraction h as been shown to be distinct from the signals associated with mitochon drial NADH dehydrogenase and succinate dehydrogenase, and has therefor e been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 2 0-hydroxylation comprises both ferredoxin and cytochrome P-450 compone nts, and thus resembles the enzyme systems of adrenocortical mitochond ria. EPR signals characteristic of the respiratory chain were also obs erved from fat-body mitochondria and assigned to the iron-sulphur clus ters associated with Complex I (Centres N1, N2), Complex II (Centres S 1, S3), Complex III (the Rieske centre), and the copper centre of Comp lex IV, demonstrating similarities to mammalian mitochondria. The redu ced membrane fraction also yielded a major resonance at g = 2.09 and 1 .88 characteristic of the [4Fe-4S] cluster of electron-transferring fl avoprotein: ubiquinone oxidoreductase. As the fatbody is the major met abolic organ of insects, this protein is presumably required for the b eta-oxidation of fatty acids in mitochondria. High-spin haem signals i n the low-held region of spectra also demonstrated that the mitochondr ial fraction contains relatively high concentrations of catalase.