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.

Treatment of Cells and Tissues with Chromate Maximizes Mitochondrial 2Fe2S EPR Signals

“…As such, little or no increase in the EPR signal was expected upon addition of chromate. The EPR signals we observed in the liver tissue are similar to signals observed by others [11]. The g-values on our figures are the same as g-values obtained by others [4,14].…”

The pro-oxidant chromium(VI) inhibits mitochondrial complex I, complex II, and aconitase in the bronchial epithelium: EPR markers for Fe–S proteins

“…1B), which demonstrates a contribution from the low field line of mitochondrial 2Fe-2S centers at 10 K ([73-75] and references therein). Also, the line shape of g = 2.02 is narrower at 10 K than at 20 or 30 K, supporting an additional component to the signal at 10 K. By contrast, the intensity of the signal at g = 1.94 [76] progressively increases as the temperature goes from 30 K to 20 K to 10 K, which is consistent with mitochondrial 2Fe-2S centers [73, 77]. The difference in the temperature dependence for the line at g = 2.02 versus the line at g = 1.94 therefore indicates that both [3Fe-4S] 1+ (probably from aconitase and/or aconitase-like signals), and the signals for reduced [2Fe-2S] 1+ centers contribute to the g = 2.02 signals in Cr(VI)-treated cells (Fig.…”

“…the FAD (which is reduced by succinate) and S1, the 2Fe/2S center that is reduced by the FADH 2 and that then reduces INT [80]. The second assay for complex II measured the succinate-dependent reduction of ubiquinone which requires electron flow through all of its redox centers, including the FAD, and the S1 (2Fe-2S) and S3 (3Fe4S) centers [77]. This measure of complex II activity was significantly inhibited by an average of 35% (P = 0.0157) in mitochondria isolated from cells treated with 25 μM Cr(VI) for 3 hr (Fig.…”

A Combined Far‐Infrared Spectroscopic and Electrochemical Approach for the Study of Iron–Sulfur Proteins

“…The ligation by His, Ser, Asp or a backbone amide occurs less frequently 7. ISPs have been extensively studied by a survey of techniques such as EPR spectroscopy,8 NMR,9 Raman spectroscopy,10, 11, 12 as well as by FTIR spectroscopy 13. In addition, synthetic FeS clusters were also produced to mimic the active sites of several ISPs 14.…”