Spectroscopic studies of flavoproteins and non-haem iron proteins of submitochondrial particles of Torulopsis utilis modified by iron- and sulphate-limited growth in continuous culture

Abstract

1. A spectroscopic resolution has been made of the components contributing to the ;iron-flavoprotein' trough extending from 450 to 520nm in the reduced-minus-oxidized difference spectrum of submitochondrial particles of Torulopsis utilis. 2. Seven components were identified other than cytochrome b, ubiquinone and succinate dehydrogenase. On the basis of the effects of iron- and sulphate-limited growth of cells on their subsequently derived electron-transport particles, and also by consideration of analytical measurements of the concentration of FMN, FAD, non-haem iron and acid-labile sulphide in the electron-transport particles in relation to the magnitude of the spectroscopic changes, it was possible to identify five of these components as follows: species 1a, the flavin of NADH dehydrogenase ferroflavoprotein; species 1b, the iron-sulphur component of NADH dehydrogenase ferroflavoprotein; species 1', the flavin of an NADPH dehydrogenase; species 2, an iron-sulphur or ferroflavoprotein component; species 3, the flavin of l-3-glycerophosphate dehydrogenase. Two additional components were a fluorescent flavoprotein, probably lipoamide dehydrogenase, and a b-type cytochrome reducible by NADH or NADPH but not reoxidizable by the respiratory chain. 3. Species 1b and 2 were undetectable in electron-transport particles from iron- or sulphate-limited cells, but could be recovered in vivo under non-growing conditions. 4. The recovery in vivo of species 2 but not species 1b was inhibited by cycloheximide. 5. The recovery of species 1b correlates with the recovery of site 1 conservation. 6. The recovery of species 1b with species 2 correlates with the recovery of piericidin A sensitivity. 7. Evidence is presented for an NADPH dehydrogenase distinct from NADH dehydrogenase. The oxidation of NADH and NADPH by the respiratory chain is sensitive to piericidin A, and an iron-sulphur protein common to both pathways (species 2) is suggested as the piericidin A-sensitive component. 8. The approximate E'(0) (pH7.0) values of species 1 (a and b, low potential) and species 2 (high potential) indicate that site 1 energy conservation occurs between the levels of species 1 (a and b) and species 2.

A Theoretical Analysis of NADPH Production and Consumption in Yeasts

“…Schuurmans Stekhoven (1 966) showed that yeast mitochondria can oxidize NADPH with respiratory control. Also submitochondrial particles can oxidize NADPH (Ragan & Garland, 1971 ;Djavadi et al, 1980). Direct oxidation of exogenous NADPH seems a general property of plant and fungal mitochondria (Palmer & Msller, 1982).…”

From identification of fluorescent flavoproteins to mitochondrial redox indicators in intact tissues

“…4 According to the substrate screening performed by Kunz and Kunz,18 additions of glycerol-3-phosphate, proline and choline to rat liver mitochondria did not result in noticeable decrease in the°uorescence of non-NADlinked°avoproteins, which indicates that the°a-voenzymes glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:ubiquinone oxidoreductase), proline dehydrogenase and choline oxidase cannot be monitored by°uorescence. The experiments by Ragan and Garland 21 corroborate this by showing that, although the glycerol phosphate oxidase activity of submitochondrial particles from Torulopsis utilis is high, its substrate only causes a paradoxical°a vin°uorescence increase, although the concomitant absorbance decrease in the \°avin through" region denotes°avin reduction. A plausible explanation is that the°uorescence increase was due to a decrease in the internal¯lter e®ect at the°avin excitation band, revealing the°uorescence of other (oxidized)°avoproteins.…”

“…A plausible explanation is that the°uorescence increase was due to a decrease in the internal¯lter e®ect at the°avin excitation band, revealing the°uorescence of other (oxidized)°avoproteins. 21 The rest of°avoproteins are reducible with dithionite only. [18][19][20][21] An additive contribution of FpL and ETF on°avin°uorescence is probably the cause of the extreme°avin°uo-rescence decrease (by taking the anoxia response as a reference) upon infusion on medium chain fatty acid into an isolated perfused rat heart, because -oxidation will feed electrons both via the NAD þ pool and ETF.…”

“…21 The rest of°avoproteins are reducible with dithionite only. [18][19][20][21] An additive contribution of FpL and ETF on°avin°uorescence is probably the cause of the extreme°avin°uo-rescence decrease (by taking the anoxia response as a reference) upon infusion on medium chain fatty acid into an isolated perfused rat heart, because -oxidation will feed electrons both via the NAD þ pool and ETF. 22,23 Free ribo°avin, FMN and FAD have three main absorbance bands centered on 268, 374 and 449 nm, and the°uorescence excitation spectrum is identical to the absorbance spectrum.…”

Electron-paramagnetic-resonance spectroscopy studies of iron-sulphur centres of submitochondrial particles from iron- and sulphur-deficient. Candida utilis

“…40g molVI indicated iron or sulphate limitation with loss of ATP synthesis coupled to the NADH-ubiquinone oxidoreductase segment of the respiratory chain (Clegg & Light, 1971). Further confirmation of iron limitation was obtained by measuring the sensitivity of the NADH oxidase activity of submitochondrial particles to the respiratory inhibitor piericidin A; glycerol-limited cells yield submitochondrial particles that were sensitive to piericidin A, whereas those from iron-or sulphate-limited cells were insensitive (Ragan & Garland, 1971 These were made with a Varian E9 spectrometer operating at 9GHz (Varian Associates Ltd., Walton-on-Thames, Surrey, U.K.), with temperature control and measurement as described by Lowe et al (1972). Spectra were generally measured at a sample temperature of 12 ± 0.2°K and at 20 mW microwave power.…”