Stepwise reduction of cytochromesb-562,b-566 andb-558 in rat liver mitochondria

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria without addition of substrate caused reduction of cytochromesb-562(having an α-band at 562 nm at room temperature),c + c₁anda + a₃. The effect of KCN on cytochromeb-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromesc+c₁ anda+a₃ was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 μM KCN under conditions where cytochromesb-562,c+c₁ anda+a₃ were reduced, caused reduction of cytochromeb-566. The absorbance spectrum of cytochromeb-566 had an α-band at 565.5 nm, a β-band at 538 nm and a γ-band at 431 nm, but no shoulder around 558 nm at room temperature.3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromesb-566,b-562,c+c₁ anda+a₃ were already fully reduced, caused reduction of cytochromeb-558 (having an α-band at 558 nm, a β-band at 527 nm and a γ-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochromeb-566).4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochromeb-566, but is due to a different species from cytochromeb-566.     

Electron flow and heme-heme interaction between cytochromes b-558, b-595 and d in a terminal oxidase of Escherichia coli

The ESR signals of the cytochromes in the Escherichia coli terminal oxidase cytochrome d complex were studied at cryogenic temperature. The intensities and g values of the rhombic high-spin signals changed when the electronic state of cytochrome d was changed from the oxidized state to the reduced or oxygen-binding or CO-binding state. These rhombic signals were therefore assigned to cytochrome b-595, which is located near cytochrome d in the oxidase complex. This assignment was supported by the finding that the Em value of the rhombic signals differed from that of cytochrome d (Hata, A. et al. (1985) Biochim. Biophys. Acta 810, 62-72). Photolysis and ligand-exchange experiments with the reduced CO complex of the oxidase were performed in the presence of oxygen at -140 degrees C. The ESR spectra of three intermediate forms trapped by controlled low temperatures were detected. These forms were designated as the oxygen-binding intermediate I (ESR-silent), oxygen-binding intermediate II (giving ESR signals at g = 6.3, 5.5 and 2.15), and oxygen-binding intermediate III (giving signals at g = 6.3, 5.5 and 6.0). From these results, electron flow in the cytochrome d complex is proposed to proceed in the order, cytochrome b-558----cytochrome b-595----cytochrome d----O2. A model of the mechanism of four-electron chemistry for oxidation of ubiquinol-8 and formation of H2O by the cytochrome d complex is presented.     

Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite.

The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 X 10(6) M-1 X S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 X 10(6) M-1 X S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.     

Growth hormone and liver mitochondria. Effects on cytochromes and some enzymes.

A reliable and reproducible assay was developed for measuring mitochondrial α-keto acid decarboxylase activity using ferricyanide as the electron acceptor. This method permitted the functional isolation and investigation of the decarboxylase step of the branched-chain α-keto acid dehydrogenases in rat liver mitochondria. Pyruvate and α-ketoglutarate decarboxylases are known to be separate and distinct enzymes from the branched-chain α-keto acid decarboxylases and were studied as controls. The relative specific activities of rat liver mitochondrial decarboxylases as measured by the ferricyanide assay showed that pyruvate and α-ketoglutarate were decarboxylated twice as rapidly as α-ketoisovalerate and four to ten times as fast as α-keto-β-methylvalerate and α-ketoisocaproate. The three branched-chain α-keto acids individually inhibit pyruvate and α-ketoglutarate decarboxylases. Inactivation of mitochondrial branched-chain α-keto acid decarboxylase activity by freezing and thawing and by prolonged storage resulted in a proportional decrease in decarboxylase activity toward each of the three branched-chain α-keto acids. However, hypophysectomy was found to increase decarboxylase activity with α-keto-β-methylvalerate to four times normal and with α-ketoisovalerate to three times normal, but the activity with α-ketoisocaproate was not changed. Hypophysectomy did not alter mitochondrial decarboxylase activity with pyruvate, α-ketoglutarate, or α-ketovalerate. The finding that hypophysectomy differentially alters the mitochondrial decarboxylase activity with the three branched-chain α-keto acids suggests either that there is more than one substrate-specific enzyme with branched-chain α-keto acid decarboxylase activity or that there is a modification of one enzyme such that the catalytic activity is selectively altered toward the three substrates.     

Ubiquinone biosynthesis by mitochondria, sonicated mitochondria, and mitoplasts of rat liver.

Ubiquinone was biosynthesized when rat liver mitochondria were incubated with S-adenosyl-L-methionine, solanesyl diphosphate, and [U-14C]p-hydroxybenzoate. The intermediates of ubiquinone biosynthesis but not ubiquinone were accumulated in mitochondria incubated without S-adenosyl-L-methionine and the accumulated intermediates were converted to ubiquinone by the addition of the methyl group donor and an excess of cold p-hydroxybenzoate. No solaneylated compounds except nonaprenyl p-hydroxybenzoate were found in sonicated mitochondria, while the biosynthesis of ubiquinone was observed in the sonicated preparation of mitochondria in which the intermediates accumulated. The results indicate that the initial decarboxylation reaction is completely abolished and the subsequent reactions of hydroxylation and methylation are not completely inhibited by the sonication treatment and therefore the decarboxylation reaction is the next step after nonaprenylation of p-hydroxybenzoate. Mitoplasts could biosynthesize ubiquinone with activity comparable to that of intact mitochondria, suggesting that components of the outer membrane and the intermembranous space of mitochondria are not involved in ubiquinone biosynthesis.     

The reduction of diamide by rat liver mitochondria and the role of glutathione.

Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a) thiol-blocking reagents (N-ethylmaleimide, p-hydroxymercuribenzoate, mersalyl and 2,6-dichlorophenol-indophenol);(b) respiratory inhibitors (arsenicals, malonate and antimycin, but not cyanide or oligomycin; inhibition by antimycin is reversed by ATP); (c) uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol and valinomycin with K+; inhibition by the first of these uncouplers is not reversed by cyanide); (d) reagents affecting energy conservation (Ca2+, increasing pH, phosphate; phosphate inhibition is augmented by catalytic ADP or ATP and augmentation is abolished by respiratory inhibitors). Concentrations of mitochondrial glutathione are high when diamide reduction is uninhibited, but low after adding one of the above inhibitors such that the reduction rate is roughly proportional to the glutathione concentration. Endogenous ATP concentrations are lower in the presence of diamide than without, but the difference is abolished by respiratory inhibitors. With oligomycin added, however, ATP concentrations are higher in the presence of diamide and this positive increment is decreased by antimycin, N-ethylmaleimide and malonate. In the presence of diamide and an uncoupler, the mitochondrial glutathione content does not fall if various reducible substrates are present, although the inhibition of diamide reduction is not relieved. Some of these substrates prevent the fall in reduced glutathione concentration found with diamide and phosphate. They also relieve the inhibition of diamide reduction and the relief is sensitive to butylmalonate. The inhibition of diamide reduction by N-ethylmaleimide, mersalyl or p-hydroxymercuribenzoate is not relieved by reducible substrates, but the latter mitigate the fall in the concentration of glutathione. Inhibitors of carriers of tricarboxylic acid-cycle intermediates also inhibit reduction of diamide. The reduced glutathione concentration remains high when they are added singly, but falls when two of them are combined. It is proposed that diamide may enter the matrix as a protonated adduct formed with the thiol groups of mitochondrial carriers and then be reduced in the matrix by glutathione, which is regenerated via NADH, energy-dependent transhydrogenase and NADP+-specific glutathione reductase. Some of the high-energy equivalents required for the transhydrogeneration may be generated by the substrate phosphorylation step of the tricarboxylic acid cycle.     

Changes in respiratory control and cytochromes in liver mitochondria during hibernation

Liver mitochondria from the ground squirrel, Citellus lateralis, were compared during states of hibernation and non-hibernation. During hibernation a decreased total amount of mitochondrial cytochrome was noted. In addition changes in the relative amounts of cytochromes b and c and a pronounced decrease in cytochrome a–a₃ was shown by difference spectra. The rate of oxygen uptake with succinate and ADP was less in the hibernator. However, addition of the uncoupler, salicylanilide XIII, stimulated oxygen consumption of the hibernator to a rate greater than that observed in the non-hibernating animal indicating that oxygen uptake was not limited by the cytochrome concentration. It is postulated that the sluggish rate of oxygen uptake under phosphorylating conditions by liver mitochondria of the hibernator may be caused by a change in the penetration of ADP and/or P_i, or an alternation in some parameter of the mechanism of coupled phosphorylation.     

ATP-synthetase activity, respiration and cytochromes of rat heart mitochondria in aging and hyperthyroidism

The ATP-synthetase activity, the rate of oxygen uptake under different metabolic conditions, the tightness of coupling of respiration to oxidative phosphorylation and the cytochrome contents in heart mitochondria of rats from different age groups were studied under normal conditions and in hyperthyroidism. It was found that heart mitochondria of aged animals did not practically differ in terms of their functional activity from those of the young animals. Administration of thyroxin to the animals from all age groups produced no significant effects on the state of mitochondria, increasing the rate of ATP synthesis on alpha-glycerophosphate, which was especially well-pronounced in aged animals, and the cytochrome content in 1-month-old rats.     

Resonance Raman spectra of cytochromes C557 and C558.

Resonance Raman spectra of cytochromes c₅₅₇ and c₅₅₈ have been recorded and compared to other low-spin ferrous cytochromes. The data support the chemical evidence that there is one vinyl group on the heme and one thioether linkage to the protein.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Proline transport in rat liver mitochondria.

Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of l-proline is two times faster than the penetration of dl-proline. (b) Proline is accumulated against a concentration gradient, (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl and p-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.     

CDP-diacylglycerol synthesis in rat liver mitochondria.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.     

Cytochrome b reduction by hexaammineruthenium in mitochondria and submitochondrial particles: Evidence for heme b-562 localization at the M-side of the mitochondrial membrane

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b-562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH₃)²⁺₆ with b cytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769-783) redox centre of at least one of the oxidized cytochromes b, most probably of b-562, is exposed to the M-aqueous phase.