Steady-state Levels Of Cytochrome Research Articles

The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m-AAA protease.

Yta10p (Afg3p) and Yta12p (Rcal1p), members of the conserved AAA family of ATPases, are subunits of the mitochondrial m-AAA protease, an inner membrane ATP-dependent metallopeptidase. Deletion of YTA10 or YTA12 impairs degradation of non-assembled inner membrane proteins and assembly of respiratory chain complexes. Mutations of the proteolytic sites in either YTA10 or YTA12 have been shown to inhibit proteolysis of membrane-integrated polypeptides but not the respiratory competence of the cells, suggesting additional activities of Yta10p and Yta12p. Here we demonstrate essential proteolytic functions of the m-AAA protease in the biogenesis of the respiratory chain. Cells harbouring proteolytically inactive forms of both Yta10p and Yta12p are respiratory deficient and exhibit a pleiotropic phenotype similar to Deltayta10 and Deltayta12 cells. They show deficiencies in expression of the intron-containing mitochondrial genes COX1 and COB. Splicing of COX1 and COB transcripts is impaired in mitochondria lacking m-AAA protease, whilst transcription and translation can proceed in the absence of Yta10p or Yta12p. The function of the m-AAA protease appears to be confined to introns encoding mRNA maturases. Our results reveal an overlapping substrate specificity of the subunits of the m-AAA protease and explain the impaired assembly of respiratory chain complexes by defects in expression of intron-containing genes in mitochondria lacking m-AAA protease.

Metabolic control analysis of the bc1 complex of Saccharomyces cerevisiae: effect on cytochrome c oxidase, respiration and growth rate.

A number of strains varying in steady-state level of assembled bc1 complex were used to test the conclusions from inhibitor titration experiments with isolated mitochondria that, in cells of Saccharomyces cerevisiae grown on non-fermentable carbon sources, the control coefficient of the bc1 complex on the mitochondrial respiratory capacity equals 1 and the respiratory chain consists of supermolecular respiratory units [Boumans, Grivell and Berden (1998) J. Biol. Chem. 273, 4872-4877]. In addition, the control coefficient of mitochondrial respiration on the growth rate was determined. It was found that a reduced level of bc1 complex is accompanied by an almost parallel decrease in steady-state level of cytochrome c oxidase. Since the linear relationship between level of active bc1 complex and respiratory capacity still holds, it is concluded that cytochrome c oxidase has disappeared from respiratory units that are already deficient in the bc1 complex and that the cytochrome c oxidase in a respiratory unit is destabilized when the bc1 complex is deficient. The control coefficient of the bc1 complex, and thus of mitochondrial electron-transfer capacity, on respiration of intact cells (without uncoupler added) is 0.20. Addition of uncoupler results in an increase in the coefficient to 0.36. Thus changing the respiratory state changes the distribution of control, increasing the control coefficient of electron-transfer activity as the respiratory state goes towards State 3u. Rates of growth of the strains on different carbon sources were determined and subsequently fitted to calculate control coefficients of the bc1 complex (and therefore of the respiratory capacity) on growth. Little variation was found between lactate-, ethanol- and glycerol-containing media, control coefficients being around 0.18 at pH 5. At pH 7 the control coefficient increased to 0.57, indicative of a higher dependence of the cell on ATP derived from oxidative phosphorylation. During growth on glucose-containing medium, the bc1 complex has no control on the growth rate, as indicated by the fact that all strains, including a respiratory-deficient strain, grow as fast as the wild-type. However, the presence of respiratory capacity in the wild-type does result in a higher growth yield compared with the respiratory-deficient strain, indicating that, in contrast with what is generally assumed, in S. cerevisiae the 'Pasteur effect' is not restricted to special experimental conditions.

Protein stability regulates the expression of cytochrome c during the developmental cycle of Trypanosoma brucei

The expression of cytochrome c is developmentally regulated during the life cycle of Trypanosoma brucei. The level of regulation appears to be post-transcriptional since cytochrome c mRNA is present in all life stages of the parasite. We have used RNA from each life stage to prime in vitro translation systems and found that the cytochrome c mRNAs are equally translatable. Continuous labeling experiments conducted in vivo indicate that cytochrome c is synthesized at similar rates in both bloodstream and procyclic trypanosomes. Western blots, however, confirm that steady-state levels of cytochrome c are severely depressed in bloodstream forms. In a series of pulse/chase experiments we demonstrate that the half-life of cytochrome c is approximately 1 h in the bloodstream form and no detectable turnover occurred in the procyclic form. We conclude that a major step in the developmental regulation of cytochrome c expression in T. brucei occurs post-translationally due to rapid turnover of the protein in the bloodstream trypanosomes.

Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes.

Addition of exogenous NADH to rotenone- and antimycin A-treated mitochondria, in 125 mM KCl, results in rates of oxygen uptake of 0.5-1 and 10-12 nanoatoms of oxygen X mg protein-1 X min-1 in the absence and presence of cytochrome c, respectively. During oxidation of exogenous NADH there is a fast and complete reduction of cytochrome b5 while endogenous or added exogenous cytochrome c become 10-15% and 100% reduced, respectively. The reoxidation of cytochrome b5, after exhaustion of NADH, precedes that of cytochrome c. NADH oxidation is blocked by mersalyl, an inhibitor of NADH-cytochrome b5 reductase. These observations support the view of an electron transfer from the outer to the inner membrane of intact mitochondria. Both the rate of exogenous NADH oxidation and the steady state level of cytochrome c reduction increase with the increase of ionic strength, while the rate of succinate oxidation undergoes a parallel depression. These observations suggest that the functions of cytochrome c as an electron carrier in the inner membrane and as an electron shuttle in the intermembrane space are alternative. It is concluded that aerobic oxidation of exogenous NADH involves the following pathway: NADH leads to NADH-cytochrome b5 reductase leads to cytochrome b5 leads to intermembrane cytochrome c leads to cytochrome oxidase leads to oxygen. It is suggested that the communication between the outer and inner membranes mediated by cytochrome c may affect the oxidation-reduction level of cytosolic NADH and the related oxidation-reduction reactions.

Alteration of Mycobacterium phlei membrane structure by freezing and thawing; reversal by heating

Membrane vesicles from Mycobacterium phlei which contain the electron transport chain, when subjected to freezing to −70 °C followed by slow thawing, exhibited a decreased level of phosphorylation coupled to the oxidation of substrates. This loss in oxidative phosphorylation was restored following heat treatment (50 °C for 10 min) of the membranes. Freeze treatment (−70 °C for 10 min) of membrane vesicles also resulted in a decrease in membrane bound coupling factor-latent ATPase activity. The soluble coupling factor(s) or cryoprotective agents (i.e., glycerol or dimethyl sulfoxide) were found to protect the membrane vesicles from the effects of freezing. Membrane vesicles depleted of particulate bound coupling factor were sensitive to exposure to low temperatures; however, complete protection was afforded by the addition of coupling factor. In addition, prolonged sonication of electron transport particles resulted in lowered P O ratios, and heat treatment of these sonicated particles restored P O ratio. Therefore, it appears that the effects of heat treatment and freeze treatment on membrane vesicles are reversible. The steady state level of reduced cytochrome b was considerably higher (40%) in heat-treated electron transport particles as compared to untreated particles (28.5%); electron transport particles subjected to freeze treatment showed a lower steady state level of cytochrome b (16.6%) as compared to electron transport particles. The steady state level of cytochrome b in freeze-treated particles returned to the original level (27.5%) for electron transport particles when subjected to heat treatment. Nevertheless, the total amount of enzymatically reducible cytochrome b was the same for all membranes after subjection to the various types of treatment. In contrast to cytochrome b, the reduced steady state levels of cytochrome c, and a + a 3 were not altered by heat or freeze treatment.

The mechanism of cytochrome b5 reduction by NADPH-cytochrome c reductase

Microsomal suspensions of liver can catalyze the reduction of cytochrome b 5 by the membrane-bound NADPH-cytochrome c reductase but the highly purified NADPH-cytochrome c reductase obtained by lipase solubilization does not reduce cytochrome b 5 under normal assay conditions. However, the purified reductase does reduce cytochrome b 5 in solutions containing high concentrations of salt. Although the flavoprotein rapidly generates superoxide anion in the presence of high concentrations of salt, the reduction of cytochrome b 5 is not dependent upon superoxide anion generation but possibly occurs via a direct interaction between the flavoprotein and the cytochrome. Further, the mechanism of cytochrome b 5 reduction involves the cycling of the enzyme between its fully reduced and half-reduced forms during the steady-state reduction of cytochrome. The reduction of cytochrome b 5 by detergent- and proteolytically solubilized NADPH-cytochrome c reductase is similar in the presence of high concentrations of KCl. However, with low salt concentrations, only the detergent-solubilized NADPH-cytochrome c reductase reduces cytochrome b 5, albeit slowly. No apparent differences in rate of reduction or steady-state level of cytochrome b 5 reduction is seen when either the detergent- or proteolytically prepared cytochrome b 5 is employed.

The preparation and properties of a solubilized respiratory complex from Escherichia coli

1. 1. Treatment of the respiratory particle fraction from Escherichia coli with cholate in the presence of ammonium sulfate liberated a stable, soluble, cytochrome-containing moiety (“soluble respiratory complex”) of “molecular weight” of about 2×10 6. 2. 2. The soluble respiratory complex contained nonheme iron, ubiquinone, cytochrome b 1, cytochrome o, acid-resistant flavin and acid-released flavin in a ratio of 56:18:3.7:0.96:0.7:1. 3. 3. The soluble respiratory complex was devoid of NADH and succinate oxidase activities until ubiquinone isoprenologues were added. Ubiquinone Q-2 was the most effective derivative in restoring oxidase activities. 4. 4. Reactivation of succinate oxidase by Q-2 was produced by a 10-fold lower concentration than that required for NADH oxidase. 5. 5. Addition of Q-2 lowered the aerobic steady state level of cytochrome b 1 reduction in the soluble respiratory complex with succinate but not with NADH as substrate. The inhibitor 2- n-heptyl 4-hydroxyquinoline N-oxide (HQNO) increased this value with the former but not with the latter substrate. Thus, deficiency of ubiquinone and addition of HQNO give a similar effect on the succinate-reducible cytochrome b 1. 6. 6. It is proposed that in the soluble respiratory complex part at least of the cytochrome b 1 reducible by succinate is either on a side-branch from the main respiratory chain or that Q-2 interacts between succinate-reducible cytochrome b 1 and oxygen.

The reoxidation of cytoplasmic reducing equivalents in Ehrlich ascites tumor cells

1. Addition of glucose to ELD (Ehrlich-Lettre' hyperdiploid) ascites tumor cells inhibited by rotenone caused an oxidation-reduction cycle of cytochrome b and oxidation of cytochrome c but it did not affect the inhibition of the endogenous respiration. 2. The effect of substrates, inhibitors, and uncouplers of oxidative phosphorylation on the transition between the oxidized and the reduced steady state of cytochrome b induced by glucose in rotenone-inhibited cells was tested. The spectrophotometric data obtained support the hypothesis that this transition is determined by the initial lowering and the subsequent increase in the phosphate potential in the cytoplasmic compartment of the glycolyzing cell. 3. The reduction of cytochromes b and c induced by glucose in rotenone-inhibited cells in the presence of vitamin K 3 was shown to be biphasic. This reduction, like that produced by vitamin K 3 alone, was significantly increased by oligomycin or aurovertin, while the biphasicity of the response was no longer apparent with these inhibitors. 4. Pretreatment with pyruvate of rotenone-inhibited cells made it possible to show, upon addition of glucose, a rapid translocation ( t 1 2 about 700 msec) of reducing equivalents to a cytochrome b-type pigment with small or undetectable changes in the steady-state level of cytochrome c. This reaction, which may be tentatively assigned to the microsomal cytochrome b 5, was not competitive with the transfer of reducing equivalents to the mitochrondria in the presence of vitamin K 3. 5. It can be concluded that in the strain of ascites cells used no direct transport of reducing equivalents occurs under physiological conditions from the cytosol to the mitochondrial cytochromes. On the other hand a pathway for the extramitochondrial oxidation of the cytosolic reducing equivalents has been evidentiated.

The effect of chlorobutanol on the respiratory metabolism and on the normal properties of isolated mitochondria

The possible site of action of chlorobutanol upon the respiratory metabolism was investigated in rat heart, cerebrum, and cerebellum preparations. The behavior of this compound on respiration of slices, homogenates, and mitochondria showed that its inhibitory effect was more evident when NAD-dependent dehydrogenases were involved in the primary oxidation of substrates, although a more drastic effect of chlorobutanol on the mitochondria could be obtained, since chlorobutanol is also an antiseptic. Nevertheless, chlorobutanol in suitable amounts was used to determine its effect on the normal properties of mitochondria-respiration, oxidative phosphorylation, respiratory control coefficient—and on the steady-state levels of cytochrome b from heart sarcosomes.