Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.     

Cytochrome c-linked reactions in Rhodopseudomonas palustris grown photosynthetically on thiosulfate

The nonsulfur purple bacterium Rps. palustris was adapted to grow photoautotrophically with thiosulfate as substrate. An isolated cell-free fraction catalyzed the enzymatic transfer of electrons from thiosulfate to endogenous and/or added mammalian cytochrome c. Antimycin A, NOQNO, rotenone, amytal and atebrin did not inhibit the thiosulfate-cytochrome c reductase. The products of thiosulfate oxidation were primarily tetrathionate, trithionate, and sulfate, suggesting oxidation via the polythionate pathway. Succinate, formate and NADH were also effective electron donors in this system showing Michaelis constants of 40, 30 and 0.025 mm, respectively for cytochrome c reduction. The NADH-cytochrome c reductase was not inhibited by flavoprotein inhibitors and by Antimycin A or NOQNO. The cell-free extracts also contained an active cytochrome c-O₂ oxidoreductase which was inhibited by cyanide, azide and EDTA, and these inhibitions were overcome by the addition of Cu²⁺. The oxidase activity was stimulated by the addition of uncoupling agents such as CCCP and DNP, as well as by Antimycin A and NOQNO. Reduced + CO minus reduced difference absorption spectra revealed the presence of cytochrome components of the a and o types which may function as the terminal oxidase(s).     

Characterization of the Proton-Translocating Cytochrome c Oxidase Activity in the Plasma Membrane of Intact Anacystis nidulans Spheroplasts

Intact spheroplasts of the cyanobacterium (blue-green alga) Anacystis nidulans oxidized various exogenous c-type cytochromes with concomitant outward proton translocation while exogenous ferricytochrome c was not reduced. The H⁺/e⁻ stoichiometry was close to 1 with each of the cytochromes and did not depend on the actual rate of the oxidase reaction. Observed proton ejections were abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Cyanide, azide, and carbon monoxide inhibited cytochrome c oxidation and proton extrusion in parallel while dicyclohexylcarbodiimide affected proton translocation more strongly than cytochrome c oxidation. The cytoplasmic membrane of A. nidulans appears to contain a proton-translocating cytochrome c oxidase similar to the one described for mitochondria.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria

1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP⁺-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD⁺- and NADP⁺-linked), glutamate dehydrogenase (NAD⁺-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.     

Biochemistry and physiological role of methionine sulfoxide residues in proteins

The cytochrome system in eggs and embryos of the sea urchin, Hemicentrotus pulcherrimus, was investigated. Difference spectra of the mitochondrial fraction demonstrated the presence of a complete cytochrome system in unfertilized eggs. Cytochrome levels and the activities of respiratory enzymes were measured in crude extracts of eggs both before and after fertilization. Unfertilized eggs contained cytochromes aa₃, b, and c + c₁ in a ratio of 1.0:1.8:0.7. Gastrulae contained almost the same amount of cytochromes aa₃and b as unfertilized eggs. However, the amount of cytochrome c + c₁ in gastrulae was 1.5 times greater than that in unfertilized eggs. The activity of cytochrome oxidase remained unchanged during development. No cytochrome oxidase inhibitor was found in unfertilized eggs. Both antimycin A-sensitive and insensitive NADH-cytochrome c reductase activities increased during development. The activity of succinate-cytochrome c reductase increased during early development, reached a temporary plateau, and then declined at the pluteus stage. These results are discussed in relation to the increase of respiration during early development.     

Molybdenum Oxidation by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans AP19-3 oxidized molybdenum blue (Mo⁵⁺) enzymatically. Molybdenum oxidase in the plasma membrane of this bacterium was purified ca. 77-fold compared with molybdenum oxidase in cell extract. A purified molybdenum oxidase showed characteristic absorption maxima due to reduced-type cytochrome oxidase at 438 and 595 nm but did not show absorption peaks specific for c-type cytochrome. The optimum pH of molybdenum oxidase was 5.5. The activity of molybdenum oxidase was completely inhibited by sodium cyanide (5 mM) or carbon monoxide, and an oxidized type of cytochrome oxidase in a purified molybdenum oxidase was reduced by molybdenum blue, indicating that cytochrome oxidase in the enzyme plays a crucial role in molybdenum blue oxidation.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N′,N′-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827–5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(l-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.     

Bovine cytochrome c oxidases,purified from heart,skeletal muscle,liver and kidney,differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.     

Methionine-oxidized horse heart cytochromes c. I. Reaction with Chloramine-T,products, and their oxidoreduction properties

Spectroscopically, the modification of horse heart ferricytochrome c with N-chloro-4-toluolsul-fonamide (Chloramine-T, CT) occurs through a two-step process, the disruption of the methionine-80 sulfur-iron linkage and a reagent-independent change, an intramolecular rearrangement. Chromatographic purification of the preparation at a 2.5:1 reagent-to-protein ratio, pH 8.0–8.5, yields two major products, the F^II and F^III CT-cytochromes c. Both products contain modification of only the methionines, 80 and 65, to sulfoxides; both are monomeric, reduced by ascorbate, and the ferrous forms are oxidized by molecular oxygen and bind carbon monoxide. The redox potentials of F^II and F^III are 135 and 175±15 mV. The F^III is indistinguishable from the native protein in its binding and the electron donor property toward mammalian cytochrome c oxidase. It also binds nearly as effectively as the native protein to yeast cytochrome c peroxidase, but is a less efficient donor. It is, however, a poor electron acceptor from both mammalian cytochrome c reductase and chicken liver sulfite oxidase. F^II lacks cytochrome c oxidase activity and is also a poorer substrate for the other three enzymes. Both the derivatives are consistently better electron donors than acceptors. It is concluded that the binding of cytochrome c to cytochrome c oxidase and to cytochrome c peroxidase does not require the integrity of the methionine-80 sulfur linkage and that the complexation process has a finite degree of freedom with regard to the state of the heme crevice opening. The alterations of the oxidoreduction function have been analyzed in light of both prevailing models of cytochrome c function, the two-site model (one site for oxidizing and the other for reducing enzymes) and the single-site model (the same site for the oxidizing and reducing enzymes). These observations can be accommodated by either model, given the latitude that the binding domains for the oxidizing and the reducing enzymes have finite overlapping and nonoverlapping regions.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Short-term effects of carbon dioxide on carnation callus cell respiration

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O₂ uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO₂ in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO₂ again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO₂/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO₂/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO₂/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO₂) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.     

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.     

The effect of phenolic compounds on the activity of respiratory chain enzymes and on respiration and phosphorylation activities of potato tuber mitochondria

The effect of derivatives of benzoic and cinnamic acids, quereetin,p-benzoquinone, and 2,5-dimethylbenzoquinone on oxygen consumption mitoehondrial suspensions and on the activity of some respiratory chain enzymes was studied. Benzoquinone and 2,5-dimethylbenzoquinone highly significantly inhibited the respiration and phosphorylation rates and malate- and succinate dehydrogenase activities. Chlorogenic acid, similarly as the quinones, very significantly inhibited the activities of the studied dehydrogenases but did not affect cytochrome oxidase. Oxygen consumption by intact mitochondria was not inhibited, only the oxidativo phosphorylation was significantly uncoupled. Quereetin significantly enhanced dehydrogenase activities and completely inhibited cytochrome oxidase activity. The respiration and phosphorylation activities of the mitochondria were significantly inhibited by quereetin. The effect of the other phenolic compounds studied on respiration and phosphorylation activities was not significant. Succinate dehydrogenase activity was the most affected enzyme among the respiratory chain enzymes. It was significantly inhibited by all the above phenolic compounds at 1^-4M or 5 10^-5M concentrations with the exception of gallic acid.     

Renal enzyme changes in pekin ducks (Anas platyrynchos) after combined administration of methylmercury,cadmium and lead

1. The combined effect of methylmercury, cadmium and lead on renal enzymes of pekin ducks was studied.2. Renal acid phosphatase and glutathione S-transferase activities were not affected by heavy metal treatment.3. Renal δ-aminolevulinic acid dehydratase activity was decreased significantly in ducks treated with lead alone or when lead was co-administered with methylmercury.4. Renal cytochrome c oxidase activity was decreased significantly when methylmercury was co-administered with cadmium and/or lead.5. The findings suggest that lead had the main effect on 5-aminolevulinic acid dehydratase and methylmercury had the main effect on cytochrome c oxidase activity. Interaction effect was also observed in cytoehrome c oxidase activity.     

The morphological site of synthesis of cytochrome c in mammalian cells (Krebs cells)

In Krebs ascites-tumour cells, cytochrome c is segregated in the mitochondria and the level in microsomes could not be measured. At 22° in glucose–buffer Krebs cells synthesized a spectrum of proteins including cytochrome c. Mild osmotic shock in the presence of ribonuclease had little effect on incorporation of [¹⁴C]-leucine or [¹⁴C]valine into mixed mitochondrial protein but strongly inhibited synthesis of non-mitochondrial cytoplasmic proteins. Under these conditions, labelling of cytochrome c was also strongly inhibited. After pulse labelling of Krebs cells at 22° for 10min. the cytcchrome radioactivity found in mitochondria was higher than in microsomes. After addition of unlabelled amino acid as `chase' there was 137% increase in radioactivity of cytochrome c but only a 3% increase in radioactivity of whole-cell protein. It is concluded that the peptide chain of cytochome c is synthesized on cytoplasmic ribosomes. Mitochondria therefore do not have the character of self-replicating entities, but are formed by the cooperative function of messenger RNA of cytoplasmic ribosomes and, possibly, of intramitochondrial messenger derived from the mitochondrial DNA.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ? 20% of the free amines are modified, inhibits ascorbate-N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ? 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc₁ and aa₃ complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.     

Derepression of mitochondria and their enzymes in yeast: regulatory aspects

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l₈-isocitrate. Cytochromes b, c₁, and aa₃ are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa₃, b, and c₁; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.