Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species

Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome.     

Effects of selected inhibitors on electron transport in Neisseria gonorrhoeae.

The electron transport system of Neisseria gonorrhoeae was partially characterized by using spectrophotometric, spectroscopic, and oxygen consumption measurements. The effects of selected electron transport inhibitors (amytal, rotenone, 2-heptyl-4-hydroxyquinoline, antimycin A1, and potassium cyanide [KCN]) on electron transfer in whole-cell and sonically treated whole-cell preparations of N. gonorrhoeae were examined. The oxidation of reduced nicotinamide adenine dinucleotide, measured as a decrease in absorbance at 340 nm, was inhibited by each of the compounds tested. Oxygen consumption stimulated by reduced nicotinamide adenine dinucleotide was also inhibited, whereas oxygen uptake stimulated by succinate and malate was inhibited by KCN alone, suggesting the presence of a KCN-sensitive terminal oxidase. Room temperature optical difference spectra indicate an operational electron bypass around the amytal-rotenone-binding site. Difference spectra in the presence of 2-heptyl-4-hydroxyquinoline suggest a possible site of interaction of this compound at the substrate side of cytochrome b. Reduced-minus-oxidized spectra of ascorbate-tetramethyl-p-phenylenediamine suggest the participation of b-, a-, and d-type cytochromes in terminal oxidase activity. Hence, N. gonorrhoeae appears to have an electron transport chain containing cytochrome c, two b-type cytochromes (one of which has an oxidase function), and possibly a- and d-type cytochromes. An abbreviated chain exists through which succinate and malate can be oxidized directly by a KCN-sensitive component.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : V. On the Reduction of Nonheme Iron and the Cytochromes by Nicotinamide Adenine Dinucleotide and Succinate

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Influences of growth substrates and oxygen on the electron transport system in Acinetobacter sp. HO1-N.

The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Metabolism of Pipecolic Acid in a Pseudomonas Species IV. Electron Transport Particle of Pseudomonas putida

Baginsky, Marietta L. (University of California, San Francisco Medical Center, San Francisco), and Victor W. Rodwell. Metabolism of pipecolic acid in a Pseudomonas species. IV. Electron transport particle of Pseudomonas putida. J. Bacteriol. 92:424-432. 1966.-Enzymes of Pseudomonas putida P2 catalyzing oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate are located in a subcellular fraction sedimenting at 105,000 x g. Since this fraction resembles the mammalian electron transport particle in both chemical composition and enzymatic activities, it was termed Pseudomonas P2 electron transport particle (P2-ETP). P2-ETP contains flavin adenine dinucleotide, flavin mononucleotide, iron, copper, and both b- and c-type cytochromes. The reduced type b cytochrome has absorption maxima at 558 to 559, 530, and 427 mmu. Its oxidized pyridine hemochromogen has an absorption maximum at 406 mmu, with a shoulder at 564 mmu. On dithionite reduction, absorption bands with maxima at 556, 522, and 418 mmu are obtained. The reduced type c cytochrome has absorption maxima at 552, 520, and 422 mmu; its reduced pyridine hemochromogen has maxima at 551, 516 to 519, and 418 mmu. No type a cytochrome was detected. P2-ETP catalyzes oxidation of pipecolate and of reduced nicotinamide adenine dinucleotide (NADH(2)) by oxygen. It can also oxidize these compounds, as well as succinate and reduced nicotinamide adenine dinucleotide phosphate, with 2,6-dichlorophenol-indophenol as electron acceptor. Mammalian cytochrome c can be used as an alternate artificial electron acceptor for the oxidation of pipecolate and succinate, but not for oxidation of NADH(2).     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.

Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.     

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.     

Electron Transport System Associated with Membranes of Bacillus cereus During Vegetative Growth and Sporulation

Membranes isolated from Bacillus cereus ATCC 4342 during vegetative growth and during sporulation contained cytochromes b, c and a + a(3) as well as flavoprotein as determined from reduced-minus-oxidized difference spectra. Although there appeared to be no qualitative change in the cytochromes, there was a significant increase in the amount of cytochromes associated with membranes isolated from sporulating cells. Succinate and nicotinamide adenine dinucleotide (reduced form) (NADH) reduced the same cytochromes indicating similar pathways of electron transport. The electron transport inhibitors-cyanide, azide, 2-heptyl-4-hydroxyquinoline-N-oxide, dicumarol and atebrine-were examined for their effect on succinate oxidase (succinate: [O(2)] oxidoreductase) and NADH oxidase (NADH: [O(2)] oxidoreductase). NADH oxidase associated with vegetative cell membranes was less sensitive to certain inhibitors than was succinate oxidase, suggesting a branched electron transport pathway for NADH oxidation. In addition to electrons being passed to O(2) through a quinone-cytochrome chain, it appears that these intermediate carriers can be bypassed such that O(2) is reduced by electrons mediated by NADH dehydrogenase. Both oxidases associated with sporulating cell membranes were inhibited to a lesser degree than were the oxidases associated with vegetative cell membranes.     

Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

A succinate-coenzyme Q reductase (complex II) was isolated in highly purified form from Ascaris muscle mitochondria by detergent solubilization, ammonium sulfate fractionation and gel filtration on a Sephadex G-200 column. The enzyme preparation catalyzes electron transfer from succinate to coenzyme Q1 with a specific activity of 1.2 mumol coenzyme Q1 reduced per min per mg protein at 25 degrees C. The isolated complex II is essentially free of NADH-ferricyanide reductase, reduced CoQ2-cytochrome c reductase and cytochrome c oxidase and consists of four major polypeptides with apparent molecular weights of 66 000, 27 000, 12 000 and 11 000 and two minor ones with Mr of 36 000 and 16 000. The complex II contained cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria, at a concentration of 3.6 nmol per mg protein, but neither other cytochromes nor quinone. The cytochrome b-558 in the complex II was reduced with succinate. In the presence of Ascaris NADH-cytochrome c reductase (complex I-III) (Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134), the cytochrome b-558 in complex II was also reduced with NADH and reoxidized with fumarate. These results suggest the cytochrome b-558 to function as an electron carrier between NADH dehydrogenase and succinate dehydrogenase in the Ascaris NADH-fumarate reductase system.     

Physiological Effects of Menaquinone Deficiency in Bacillus subtilis

Several aspects of the respiratory physiology of a mutant of Bacillus subtilis deficient in menaquinone-7 (MK-7) and in cytochromes were investigated. The mutant, an aromatic amino acid auxotroph blocked at dehydroshikimate reductase, is unable to synthesize MK-7 unless grown in the presence of the common aromatic amino acid intermediate, shikimate. The inability to synthesize MK-7 prevents the mutant from expressing the normal postexponentialphase cytochrome phenotype. When grown in the presence of shikimate, normal levels of these electron transport components are formed. It was found that the intracellular concentration of MK-7 could be predictably regulated by growing the cells with known concentrations of exogenous shikimate. When the mutant was grown under conditions where MK-7 biosynthesis was severely limited, there was a decrease in oxygen uptake and in membrane-associated reduced nicotinamide adenine dinucleotide (NADH) oxidase and succinate oxidase activity. NADH oxidase, but not succinoxidase, could be restored in membrane preparations by the addition of menadione to the reaction mixture. Reduced-minus-oxidized cytochrome difference spectra indicate that an MK-7 deficiency limits electron flow through the cytochrome chain. Furthermore, oxidation-reduction patterns suggest that MK-7 functions between the primary dehydrogenases and the cytochromes. Although the mutant is asporogenous when grown under conditions where MK-7 biosynthesis is limited, the inability to sporulate does not appear to result from lesions in the electron transport system.     

Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus.

Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide-insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha-ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger.     

The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris.

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically growth Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three beta-types cytochromes b561, b560 and b558, and at least two c-type cytochromes c556 and c2 as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b560, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c'. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b561 with associated beta and gamma bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c2 may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.     

Respiratory Development in Saccharomyces cerevisiae Grown at Controlled Oxygen Tension

Saccharomyces cerevisiae was grown in batch culture over a wide range of oxygen concentrations, varying from the anaerobic condition to a maximal dissolved oxygen concentration of 3.5 muM. The development of cells was assayed by measuring amounts of the aerobic cytochromes aa(3), b, c, and c(1), the cellular content of unsaturated fatty acids and ergosterol, and the activity of respiratory enzyme complexes. The half-maximal levels of membrane-bound cytochromes aa(3), b, and c(1), were reached in cells grown in O(2) concentrations around 0.1 muM; this was similar to the oxygen concentration required for half-maximal levels of unsaturated fatty acid and sterol. However, the synthesis of ubiquinone and cytochrome c and the increase in fumarase activity were essentially linear functions of the dissolved oxygen concentration up to 3.5 muM oxygen. The synthesis of the succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase complexes showed different responses to changes in O(2) concentration in the growth medium. Cyanide-insensitive respiration and P(450) cytochrome content were maximal at 0.25 muM oxygen and declined in both more anaerobic and aerobic conditions. Cytochrome c peroxidase and catalase activities in cell-free homogenates were high in all but the most strictly anaerobic cells.     

Chlorpromazine inhibition of electron transport in Azotobacter vinelandii membranes

Chlorpromazine was a potent inhibitor of O2-dependent malate oxidation, but not of H2 oxidation in Azotobacter vinelandii membranes. However, chlorpromazine did not significantly affect the activity of malate reductase or the reduction of cytochromes c and d. In the presence of chlorpromazine, cytochrome o failed to form a complex with CO. The site of action of chlorpromazine seems to be in the cytochromes c to cytochrome o branch, the pathway utilized by malate, succinate and NADH, but not by H2.     

Phenotype of a Temperature-Sensitive, Respiration-Deficient (cyt) Mutant of Yeast

A temperature-sensitive respiration-deficient mutant of yeast lacks hemoproteins and accumulates coproporphyrin III when cultivated at elevated temperatures. Cells grown at 20 C respired normally and contained cytochromes a, b, and c. Cells grown at 35 C showed respiration-deficient mutant characters; they did not respire, lacked cytochromes, and accumulated coproporphyrin III. Addition of protoporphyrin IX or protohemin IX to the culture medium restored the respiratory activity of this mutant during growth at 35 C. The activities of various enzymes, including succinate-2,6-dichlorophenol indophenol (DCPIP), reduced nicotinamide adenine dinucleotide (NADH(2))-DCPIP, succinate-cytochrome c, and NADH(2)-cytochrome c oxidoreductase, and cytochrome oxidase, and the cytochrome c content of cells cultured in various conditions were determined. Changes in the number and structure of mitochondria were associated with changes in respiratory activity.     

Effect of aeration intensity on the biochemical composition of baker's yeast. II. Activities of the oxidative enzymes

When the effect of catabolite repression is eliminated Saccharomyces cerevisiae prefers an aerobic metabolism. The potential for completely aerobic catabolism exists even in circumstances where its action is limited by the oxygen available. When the oxygen absorption in the medium is adequate, yeast uses a solely oxidative metabolism for energy-yielding reactions. The changes observed in the activity of malate dehydrogenase can be described as a function of two isoenzymes, both of which are affected by oxygen; the isoenzyme participating in the glyoxylate cycle shows variations in activity similar to that observed in isocitrate lyase. NAD-linked glutamate dehydrogenase activity roughly follows that of malate dehydrogenase and isocitrate lyase; in cultivations with the same growth rate the NADP-linked dehydrogenase is insensitive to the oxygen level. The cytochromes aa₃, b, and c have a clear maximum at low oxygen tension, the most sensitive being cytochrome aa₃. The imbalance between cytochrome c:oxygen oxidoreductase activity and the amount of cytochrome aa₃, and the correlation observed between respiration rate and the activities of cytochrome c oxidase and NADH₂:cytochroine c oxidoreductase are discussed. Methods used for estimation of cytochromes are compared.