Cytochrome synthesis in synchronous cultures of the yeast, Saccharomyces cerevisiae.

The synthesis of cytochromes aa3, b, and c has been investigated during synchronous growth in the yeast, Saccharomyces cerevisiae. These cytochromes increase in concentration continuously throughout each cell cycle, with an approximate doubling in rate during successive cycles. The rates of cytochrome formation are considerably higher in galactose-grown cultures than in cells grown in glucose. Although cytochrome aa3 increases at a continuous rate, its functional counterpart, cytochrome c oxidase, increases in stepwise fashion, with the increments occurring at the beginning of each new cell cycle. Chloramphenicol, a specific inhibitor of intramitochondrial protein synthesis, inhibits the formation of cytochrome aa3 at all stages of the cell cycle, but does not inhibit cytochrome c. Chloramphenicol exhibits a somewhat intermediate effect on cytochrome b synthesis, with transient inhibition occurring only when the drug is added prior to or during the initial part of the first cell cycle. After this time, chloramphenicol had no effect on the rate of cytochrome b synthesis. The data indicate that under our conditions of cell synchrony mitochondrial membrane formation as reflected by increments in mitochondrial cytochromes occurs by continuous accretion of new material throughout the cell cycle. Intramitochondrially synthesized polypeptide products, responsible for the formation of new cytochrome aa3, appear to be synthesized throughout the cell cycle.     

The "SUN" family: UTH1, an ageing gene, is also involved in the regulation of mitochondria biogenesis in Saccharomyces cerevisiae

Since it was shown in previous work that NCA3 (one of the four genes of the SUN family) is involved in mitochondrial protein synthesis regulation, the effect of the other members of this gene family was tested. UTH1 (but not SUN4 or SIM1) was also shown to interfere with mitochondria biogenesis. In Deltauth1 cells, cytochromes aa(3), c, and b were lowered by 25 and 15%, respectively. In the double-null mutant Deltauth1Deltanca3, only cytochrome aa(3) was lowered by 50% relative to the wild type. However, the ratio of cellular respiration to cytochrome oxidase was greatly enhanced in the double-null mutant. Measurements on whole lysed cells showed that another mitochondrial enzyme, citrate synthase, was also lowered in Deltauth1 and Deltauth1Deltanca3 whereas hexokinase was not. Electron micrographs showed no difference in global mitochondria content in Deltauth1Deltanca3, but mitochondria appeared less dense to electrons compared to the wild type. Cardiolipin and mtDNA were equivalent in parental and mutant strains. Measurements on isolated mitochondria showed that the cyt aa(3)/cyt b ratio was also lowered in Deltauth1Deltanca3, but the control exerted by the oxidase on the respiratory flux was higher. The activity of other mitochondrial complexes versus oxidase was equivalent in mutants compared to the wild type. These results suggest that the protein equipment could be lowered in mitochondria from strains inactivated for UTH1.     

Terminal oxidases in the trypanosomatid Trypanosoma cruzi

Titration of Trypanosoma cruzi respiration with cyanide, with results treated as Dixon plots, indicated the presence of several terminal oxidases. The inhibitions obtained at low cyanide concentrations (0-300 microM), taken together with cyanide effects on cytochrome aa3-deficient, dyskinetoplastic epimastigotes, supported cytochrome aa3 as T. cruzi main terminal oxidase. By increasing cyanide concentration to 1.0 mM, two alternative terminal oxidases could be detected. One of these was active in both kinetoplastic and dyskinetoplastic (cytochrome aa3-deficient) epimastigotes, and azide- and antimycin-insensitive. Complementary cytochrome studies with intact epimastigotes and mitochondrial membranes revealed the presence of cytochromes aa3, b, c558, o and possibly d, as components of the parasite electron transport system. Fractionation studies demonstrated that both o and d were bound to the mitochondrial membrane. Reduction by endogenous substrates and complex formation with cyanide supported cytochrome o as alternative terminal oxidase. EB-cultured, dyskinetoplastic epimastigotes showed the same respiration rate as the kinetoplastic cells, despite the significant decrease of cytochrome aa3, thus indicating adaptive mechanisms that determine the expression of alternative oxidases, whenever the main terminal activity is depressed.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.     

Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.

Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.     

Postnatal development of energy metabolism in the rat brain

The activities of cytochrome c oxidase and F0F1-ATPase as well as the content of cytochromes cc1, aa3, and b were investigated in free brain mitochondria in the course of postnatal development and aging. The results show an increase of Vmax of both enzymes during postnatal development (between day 5 and 30). During the following phase ending at the age of 6 months, a decrease of F0F1-ATPase and cytochrome c oxidase activity occurs. From 6 to 12 months of age the activity of these enzymes did not change. The KM for both enzymes remained unchanged during the whole period observed. The content of cytochromes increased from the low values found in young rats, reached the highest values at around one month, and decreased till the age of 3 months. Later, their content in brain mitochondria did not markedly change. Our results suggest that the metabolic maturation of brain mitochondria differs in several aspects from the same process in other tissues, mainly in the time course. This is probably due to the unique role of neural tissue in the organism.     

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.     

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.     

Membrane-bound cytochromes in a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F

Cytoplasmic membranes were isolated from the cells of a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F and membrane-bound cytochromes were characterized. Redox difference spectra at 77 K revealed the presence of cytochromes with the alpha peaks at 552 and 556 nm while CO-binding difference spectra showed the presence of o-type cytochrome(s). Partial purification of the cytochromes demonstrated that the membranes contain cytochromes c550, c551, c556 and possibly d1 besides high molecular mass cytochrome c and cytochrome c3. It turned out that two kinds of novel CO-binding c-type cytochromes are present in the membrane. The membranes and a partially purified fraction showed weak ubiquinol-1 oxidase activity but no cytochrome c oxidase activity. Results suggest that D. vulgaris does not express the heme-copper terminal oxidase under our growth conditions in spite of the presence of the col gene, which is homologous to the gene of subunit I of the aa3-type oxidase.     

Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross-immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side.     

Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation.

Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli.     

Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration

The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3). The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C. At liquid nitrogen temperature, CO-rebinding did not occur. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.     

Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1

The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE). The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3). Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3). If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.     

Regulation of mitochondrial membrane assembly in Neurospora crassa. Transient expression of a respiratory mutant phenotype.

Cultures of mutant cni-1, a chromosomal mutant of Neurospora crassa, undergo a marked change in respiratory properties as the age of the culture increases. Early log phase cultures have a high level of respiration that is insensitive to inhibition by cyanide or antimycin A. Late log and stationary phase cultures have reduced rates of respiration. A high percentage of this respiration is inhibited by cyanide. Mitochondria from early log phase cni-1 have an excess of cytochrome c and little or no detectable cytochrome aa3. Mitochondria from late log and stationary phase cultures have levels of c-, b-, and a-type cytochromes that are not significantly different in concentration from those found in wild type cells. The cytochrome aa3 content and the cytochrome oxidase activity of cni-1 mitochondria increase 5- to 10-fold as the age of the culture increases. Mitochondria from early log phase cells of cni-1 synthesize only polypeptides of apparent molecular weights 7,000 to 10,000 and donot synthesize any of the mitochondrial components of cytochrome oxidase. Mitochondria from late log and stationary phase cells synthesize the normal complement of mitochondrial translation products including the mitochondrial components of cytochrome oxidase. The assembly of cytochrome oxidase is likely due to the availability of the mitochondrially synthesized components of the enzyme. The regulation of mitochondrial translation in the cni-1 mutant is independent of the nutrient content of the growth medium and is due to the accumulation or depletion of some component within the cell.     

Altered Mitochondrial Respiration in a Chromosomal Mutant of Neurospora crassa

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.     

Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants

Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551. Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their K(m) values were 0.04-0.12 microM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa(3) oxidase has already been established.     

Nuclear suppressors of the (poky) cytoplasmic mutant in Neurospora crassa. II. Mitochondrial cytochrome systems.

The mitochondrial cytochrome aa3 and b deficiencies of the [poky] cytoplasmic mutant of Neurospora crassa are partially suppressed by mutant alleles of any one of six nuclear genes, namely sup-1, sup-3, sup-4, sup-5, sup-10 and sup-14. The suppressor-induced increases in the concentration of both cytochromes are detected in the mitochondria from exponentially growing [poky] cultures, and, thus, are clearly distinguishable from the age-dependent changes in the cytochrome system that occur in cultures that approach, or have reached, the stationary phase of growth. The relative amounts of mitochondrial cytochromes aa3 and b show a direct correlation with the relative efficiency of the various sup genes as suppressors of the slow-growth phenotype of [poky]. Since [poky] is defective in mitochondrial protein synthesis due to a lack of 30 S mitochondrial ribosomal subunits, it is proposed that the six suppressors promote the assembly of functional mitochondrial ribosomes.     

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.     

Studies on the orientations of the mitochondrial redox carriers. II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated, oriented mitochondrial multilayers.

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c1, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the "tightly bound" mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90 degrees.