Yeast Cytochrome Oxidase Research Articles

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K). In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration. Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined. Sequence determinations of subunits IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids. The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.

Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c.

To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.

Translation of RNA that contains polyadenylate from yeast mitochondria in an Escherichia coli ribosomal system.

RNA that contains poly(A) [poly(A)-RNA] has been isolated from yeast mitochondria by poly(U) Sepharose-4B column chromatography. Pulse-labeled poly(A)-RNA shows 8-10 discrete peaks by acrylamide gel electrophoresis. The specific activity of mitochondrial poly(A)-RAN is six to eight times greater than that of mitochondrial rRNA after pulse labeling of protoplasts with [3H-]uridine. Ethidium bromide inhibits incorporation by over 90%. The total mitochondrial RNA preparation was contaminated with 5-15% cytoplasmic rRNA as determined by gel electrophoresis, but RNA exhaustion hybridization experiments indicated little or no cytoplasmic contamination of the mitochondrial poly(A)-RNA. The poly(A)-RNA stimulates [3H]leucine incorporation into protein in an E. coli cell-free system. A fraction of the labeled product is precipitated with antibody directed toward yeast cytochrome oxidase, but not with antibody directed toward bovine serum albumin. Sodium dodecyl sulfate gel electrophoresis of the immunoprecipitated material reveals labeled peptides having the mobility of the three larger cytochrome oxidase peptides, which are known to be translated by mitochondrial ribosomes.

Studies on the mechanism of the oxygen-induced synthesis of cytochrome oxidase in yeast. Specific effects of benzimidazole on cytochrome-oxidase anabolism.

At concentrations where benzimidazole inhibited partially total protein synthesis in yeast, the drug also inhibited protein synthesis mediated by cytoplasmic ribosomes and by isolated mitochondria in vitro. Except for cytochrome oxidase, the effects of benzimidazole on the anabolism of hemoproteins were also partial and unspecific; the induction by oxygen of the particulate cytochromes b and c1, the synthesis of which is determined by the obligate co‐operation of both protein‐synthesizing systems, was not affected more than the induction of catalase, an extramitochondrial hemo‐protein, which is synthesized exclusively in cytoplasm. The drastic inhibition of cytochrome oxidase was the consequence of a specific blocking of cytochrome (a, a3) production. The delayed adaptation to oxygen, following the exclusion of benzimidazole, was characterized by its exceptionally high rate and correlated with the simultaneous increase of hemo‐protein (a, a3) content and the development of cytochrome oxidase activity; the latter activity ruled respiration rate. The process was largely unsensitive to the concerted actions of cycloheximide and chloramphenicol; under identical conditions, the synthesis of other hemoproteins was immediately stopped. The reconstitution of cytochrome oxidase in vivo at the expense of the double set of cytoplasmic and mitochondrial adaptation products, which were simultaneously accumulated, was established by reference to the specific effects of cycloheximide and chloramphenicol. As the prevailing effect of benzimidazole was operationally to switch off the assembly of cytochrome (a, a3), it is inferred that the drug will afford new experimental approaches in further studies of cytochrome oxidase anabolism.

Assembly of the Mitochondrial Membrane System: IX. PURIFICATION, CHARACTERIZATION, AND SUBUNIT STRUCTURE OF YEAST AND BEEF CYTOCHROME OXIDASE

Abstract Yeast cytochrome oxidase has been highly purified by a procedure involving solubilization in deoxycholic acid, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Standard preparations of beef cytochrome oxidase have been further purified by glycerol gradient centrifugation. The heme contents of the yeast and beef enzymes, respectively, were 15 and 12 nmoles of heme a per mg of protein. Yeast oxidase is composed of at least seven polypeptides when examined on polyacrylamide gels in the presence of sodium dodecyl sulfate. The molecular weights of the component polypeptides on 7.5% acrylamide gels range from 35,000 to 8,600. The beef enzyme has six subunits of molecular weights 34,500 to 7,300. The molecular weight of yeast cytochrome oxidase, determined by a combination of sucrose gradient centrifugation and gel filtration, was 226,000 after correction for bound detergent.

Mechanism of Induction of Cytochrome Oxidase in Yeast: I. Kinetics of Induction and Evidence for Accumulation of Cytoplasmic and Mitochondrial Precursors

Abstract The induction of cytochrome oxidase in Saccharomyces carlsbergensis was studied by following the time course of elaboration of (a) hemeprotein (or hemeproteins) having heme a as prosthetic group, measured spectrophotometrically at 605 mµ, and (b) the cytochrome oxidase activity, measured in cell-free extracts. The kinetics of induction was investigated after removal of inducer (air) and after the separate addition of cycloheximide, chloramphenicol, and acriflavine at different times during respiratory adaptation. The following results were obtained. 1. Anaerobically grown cells harvested from early stationary phase form hemeprotein a and cytochrome oxidase activity at a constant rate from the onset of aeration. Hemeprotein a reaches its maximal inducible level after 2 hours, whereas cytochrome oxidase activity is at a maximum after 3 hours of aeration. These levels are 33 to 37% of those of aerobically grown cells. The enzyme of induced or aerobically grown cells exhibits the same Km with respect to ferrocytochrome c. 2. Cells induced for periods up to 25 min possess the capacity to form additional cytochrome oxidase activity upon further incubation in the absence of air, whereas cells induced for longer periods are unable to do so. Cycloheximide added prior to aeration prevents the expression of this capacity, but chloramphenicol and acriflavine are without effect. 3. Separate addition of cycloheximide, chloramphenicol, and acriflavine prior to aeration allows the formation of different amounts of hemeprotein a and cytochrome oxidase activity. The amount of inhibitor-resistant synthesis of hemeprotein a is greater than that of cytochrome oxidase activity, and the kinetics of these syntheses varies with the inhibitor used. 4. Cells undergoing respiratory adaptation exhibit the capacity to form additional cytochrome oxidase activity upon further incubation with cycloheximide. This capacity is seen only during the 1st hour of induction, and its expression is inhibited by chloramphenicol. Conversely, cells induced for periods longer than 80 min can form additional cytochrome oxidase when further incubated with chloramphenicol, and the expression of this capacity is blocked by cycloheximide. During the 1st 80 min of induction, the cells can synthesize additional amounts of hemeprotein a when further incubated with either cycloheximide or chloramphenicol. 5. Cells induced for periods longer than 40 min form additional cytochrome oxidase when further incubated with acriflavine, and the expression of this capacity is inhibited by cycloheximide (between 60 and 100 min) and by chloramphenicol (between 40 and 80 min). 6. Cycloheximide inhibits the induction of cytochrome c, anaerobic growth, and incorporation of 14C-leucine into the proteins of adapting yeast, whereas chloramphenicol and acriflavine are not inhibitory.

The incorporation of [2- 14C]glycine and δ-amino[4- 14C]- laevulinic acid into the prosthetic groups of cytochrome oxidase and other cytochromes in yeast adapting to oxygen

1. 1. The concentration of cytochrome a + a 3 and other cytochromes and their prosthetic groups have been measured in resting anaerobically grown Sacchromyces cerevisiae “yeast foam” before and after adaptation to O 2. The increase in concentration of haem a and other haems parallels the increase in the haemoproteins in the adapted yeast. 2. 2. The incorporation of [2- 14C]glycine and δ-amino[4- 14C]laevulinic acid into haem a and other cytochrome prosthetic groups confirms that there is substantial biosynthesis de novo of haem a, cryptohaem a and other haems in adapting yeast. The labelled glycine is equally available for incorporation into the haem and protein moieties of cytochrome c. 3. 3. The uptake of [2- 14C]glycine into the withdrawal from the amino acid pool during adaptation has been measured. The concentration of glycine and other amino acids, excepting glutamic acid, is considerably diminished during adaptation. Glutamic acid had accumulated by the end of adaptation. 4. 4. After development of the cytochromes porphyrin synthesis continued, the main porphyrin being coproporphyrin. A new tetrapyrrole, coprobiliverdin, the formation of which is likely to be different from that of biliverdin, is also excreted from the yeast cells.

Cytochrome oxidase apoprotein in "petite" mutant yeast mitochondria. Reconstitution of cytochrome oxidase by combining apoprotein with cytohemin.

1 Cytochrome oxidase extracted from mitochondria of wild-type baker's yeast by means of bile salts and fractionated with ammonium sulfate had a specific activity about 18 times that of the mitochondria. When mitochondria of a cytoplasmic “petite” mutant were subjected to the same procedure, the resulting preparation exhibited virtually no cytochrome oxidase activity. 2 Gel filtration resolved the active enzyme preparation into five protein peaks, the first of which contained copper and cytohemin and showed cytochrome oxidase activity. Bio-Gel P-300 filtration of the inactive preparation from mitochondria of the “petite” mutant likewise gave five peaks. The first peak, however, while containing copper, was free from cytohemin and devoid of cytochrome oxidase activity. 3 When the material recovered from the first gel filtration peak of the active cytochrome oxidase preparation was subjected to disc electrophoresis on polyacrylamide, there appeared two protein bands. Two bands characterized by the same mobilities were also observed upon disc electrophoresis of the first peak material derived from mutant mitochondria. 4 The evidence for the presence in “petite” mutant mitochondria of a protein which is identical with or closely similar to the apoprotein of wild-type yeast cytochrome oxidase was confirmed by the finding that the incubation of submitochondrial particles of the “petite” mutant with cytohemin produced cytochrome oxidase activity. The enzymatic activity obtained was about 30% of that encountered in submitochondrial particles of wild-type yeast.

Biochemical correlates of respiratory deficiency—VIII. A precipitating antiserum against cytochrome oxdase of yeast and its use in the study of respiratory deficiency

Immunization of a rabbit with purified yeast cytochrome oxidase resulted in the formaiton of precipitating antibodies, which were employed in the study of crossreacting material from the membrane fraction of the DP-28 (Avers) respiratory dificient mutant of yeast. The presence of cross-reacting material was demonstrated both by immuno-diffusion experiments (Ouchterlony test) and by inhibition of enzyme-amtiserum precipitation.

Electron Transport Systems of Yeast: III. PREPARATION AND PROPERTIES OF CYTOCHROME OXIDASE

Abstract Cytochrome oxidase, prepared from the electron transport particles of yeast, has been purified approximately 5- to 6-fold. The enzyme preparation contained copper, nonheme iron, labile sulfide, and cytochromes a + a3 in approximately equimolar amounts, and 25 to 30% lipid on a dry weight basis. Difference spectra (reduced minus oxidized) of the enzyme preparation showed absorption maxima at 445 and 604 mµ and a small broad peak at 650 to 750 mµ, but no change in absorbance was found in the region 800 to 900 mµ. Yeast cytochrome oxidase was strongly inhibited by cyanide, azide, diethyldithiocarbamate, and ethylenediaminetetraacetate, but not by p-chloromercuriphenyl sulfonic acid or antimycin A.