Yeast cytochrome c oxidase

Taanman, J.W. and Capaldi, R.A., Subunit Via of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity. Isolation and characterization of the nuclear-coded gene, J Biol Chem 268 (1993) 18754—18761. 

Allen LA, Zhao X-J, Caughey W, Poyton RO. Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast C3dochrome c. J Biol Chem 1995 270 110—118. 

Waterland RA, Basu A, Chance B, Poyton RO. The isoforms of yeast cytochrome c oxidase subunit V alter the in vivo kinetic properties of the holoenzyme. J Biol Chem 1991 266 4180-4186. 

Figure 12.2 Copper chaperone function, (a) Copper homeostasis in Enterococcus hirae is affected by the proteins encoded by the cop operon. CopA, Cu1+-import ATPase CopB, Cu1+-export ATPase CopY, Cu1+-responsive repressor copZ, chaperone for Cu1+ delivery to CopY. (b) The CTR family of proteins transports copper into yeast cells. Atxlp delivers copper to the CPx-type ATPases located in the post Golgi apparatus for the maturation of Fet3p. (c) Coxl7p delivers copper to the mitochondrial intermembrane space for incorporation into cytochrome c oxidase (CCO). (d) hCTR, a human homologue of CTR, mediates copper-ion uptake into human cells. CCS delivers copper to cytoplasmic Cu/Zn superoxide dismutase (SOD1). Abbreviations IMM, inner mitochondrial membrane OMM, outer mitochondrial membrane PM, plasma membrane PGV, post Golgi vessel. Reprinted from Harrison et al., 2000. Copyright (2000), with permission from Elsevier Science.

Bisson et al. (1980) labeled cytochrome oxidase with yeast cytochrome c to which an azido aryl group had been attached at Lys-13, and demonstrated that subunit II of the oxidase reacted specifically. In contrast, subunit III was attacked by a cytochrome c with an azido aryl group attached at Cys-102 (Moreland and Dockter, 1981). Lysine-13 and Cys-102 are on opposite sides of the cytochrome c molecule. 

Fig. 1. Schematic overview of copper trafficking and homeostasis inside the yeast cell. The actions of Mad and Ace 1, copper-dependent metalloregulatory transcription factors, control the production of copper import [copper transporter (Ctr) and reductase (Fre)] and detoxification/sequestration [metallothionein (MT)] machineries, respectively. Three chaperone-mediated delivery pathways are shown. Atxl shuttles Cu(I) to the secretory pathway P-type ATPase Ccc2 (right). CCS delivers Cu(I) to the cytoplasmic enzyme copper-zinc superoxide dismutase (SOD) (left). Coxl7 shuttles Cu(I) to cytochrome c oxidase (CCO) in the mitochondria (bottom). Mitochondrial proteins Scol and Sco2 may also play a role in copper delivery to the CuA and CuB sites of CCO. Copper metabolism and iron metabolism are linked through the actions of Fet3, a copper-containing ferroxidase required to bring iron into the cell (lower right) (see text).

Coxl7, an 8.1-kDa cysteine-rich protein, was the first copper chaperone to be identified. Saccharomyces cerevisiae harboring mutations in coxl 7 are respiratory deficient, a phenotype resulting from their inability to assemble a functional cytochrome c oxidase complex (Glerum et al., 1996a). coxl7 mutant yeast are, however, able to express all the subunits of the cytochrome c oxidase complex, indicating that the lesion must lie in a posttranslational step that is essential for assembly of the functional complex in the mitochondrial membrane. Unlike other cytochrome c... 

Cytochrome c oxidase, the terminal oxidase in the respiratory metabolism of all aerobic organisms, plants, animals, yeasts, algae, and some bacteria, is responsible for catalyzing the reduction of dioxygen to water. 

While the in vitro studies on assembly have provided relatively little information, in vivo data can give us some suggestions on possible pathway of assembly. For a long time it has been known that in rho yeast cells, where cytochrome b is not produced, cytochrome c, is still accumulated in the inner membrane. This agrees with the plasmid studies of overproduction. On the other hand the cytoplasmically synthesized subunits of cytochrome c oxidase accumulate in much lower quantities in the absence of subunits I, II and III, which are mitochondrial products. It is unlikely that this diminished accumulation is due to substantially reduced gene expression. This may indicate that certain subunits are stabilized by their counterparts. 

Fig. 12.6. The onset of synthesis of various mitochondrial polypeptides upon transferring anaerobically grown yeast cells to aerobic conditions. Yeast cells were grown overnight under anaerobic conditions. At time zero they were transferred to aerobic conditions, and at the indicated time periods samples of cells were removed and lysed in the presence of NaOH and mercaptoethanol. Samples containing about 50 /ig of protein were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel. The proteins were electrotransferred to nitrocellulose sheets and decorated with specific antibodies and l-labelled protein A. Paper pieces corresponding to the labelled protein spots were cut out from the immune blot and counted in a y counter. The amount of counts obtained in the samples of 8 h aerobic conditions was taken as 100%. The antibodies used were directed against the following polypeptides porin of the mitochondrial outer membrane (29 k) /8 subunit of the proton-ATPase (iS-F,) subunit IV of cytochrome c oxidase (OxIV) and subunit V of cytochrome c oxidase (OxV).

A short electron transport chain involving flavocytochrome b2, cytochrome c, and cytochrome c oxidase allows yeast to respire on L-lactate even if the main electron transport chain is blocked, for example, by antimycin (3). The topological arrangement of this respiratory pathway is shown diagrammatically in Fig. 2. 

Cytochrome c-550(s) was partially purified by Ketchum et al. (1969). Afterward it was purified to an electrophoretically homogeneous state (Yamanaka et al., 1982), and its complete amino acid sequence was determined (Tanaka et al., 1982). Its molecular mass is 12.4 kDa. The cytochrome is very similar to mitochondrial cytochrome c (similarity, 40% 19%) on the basis of the sequence, and reacts with yeast cytochrome c peroxidase at the rate of 79% as fast as mitochondrial cytochrome c. Ferrocytochrome c-550(s) is oxidized very fast with molecular oxygen by the catalysis of N. winogradskyi cytochrome c oxidase turnover number is 117 s 1 (Yamanaka et al., 1982 Nomoto et al., 1993). 

Starkeya novella cytochrome c oxidase reacts rapidly not only with native cytochrome c but also with tuna and yeast cytochromes c, while it reacts very slowly with horse and cow cytochromes c (Yamanaka and Fukumori, 1977 Yamanaka and Fujii, 1980). Cytochromes c which react rapidly with the oxidase have Tyr (46), while cytochromes c which react slowly with the oxidase have Phe (46) (Yamanaka and Fukumori, 1978). Thus, human cytochrome c reacts with the oxidase more rapidly than horse and cow cytochromes c. However, native cytochrome c which reacts rapidly with the oxidase has Phe(46). 

Yamazaki Takeshi, Fukumori Y, Yamanaka T (1988) Catalytic properties of cytochrome c oxidase purified from Nitrosomonas europaea. J Biochem 103 499-503 Yano T (1992) The oxidation system of ferrous ion in Thiobacillus ferrooxidans. Dissertation for Ph.D. degree, Tokyo Institute of Technology, Tokyo Yano T, Fukumori Y, Yamanaka T (1991) The amino acid sequence of rusticyanin isolated from Thiobacillus ferrooxidans. FEBS Lett 288 159-162 Yaoi Y (1967) Comparison of the primary structures of cytochromes c from wild and respiration-deficient mutant yeasts. J Biochem 61 54—58... 

In metazoans, the electron transport chain consists of four integral membrane complexes localized to the inner mitochondrial membrane complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III (ubiquinol-cytochrome c oxidoreductase) and complex IV (cytochrome c oxidase), plus coenzyme Q (ubiquinone) and cytochrome c. As first shown by Fry and Beesley (1991), the plasmodial electron transport chain differs from the metazoan system in lacking complex I however, a single subunit NADH dehydrogenase is present and is homologous to that found in plants, bacteria and yeast but not in animals (Krungkrai, 2004 Vaidya, 2004,2005 van Dooren et al., 2006). 

Cytochrome c oxidase is the terminal member of the respiratory chain in all animals and plants, aerobic yeasts, and some bacteria." " This enzyme is always found associated with a membrane the inner mitochondrial membrane in higher organisms or the cell membrane in bacteria. It is a large, complex, multisubunit enzyme whose characterization has been complicated by its size, by the fact that it is membrane-bound, and by the diversity of the four redox metal sites, i.e., two copper ions and two heme iron units, each of which is found in a different type of environment within the protein. Because of the complexity of this system and the absence of detailed structural information, spectroscopic studies of this enzyme and comparisons of spectral properties with 02-binding proteins (see Chapter 4) and with model iron-porphyrin and copper complexes have been invaluable in its characterization. 

The surprising consequence of the lack of PG and CL in yeast is the lack of translation of mRNAs of four mitochondria-encoded proteins (cytochrome b and cytochrome c oxidase subunits I-III) as well as cytochrome c oxidase subunit IV [13] that is nuclear encoded. These results indicate that some aspects of translation of a subset of mitochondrial proteins (those associated with electron transport complexes in the inner membrane but not ATP metabolism) require PG and/or CL. 

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