Hydrophobic photolabelling of the yeast cytochrome c oxidase subunits in contact with lipids

The hydrophobic domain of the membrane-bound enzyme yeast cytochrome c oxidase was labelled with photoactivable phosphatidylcholines.Subunits I, II and III were labelled; a minor labelling was also found on subunits V and VII.The labelling of subunit V was located in a small terminal polypeptide sequence.     

Cytochrome c oxidase in prokaryotes

Abstract Several heme aa ₃-type cytochrome c oxidases, purified from the cytoplasmic membranes of bacteria, are able to catalyze the same reactions as the structurally far more complex eukaryotic enzyme, i.e., electron transport from cytochrome c to oxygen coupled to proton translocation. However, these oxidases show a very simple subunit pattern, and moreover, individual polypeptides even have homologous amino-acid sequences. This review summarizes the present data on purified bacterial cytochrome c oxidases and relates these findings to results obtained with the mitochondrial enzymes.     

Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.     

Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits.

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.     

Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme.

Cytochrome c oxidase from baker's yeast contains three mitochondrially made subunits (I to III) which are relatively hydrophobic and four cytoplasmically made subunits (IV to VII) which are relatively hydrophilic (Mason, T. L., Poyton, R. O., Wharton, D.C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354 and Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761). In order to explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of "surface probes" was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound cytochrome c oxidase. The surface probes included iodination with lactoperoxidase and coupling with p-diazonium benzenesulfonate. In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound isocyanate group. In the membrane-bound enzyme, Subunit I was almost completely inaccessible and Subunit II was partly inaccessible to all surface probes. All of the other subunits were accessible. Similar results were obtained with the solubilized enzyme, except that the differences in reactivity between the individual subunits were less clear-cut. The results obtained with liposome-bound cytochrome c oxidase resembled those obtained with the mitochondrially bound enzyme. These data suggest that the two largest mitochondrially made subunits are localized in the interior of the enzyme and that they are genuine components of cytochrome c oxidase.     

Cytochrome c oxidase dysfunction in oxidative stress

Cytochrome c oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits of which the 3 catalytic subunits are encoded by the mitochondrial genes. The remaining 10 subunits with suspected roles in the regulation, and/or assembly, are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu(2+) centers (Cu(2+) A and Cu(2+) B) as catalytic centers and handles more than 90% of molecular O(2) respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pacesetter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme are affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases, and diabetes. Despite handling a high O(2) load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this paper we review the literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.     

Cytochrome c oxidase is not a proton pump.

We conclude that the reduction of O2 to 2 H2O by cytochrome c oxidase of rat liver mitochondria involves the translocation of 4-from cytochrome c at the outer surface of the cristae membrane per O2 reduced and protonated by 4 H+ ions that enter the reaction domain from the inner aqueous phase. This net electron-translocating function of cytochrome c oxidase plugged through the mitochondrial cristae membrane is not linked to a proton-pumping function, such as that proposed by Wikstr?m [7,8].     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Cytochrome c oxidase, ligands and electrons

We present hereby an overview of the reactions of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, with ligands (primarily oxygen) and electrons, pointing out where necessary unresolved facts or questionable interpretations.     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.