Effect of Phospholipid on Pea Cytochrome c Oxidase

Inhibitors of angiotensin I-converting enzyme were isolated from an enzymatic hydrolysate of bovine casein. The amino acid sequences of these inhibitors were Phe-Phe-Val-Ala-Pro-Phe-Pro- Glu-Val-Phe-Gly-Lys (CEI₁₂), Phe-Phe-Val-Ala-Pro (CEI₅), and Ala-Val-Pro-Tyr-Pro-Gln-Arg (CEI_β7). CEI₅ is a penta-peptide derived from the hydrolysate of CEI₁₂ with proline-specific endopeptidase, and CEI_β7 is a hepta-peptide derived from β-casein. These inhibitors potentiated bradykinin in the contraction of the uterus and the ileum of rats. The ileum was more sensitive to these inhibitors than the uterus. The bradykinin-potentiating activity of these inhibitors on the ileum lasted for more than 90 min even after washing the organ.     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Pathways of Proton Transfer in Cytochrome c Oxidase

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a ₃, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O₂ reduction.     

Evolution of the Cytochrome c Oxidase Proton Pump

The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme–copper dioxygen activating and reduction center. On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the Cu_A center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme. The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without Cu_A centers has strengthened this view. However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo ₃ complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H₂)-loop mechanism of proton translocation [Musser SM et al. (1997) Biochemistry 36:894–902]. In addition, a number of reports suggest that a Q(H₂)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa ₃ -type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes. The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc ₁ and cytochrome c oxidase complexes, is explored here. This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process. The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis. Received: 11 June 1997 / Accepted: 30 October 1997     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

Pathways for Electron Tunneling in Cytochrome c Oxidase

Warburg showed in 1929 that the photochemical action spectrum for CO dissociation from cytochrome c oxidase is that of a heme protein. Keilin had shown that cytochrome a does not react with oxygen, so he did not accept Warburg's view until 1939, when he discovered cytochrome a ₃. The dinuclear cytochrome a ₃-Cu_B unit was found by EPR in 1967, whereas the dinuclear nature of the Cu_A site was not universally accepted until oxidase crystal structures were published in 1995. There are negative redox interactions between cytochrome a and the other redox sites in the oxidase, so that the reduction potential of a particular site depends on the redox states of the other sites. Calculated electron-tunneling pathways for internal electron transfer in the oxidase indicate that the coupling-limited rates are 9×10⁵ (Cu _A a) and 7×10⁶ s^–1 (a a ₃); these calculations are in reasonable agreement with experimental rates, after corrections are made for driving force and reorganization energy. The best Cu_A-a pathway starts from the ligand His204 and not from the bridging sulfur of Cys196, and an efficient a-a ₃ path involves the heme ligands His378 and His376 as well as the intervening Phe377 residue. All direct paths from Cu_A to a ₃ are poor, indicating that direct Cu_A a ₃ electron transfer is much slower than the Cu_A a reaction. The pathways model suggests a means for gating the electron flow in redox-linked proton pumps.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

Cytochrome c Oxidase as a Proton-Pumping Peroxidase: Reaction Cycle and Electrogenic Mechanism

Cytochrome oxidase (COX) is considered to integrate in a single enzyme two consecutive mechanistically different redox activities--oxidase and peroxidase--that can be catalyzed elesewhere by separate hemoproteins. From the viewpoint of energy transduction, the enzyme is essentially a proton pumping peroxidase with a built-in auxiliary eu-oxidase module that activates oxygen and prepares in situ H₂O₂, a thermodynamically efficient but potentially hazardous electron acceptor for the proton pumping peroxidase. The eu-oxidase and peroxidase phases of the catalytic cycle may be performed by different structural states of COX. Resolution of the proton pumping peroxidase activity of COX and identification of individual charge translocation steps inherent in this reaction are discussed, as well as the specific role of the two input proton channels in proton translocation.     

Prognostic Relevance of Cytochrome c Oxidase in Primary Glioblastoma Multiforme

Patients with primary glioblastoma multiforme (GBM) have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO) promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%), and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.     

Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c₁ subunits and by inference from comigration.     

The Effect of Cytochrome Oxidase Inhibitors on the Thermotolerance of Yeasts

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubraand D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.