The cytochrome c oxidase and its mitochondrial function in the whiteleg shrimp Litopenaeus vannamei during hypoxia

Cytochrome c oxidase (COX), which is located in the inner membrane of mitochondria, is a key constituent of the electron transport chain that catalyzes the reduction of oxygen. The Pacific whiteleg shrimp Litopenaeus vannamei is constantly exposed to hypoxic conditions, which affects both the central metabolism and the mitochondrial function. The purpose of this study was to isolate shrimp mitochondria, identify the COX complex and to evaluate the effect of hypoxia on the shrimp mitochondrial function and in the COX activity. A 190 kDa protein was identified as COX by immunodetection techniques. The effect of hypoxia was confirmed by an increase in the shrimp plasma L-lactate concentration. COX activity, mitochondrial oxygen uptake and protein content were reduced under hypoxic conditions, and gradually restored as hypoxia continued, this suggests an adaptive mitochondrial response and a highly effective COX enzyme. Both mitochondrial oxygen uptake and COX activity were completely inhibited by KCN and sodium azide, suggesting that COX is the unique oxidase in L. vannamei mitochondria.     

Effect of oleic acid on mitochondrial cytochrome c oxidase activity

Both Km and Vmax values of cytochrome c oxidase for cytochrome c were elevated in oleic acid-incorporated mitochondria, whereas the amount of oleic acid incorporated into submitochondrial particles was smaller than that into mitochondria and the fatty acid had little effect on the enzyme activity. The degree of change in the bulk membrane fluidity was, however, almost the same in mitochondria and submitochondrial particles. Solubilized cytochrome c oxidase was insensitive to the effect of oleic acid. Oleic acid may act as a modifier of the interaction between cytochrome c oxidase and membrane lipids.     

Tight control of mitochondrial membrane potential by cytochrome c oxidase

In the present work we have critically examined the use of the KCN-titration technique in the study of the control of the cellular respiration by cytochrome c oxidase (COX) in the presence of the mitochondrial membrane potential (Δψ(mito)) in HepG2 cells. We clearly show that the apparent high inhibition threshold of COX in the presence of maximal Δψ(mito) is due to the KCN-induced decrease of Δψ(mito) and not to a low control of COX on the mitochondrial respiration. The tight control exerted by COX on the Δψ(mito) provides further insights for understanding the pathogenetic mechanisms associated with mitochondrial defects in human neuromuscular degenerative disorders.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.     

Ionic strength dependence of the kinetics of electron transfer from bovine mitochondrial cytochrome c to bovine cytochrome c oxidase.

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme a prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme a center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme a site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme a. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme a reduced. We presume that the second equivalent was used to reduce the Cua center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme a showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.     

Aflatoxin inhibition of rat liver mitochondrial cytochrome oxidase activity

Aflatoxins B1, B2, G1, G2, and M1 have been evaluated for activity toward cytochrome oxidase in isolated rat liver mitochondria employing ferrocytochrome c and p-phenylene diamine as reductants. The aflatoxins inhibited the cytochrome oxidase activity to a greater extent when monitored by O2 uptake measurements than by substrate oxidation. AFG2 and AFM1 were the most potent (50-70%). Using oligomycin and 2,4-DNP as respiratory inhibitor and uncoupler, respectively, the aflatoxins appear to inhibit e- rather than energy transfer reactions. These toxins did not uncouple cytochrome oxidase activity.     

Spatial diversity in mitochondrial cytochrome c oxidase in house flies

DNA sequence analysis at mitochondrial gene COI was surveyed in 293 house flies, Musca domestica Linneaus (Diptera: Muscidae), in 29 populations from North, Central and South America, Europe, Asia, Africa, and the Western Pacific. Nei's gene diversity index (H(S)) was 0.47, the chance that two randomly chosen flies have different COI haplotypes. Haplotype diversity was greater in the Old World (H(S) = 0.58) than the New World (H(S) = 0.31). The hierarchical partition of the total diversity indicated substantial differentiation at all levels (G(ST) = 0.30), and highly structured populations. All pairwise estimates of gene flow between zoogeographical regions were less than 0.70 reproducing females per generation. The results are compared to those of a similar study based on the single-strand conformation polymorphism method. Probable colonization scenarios for house flies into the New World are discussed and it is concluded that house flies are a recent addition to the fauna of the Western Hemisphere.     

Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation

Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.     

Purification and characterization of cytochrome c oxidase from rat liver mitochondria.

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.     

EPR signals from cytochrome c oxidase.

1. The major EPR signals from native and cytochrome c-reduced beef heart cytochrome c oxidase (EC 1.9.3.1) are characterized with respect to resonance parameters, number of components and total integrated intensity. A mistake in all earlier integrations and simulations of very anisotropic EPR signals is pointed out. 2. The so-called Cu2+ signal is found to contain at least three components, one "inactive" form and two nearly similar active forms. One of the latter forms, corresponding to about 20% of the total EPR detectable Cu, has not been observed earlier and can only be resolved in 35 GHz spectra. It is not reduced by cytochrome c and is thought to reflect some kind of inhomogeneity in the enzyme preparation. The 35 GHz spectrum of the cytochrome c reducible component shows a rhombic splitting and can be well simulated with g-values 2.18, 2.03 and 1.99. The origin of such a unique type of Cu2+ spectrum is discussed. 3. The low-spin heme signal in the oxidized enzyme (g = 3.03, 2.21, 1.45) is found to correspond closely to one heme and shows no signs of interaction with other paramagnetic centres. 4. The high-spin heme signals appearing in partly reduced oxidase are found to consist of at least three species, one axial and two rhombic types. An integration procedure is described that allows the determination of the total integral intensity of high-spin heme EPR signals only by considering the g = 6 part of the signals. In a titration with ascorbate and cytochrome c the maximum intensity of the g = 6 species corresponds to 23% of the enzyme concentration.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Charge interactions of cytochrome c with cytochrome c oxidase

• 1.1. The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase.
• 2.2. The rate constants for (PLP);-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, 1=0.02) are 3.6 × 10⁻³'sec-', 5.5 × 10⁻³, 5.2 × 10⁻³-'sec⁻¹ and 9.8 × 10⁻³sec⁻¹, respectively.
• 3.3. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c.
• 4.4. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c.
• 5.5. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.

     

Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent.

The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c: O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr approximately 130 000) consisting of a flavin subunit (Mr 72 000) an iron-sulfur subunit (Mr 28 000) and a cytochrome b subunit (Mr probably 14 000). Cytochrome c reductase was obtained in a dimeric form (Mr approximately 550 000), the monomeric unit comprising the cytochromes b (Mr each 30 000), a cytochrome c1 (Mr 31 000), the iron-sulfur subunit (Mr 25 000), and six subunits without known prosthetic groups (Mr 9000, 11 000, 14 000, 45 000, 45 000, and 52 000). Cytochrome c oxidase was also isolated in a dimeric form (Mr approximately 320 000) comprising two copies each of seven subunits (Mr 9000, 12 000, 14 000, 18 000, 21 000, 29 000, and 40 000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr approximately 90 000). After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.     

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase

Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708-713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569-1579).     

Turnover of cytochrome c oxidase from Paracoccus denitrificans

The heme aa3 type cytochrome oxidase from Paracoccus denitrificans incorporated into vesicles with phospholipid reacts during turnover much as the oxidase from mitochondria does. The spectrophotometric changes observed at various wavelengths are closely similar, and the rate is about one-half of that for beef heart oxidase under the same conditions. The rate of appearance of oxidized cytochrome c on initiation of the reaction is also similar and depends on the previous treatment of the oxidase as described by Antonini, E., Brunori, M., Colosimo, A., Greenwood, C. and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132. In terms of their model the resting Paracoccus enzyme is converted to the pulsed form during turnover. The effect is observed with both cytochrome c and hexamine ruthenium as reductants. With the latter a 60-fold increase in rate is observed.