Spinal muscular atrophy-like picture, cardiomyopathy, and cytochrome c oxidase deficiency

The authors report a child with a spinal muscular atrophy (SMA)-like picture, cardiomyopathy, and cytochrome c oxidase (COX) deficiency. Electromyography and muscle biopsy showed findings typical of SMA. However, COX staining of the muscle was negative. DNA analysis did not detect deletions in the survival motor neuron (SMN) gene. The lactate and lactate-to-pyruvate ratios were increased in blood and CSF. COX activity was decreased in muscle and fibroblasts. Western blot analysis showed reduced contents for all COX subunits. Patients with clinical features resembling SMA but with an intact SMN gene should be screened for a mitochondrial disorder.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase

Determining the way in which deleterious mutations interact in their effects on fitness is crucial to numerous areas in population genetics and evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last (synergistic epistasis), then the evolution of sex and recombination may be favored to facilitate the elimination of deleterious mutations. However, there is a severe shortage of relevant data. Three relatively simple experimental methods to test for epistasis between deleterious mutations in haploid species have recently been proposed. These methods involve crossing individuals and examining the mean and/or skew in log fitness of the offspring and parents. The main aim of this paper is to formalize these methods, and determine the most effective way in which tests for epistasis could be carried out. We show that only one of these methods is likely to give useful results: crossing individuals that have very different numbers of deleterious mutations, and comparing the mean log fitness of the parents with that of their offspring. We also reconsider experimental data collected on Chlamydomonas moewussi using two of the three methods. Finally, we suggest how the test could be applied to diploid species.     

Mammalian mitochondrial DNA evolution: a comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.     

Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein

The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.     

Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.     

Degradation of cytochrome oxidase subunits in mutants of yeast lacking cytochrome c and suppression of the degradation by mutation of yme1

We have confirmed by spectral analysis that cytochrome oxidase is not present in strains of the yeast Saccharomyces cerevisiae having a primary deficiency in cytochrome c, and we have demonstrated by immunological procedures that such strains lack the mitochondrially encoded subunits I, II, and III of cytochrome oxidase. Furthermore, pulse-chase experiments demonstrated that subunit II is rapidly degraded in vivo. This degradation can be at least partially suppressed by disruption of the nuclear gene YME1, which encodes a putative ATP-Zn(2+)-dependent protease. We suggest that the cytochrome oxidase subunits are not properly assembled in the absence of cytochrome c, and that Yme1 and possibly other proteases degrade the unassembled mitochondrial-encoded subunits of cytochrome oxidase.     

The subunit location of magnesium in cytochrome c oxidase

The magnesium ion in bovine heart cytochrome c oxidase can be depleted up to 75% by heat treatment of the enzyme at 43 degrees C followed by dialysis against EDTA buffer solution. The magnesium-depleted enzyme so obtained retains 40% of the activity of the native enzyme. This is the first attempt to deplete magnesium ion from bovine heart cytochrome c oxidase without denaturation of the protein. Magnesium depletion exposes at least one carboxyl group on subunit IV for labeling by N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide (NCD-4). The NCD-4 labeling of subunit IV of the magnesium-depleted enzyme is significantly enhanced relative to what is observed for the native and heat-treated oxidase, suggesting that the magnesium ion is located in subunit IV with at least one carboxyl ligand. By comparing the activity of the magnesium-depleted enzyme with that of a control sample of heat-treated oxidase, the influence of divalent magnesium on the activity of the enzyme is assessed.     

Involvement of cytochrome c oxidase subunit III in energy coupling

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.     

Novel function of the regulatory subunit of protein kinase A: regulation of cytochrome c oxidase activity and cytochrome c release

There have been speculations that the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) may have other functions. A recent study has shown that the catalytic (C) subunit of PKA may be regulated in a cAMP- and R subunit-independent manner. However, evidence linking a function to the R subunit apart from inhibiting the C subunit has been elusive. In this report, interaction cloning experiments showed that the RIalpha subunit association with the cytochrome c oxidase subunit Vb (CoxVb) is cAMP-sensitive. Interaction was detected with a GST-RIalpha fusion protein as well as by coimmunoprecipitation. Transient treatment with cAMP-elevating agents inhibited cytochrome c oxidase in Chinese hamster ovary (CHO) cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIalpha show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIalpha with CoxVb thereby regulating cytochrome c oxidase activity as well as the cytochrome c levels.     

The post-translational modification in cytochrome c oxidase is required to establish a functional environment of the catalytic site

Mutation of tyrosine-288 to a phenylalanine in cytochrome c oxidase from Rhodobacter sphaeroides drastically alters its properties. Tyr-288 lies in the CuB-cytochrome a3 binuclear catalytic site and forms a hydrogen bond with the hydroxy group on the farnesyl side chain of the heme. In addition, through a post-translational modification, Y288 is covalently linked to one of the histidine ligands that is coordinated to CuB. In the Y288F mutant enzyme, the "as-isolated" preparation is a mixture of reduced cytochrome a and oxidized cytochrome a3. The cytochrome a3 heme, which is largely six-coordinate low-spin in both oxidation states of the mutant, cannot be reduced by cytochrome c, but only by dithionite, possibly due to a large decrease in its reduction potential. It is postulated that the Y288F mutation prevents the post-translational modification from occurring. As a consequence, the catalytic site becomes disrupted. Thus, one role of the post-translational modification is to stabilize the functional catalytic site by maintaining the correct ligands on CuB, thereby preventing nonfunctional ligands from coordinating to the heme.     

The water channel of cytochrome c oxidase: inferences from inhibitor studies

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.     

Structural water in oxidized and reduced horse heart cytochrome c

The existence of structural water in the interior of both oxidized and reduced horse-heart cytochrome c in solution is demonstrated using nuclear magnetic resonance spectroscopy. Six water molecules have been located in ferrocytochrome c and five in ferricytochrome c, with residence times greater than a few hundred picoseconds. Two water molecules are located in the haem crevice, one of which is found to undergo a large change in position with a change of oxidation state. Both of these observations indicate that buried structural waters in the haem crevice have, by microscopic dielectric effects, significant roles in the setting of the solvent reorganization energy associated with electron transfer.     

Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy

The data gained from clinical studies in the past years have indicated that the thrombolytic therapy (TL) has favourable effect on patients with acute myocardial infarction (AMI). It is aimed at reperfusion in the ischaemic area, a decrease in the extent of infarction site and a decrease in mortality. TL administered within the initial hours after the onset of AMI leads to better results than when administered after several hours. Currently, TL is not limited by age. The patients who were given streptokinase (SK) or anistreplase (APSAC) prior to more than 4 days, if necessary, urokinase or alteplase (rt-PA) should be given. There are differences in the opinions as to the optimal selection of thrombolytic drugs. However, all currently used drugs lead to a significant decrease in mortality due to AMI. The preferential use of accelerated administration of rt-PA in contrast to SK is justified in younger patients with extensive AMI of the anterior wall, in whom the therapy has begun within 4 hours since its onset. The occurrence of severe bleeding indicates that TL should be halted and coagulation factors should be replaced by freshly frozen plasma or fibrinogen concentrate, if necessary, transfusion of full blood should take place. If the severe bleeding occurs shortly after the administration of SK, the persisting plasminaemia can be arranged by antifibrinolytic drugs. An improvement in TL results can be achieved by adjuvant antithrombotic therapy. At the same time, in addition to acetylsalicylic acid, the patient treated with rt-PA should be given heparin. Heparin administration is not necessary in patients treated with SK or APSAC. However, heparin is indicated in patients at risk due to systemic embolization in congestive heart disease, extensive infarction or atrial fibrillation. (Tab. 1, Ref. 28.)     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

Suppression of constitutive and inducible cytochrome P450 gene expression by alpha-hederin in mice

The effects of alpha-Hederin, a triterpenoid saponin which exists in some oriental herbs, on the expression of liver cytochrome P450s were examined in mice. The administration of alpha-Hederin to mice significantly decreased the hepatic content of P450 and the activities of microsomal ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, and aniline hydroxylase, representative activities of cytochrome-P4501A1, -P4501A2, and -P4502E1, respectively, in a dose- and time-dependent manner. However, pentoxyresorufin O-dealkylase, a representative activity of cytochrome P4502B1/2, was decreased to a lesser extent. alpha-Hederin also decreased inducible monooxygenase activities in the same manner. Suppressions of P450 isozyme expression occurred in alpha-Hederin treated hepatic microsomes, as determined by immunoblot analysis in a manner consistent with that of the enzyme activity levels. Levels of mRNA of P4501A1/2 and P4502B1/2 were also decreased by alpha-Hederin as shown by Northern blot analysis. In contrast, the level of P4502E1 mRNA in the liver of alpha-Hederin treated mice was unchanged. These results suggest that alpha-Hederin may act as a more specific suppressor for P4501A and P4502E1 than P4502B and that the suppression involves decreases in mRNA levels except in the case of P4502E1.     

Intermediates in the reaction of fully reduced cytochrome c oxidase with dioxygen

The reduction of dioxygen to water by cytochrome c oxidase was monitored in the Soret region following photolysis of the fully reduced CO complex. Time-resolved optical absorption difference spectra collected between 373 and 521 nm were measured at delay times from 50 ns to 50 ms and analyzed using singular value decomposition and multiexponential fitting. Five processes were resolved with apparent lifetimes of 0.9 micros, 8 micros, 36 micros, 103 micros, and 1.2 ms. A mechanism is proposed and spectra of intermediates are extracted and compared to model spectra of the postulated intermediates. The model builds on an earlier mechanism that used data only from the visible region (Sucheta et al. (1997) Biochemistry 36, 554-565) and provides a more complete mechanism that fits results from both spectral regions. Intermediate 3, the ferrous-oxy complex (compound A) decays into a 607 nm species, generally referred to as P, which is converted to a 580 nm ferryl form (Fo) on a significantly faster time scale. The equilibrium constant between P and Fo is 1. We propose that the structure of P is a3(4+)=O CuB2+-OH- with an oxidizing equivalent residing on tyrosine 244, located close to the binuclear center. Upon conversion of P to Fo, cytochrome a donates an electron to the tyrosine radical, forming tyrosinate. Subsequently a proton is taken up by tyrosinate, forming F(I) [a3(4+)=O CuB2+-OH- a3+ CuA+]. This is followed by rapid electron transfer from CuA to cytochrome a to produce F(II) [a3(4+)=O CuB2+-OH- a2+ CuA2+].