The oxidation of exogenous cytochrome c by mitochondria. Resolution of a long-standing controversy

Several reports in the past have dealt with the oxidation of cytochrome c added to suspensions of rat liver mitochondria. Yet, it is generally believed that the cytochrome cannot penetrate the outer membrane. Probably it has been assumed that the permeability of the outer membrane to cytochrome c is very low but finite, and that fast oxidation may be observed if time is allowed for sufficient penetration before initiation of electron flow. Here we show that this view is false. The main fraction of rat liver mitochondria, as isolated by conventional procedures, does not catalyse any significant oxidation of added cytochrome c, even after prolonged incubation. The observed appreciable oxidation of added cytochrome c is catalysed by a very small fraction (5-12%) of the mitochondria that apparently has a damaged outer membrane. Consequently, the turnover of cytochrome oxidase is very high in this fraction during oxidation of added cytochrome c. This finding readily explains why Moyle and Mitchell (e.g., FEBS Lett. 88 (1978) 268-272; 90 (1978) 361-365) have failed to observe proton translocation by cytochrome oxidase during oxidation of ferrocytochrome c added to rat liver mitochondria, which has been their main reason for rejecting the proton-pumping function of cytochrome oxidase.     

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.     

The oxidation of exogenous NADH by mitochondria of Euglena gracilis

A novel oxidase activity of external NADH was found in mitochondria of a streptomycin-bleached mutant and the wild strain of Euglena gracilis. In contrast to higher plants the oxidation of external NADH in mitochondria of E. gracilis is sensitive to rotenone and yields the same phosphorylation efficiency as the matrix pool of NADH. Simulation of this activity by the classic complex I of the matrix side of the mitochondrial membrane, as a result of preparation-generated artefacts, is excluded. The external NADH-dehydrogenase activity is bound to the inner mitochondrial membrane with its active side facing the cytosol. State-4 enzyme activity is only slightly influenced by pH in the physiological range, whereas state-3 oxidation indicates an optimum in the physiological pH, as expected from a limitation by the ATPase. The external redox potential of NADH does not control enzyme activity. The results are discussed with respect to the metabolic status of the cells at the time of harvesting.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.     

Binding of cytochrome c to cytochrome c-oxidase in intact mitochondria. A study with radioactive photoaffinity-labeled cytochrome c

[³H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome $\text{c}$ from $\text{Saccharomyces cerevisiae}$ was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome $\text{c}$ was linked by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome $\text{c}$ bound to one of the smaller subunits of cytochrome $\text{c}$ oxidase with an apparent molecular weight of 15,000.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Reduction of cytochrome c by NADH induced by light

We have studied the behaviour of Fe(III) cytochrome c upon irradiation in the 290-360 nm wavelength range either in the presence or in the absence of NADH; in both cases the photoexcitation caused the reduction of the heme iron. When the irradiation was performed in the absence of NADH, the iron reduction was coupled to a non reversible modification in the protein structure; the photoreduction quantum yield was decreasing with the increase of the irradiation wavelength. Irradiation in the presence of NADH gave heme iron reduction coupled to NADH oxidation and the protein resulted finally unmodified; the quantum yield depended on the irradiation wavelength in a way similar to the observed in the absence of NADH, but it was tenfold higher. We propose that in both cases the active species is an electronic excited state of the heme iron.     

Mechanisms of cytochrome c release from mitochondria

In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (CL). Several proapoptotic stimuli induce the permeabilization of the outer membrane, facilitate the communication between intermembrane and intercristae spaces and promote the mobilization of Cyt c from CL, allowing for Cyt c release. In the cytosol, Cyt c mediates the allosteric activation of apoptosis-protease activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Activated caspases ultimately lead to apoptotic cell dismantling. Nevertheless, cytosolic Cyt c has been associated also to vital cell functions (i.e. differentiation), suggesting that its release not always occurs in an all-or-nothing fashion and that mitochondrial outer membrane permeabilization may not invariably lead to cell death. This review deals with the events involved in Cyt c release from mitochondria, with special attention to its regulation and final consequences.     

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase.     

Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria

The import of cytochrome c into Neurospora crassa mitochondria was examined at distinct stages in vitro. The precursor protein, apocytochrome c, binds to mitochondria with high affinity and specificity but is not transported completely across the outer membrane in the absence of conversion to holocytochrome c. The bound apocytochrome c is accessible to externally added proteases but at the same time penetrates far enough through the outer membrane to interact with cytochrome c heme lyase. Formation of a complex in which apocytochrome c and cytochrome c heme lyase participate represents the rate-limiting step of cytochrome c import. Conversion from the bound state to holocytochrome c, on the other hand, occurs 10-30-fold faster. Association of apocytochrome c with cytochrome c heme lyase also takes place after solubilizing mitochondria with detergent. We conclude that the bound apocytochrome c, spanning the outer membrane, forms a complex with cytochrome c heme lyase from which it can react further to be converted to holocytochrome c and be translocated completely into the intermembrane space.     

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.     

Membrane-bound and water-soluble cytochrome c1 from Neurospora mitochondria

Cytochrome c1 is a subunit of ubiquinol--cytochrome c reductase (EC 1.10.2.2). In Neurospora crassa wild type 74A grown in the presence of chloramphenicol, the subunit is inserted only into the bilayer of the mitochondrial inner membranes without associating with other proteins. From these modified membranes a monodisperse (cytochrome c1)-Triton complex was isolated by subjecting the Triton-solubilized membranes to affinity chromatography on immobilized cytochrome c. A water-soluble pentamer of cytochrome c1 was prepared from the (cytochrome c1)-Triton complex by removing the detergent. By limited proteolytic digestion of the cytochrome c1-Triton complex with chymotrypsin, a water-soluble monomeric cytochrome c1 was prepared which has a molecular weight of only 24 000 as compared to 31 000 of the membrane-bound cytochrome c1. The 24 000-Mr cytochrome c1 and the 31 000-Mr cytochrome c1 have same light absorption spectra and cytochrome-c-binding properties. These results are used to propose the following model. Cytochrome c1 consists of a large hydrophilic part and a small hydrophobic part. The hydrophilic part extends from the mitochondrial inner membrane into the intermembrane space. This part carries the heme and interacts with cytochrome c. The hydrophobic part anchors the cytochrome c1 to the bilayer.     

Import of cytochrome c into mitochondria. Cytochrome c heme lyase

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.     

The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle

Mitochondria isolated from minute amounts (100-500 mg) of human skeletal muscle displayed a very high rotenone-resistant NADH cytochrome c reductase activity. Moreover, compared to succinate cytochrome c reductase activity, a low rate of rotenone-sensitive NADH cytochrome c reductase activity was measured when using standard procedures to disrupt mitochondrial membranes. Only a drastic osmotic shock in distillated water as a mean to disrupt mitochondrial membrane was found to strongly increase the actual rate of the rotenone-sensitive activity. This was accompanied by a decrease in the rotenone-insensitive activity. Using such a simple procedure, the NADH cytochrome c reductase was found 70-80% inhibited by rotenone and roughly equivalent to 70-85% of the activity of the succinate cytochrome c reductase.     

Characterization of tBid-induced cytochrome c release from mitochondria and liposomes

tBid, the cleaved form of Bid, can induce cytochrome c (Cyt. c) release from rat heart mitochondria more efficiently and reproducibly than that from liver or brain mitochondria. Unlike Bax, such release was not prevented by cyclosphorin A, an inhibitor of the opening of permeability transition pore. Carbonyl-cyanide m-chlorophenyl-hydrazone or oligomycin also have no obvious effect on the release of Cyt. c. In contrast to ceramide, tBid-mediated Cyt. c release from mitochondria is independent of the redox state of Cyt. c. Furthermore, Bid or tBid can directly trigger the efflux of encapsulated Cyt. c or trypsin within liposomes without involvement of other protein factors.     

Mobility of fluorescent derivatives of cytochrome c in mitochondria

Motion of cytochrome c bound to giant (2-10-micron diam) mitochondria isolated from the waterbug Lethocerus indicus was examined using the technique of fluorescence recovery after photobleaching. Fluorescent cytochrome c was exchanged for native cytochrome c through partly damaged outer membrane. Recovery profiles were not statistically different when the fluorescence from iron-free cytochrome c or fluorescein-labeled cytochrome c was used and were essentially the same in the presence or absence of an uncoupler. In the presence of excess porphyrin cytochrome c, the apparent diffusion coefficient was 6 X 10(-11) cm2/s in 0.3 M sucrose-mannitol-EDTA and 3 X 10(-10) cm2/s in 0.10 M KCl/0.10 M sucrose. At concentrations of porphyrin cytochrome c that are stoichiometric with cytochrome c oxidase and for mitochondria in which excess cytochrome c was washed away, two components were observed in the recovery profile. The diffusion coefficient of the fast component was 1 X 10(-10) cm2/s. The second component showed no recovery during the time scale of measurement (D less than 10(-12) cm2/s). We speculate on the origin of the immobile fraction.