Cause of the activity loss of the alternative pathway of electron transport in cyanide-resistant mitochondria of the yeast Candida lipolytica

The cause of the activity loss of alternative pathway of electron transport in mitochondria of the yeast Candida lipolytica has been investigated. Incubation of cyanide-resistant mitochondria at 25 degrees was shown to cause the loss by mitochondria of their ability to oxidize substrates in the presence of 1 mM cyanide. This suggests that in the course of incubation the alternative pathway loses its activity. Repeated washing of mitochondria with a solution containing 2,5 mM EDTA inhibits, while Ca2+, Mn2+, Cu2+ and Zn2+ (but not Sr2+) enhance the process of the activity loss of the alternative pathway. The loss of the cyanide-resistant respiration is also observed during incubation of mitochondria in the presence of phospholipases A, C and D or lysolecithin. In all cases studied the reactivation of the cyanide-resistant respiration of mitochondria is attained by addition of azolectin. The loss of cyanide-resistant respiration is accompanied by the activity reduction of the main respiratory chain, which is restored by addition of cytochrome c and Mg2+. These data indicate that the activity loss of the alternative pathway is not related to inactivation of any components in the alternative pathway itself or in the main respiratory chain. The most probable cause of the activity loss in the destruction of reducing equivalents in the alternative pathway of a donor as a result of a break of the structural entity of the internal membrane of mitochondria due to the detersive action of the phospholipid lysoforms produced either by endogenic or exogenic phospholipases.     

Isolation and some properties of mitochondria from yeast Candida lipolytica 695]

Mitochondria is obtained from yeast Candida lipolytica 695 grown in the presence of glucose, lactate or citrate. Yeast mitochondria were shown to be practically indistinguishable from animal tissue mitochondria in [ADP]/[O] values and in their sensitivity to electron transport inhibitors, to inhibitors and uncoupling agents of oxidative phosphorylation. The only exception was more low value of the respiration control under succinate oxidation. Mitochondria from yeast, grown in the presence of lactate or citrate were capable of the reduction of endogenous pyridine nucleotides under succinate oxidation for the expense of the reverse electron transport. No reverse electron transport from succinate to NAD(P) was observed in mitochondria from yeast grown in the presence of glucose, but it was found under oxidation of alpha-glycerophosphate. All three types of yeast mitochondria were not capable of the reverse electron transport coupled with the pyridine nucleotides reduction under lactate oxidation.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria.

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica.

Changes in respiratory activity and in the contents of adenine nucleotides (ATP, ADP, AMP) were studied in cells of the yeast Yarrowia lipolytica during the development of cyanide-resistant respiration. The transition of the yeast from the logarithmic to the stationary growth phase due to exhaustion of glucose was associated with decreased endogenous respiration and with the activation of a cyanide-resistant oxidase. Cyanide activated cell respiration during the stationary growth phase. The cyanide-resistant respiration was inhibited by benzohydroxamic acid (BHA), an inhibitor of the alternative oxidase. In the absence of cyanide, BHA had no effect on the cells which had the cyanide-resistant oxidase. This indicates that the cells do not use the alternative pathway in vivo. The decreased endogenous respiration of the cells was accompanied by decreased contents of adenine nucleotides. Addition of cyanide resulted in a sharp decrease in the content of ATP, in a twofold increase in the content of ADP, and in a fivefold increase in the content of AMP. In the absence of cyanide, BHA had virtually no effect on the contents of adenine nucleotides. The decreased rate of oxygen consumption during the transition of the cells to the stationary growth phase was caused by the decreased activity of the main cytochrome-containing respiratory chain (2,4-dinitrophenol (DNP) stimulated respiration). The alternative oxidase was synthesized in the cell but was inactive. Cyanide stimulated respiration due to activation of the alternative oxidase via the AMP produced. The decrease in the cell content of ATP is suggested to be a factor inducing the synthesis of the alternative oxidase.     

Photosynthetic electron transfer at the level of cytochrome f and plastocyanin]

Light-induced redox conversions of cytochrome f and plastocyanin in situ and electron transporst from H2O to NADP+ were studied by a dual-wave differential spectrophotometry under identical conditions and subsequently compared. The analysis in red and far red light, treatment by inhibitors, e. g. diurone and dibromothymoquinone, and the analysis of photoreactions during the greening of etiolated seedlings demonstrated that cytochrome f functions only in the non-cyclic chain of electron transport, whereas plastocyanin--both in the non-cyclic and in the cyclic electron transport chains. The jositions of cytochrome f and plastocyanin in various electron-transport chains are proposed.     

Cardiolipin is not required for Bax-mediated cytochrome c release from yeast mitochondria

Cardiolipin (CL) is an inner mitochondrial membrane phospholipid that contributes to optimal mitochondrial function and is gaining widespread attention in studies of mitochondria-mediated apoptosis. Divergent hypotheses describing the role of CL in cytochrome c release and apoptosis have evolved. We addressed this controversy directly by comparing the spontaneous- and Bax-mediated cytochrome c release from mitochondria isolated from two strains of Saccharomyces cerevisiae: one lacking CL-synthase and therefore CL (DeltaCRD1) and the other, its corresponding wild type (WT). We demonstrated by liquid chromatography-mass spectrometry that the main yeast CL species [(16:1)2(18:1)2] differs in fatty acid composition from mammalian CL [(18:2)4], and we verified the absence of the yeast CL species in the DeltaCRD1 strain. We also demonstrated that the mitochondrial association of Bax and the resulting cytochrome c release is not dependent on the CL content of the yeast mitochondrial membranes. Bax inserted equally into both WT and DeltaCRD1 mitochondrial membranes under conditions that lead to the release of cytochrome c from both strains of yeast mitochondria. Furthermore, using models of synthetic liposomes and isolated yeast mitochondria, we found that cytochrome c was bound more "loosely" to the CL-deficient systems compared with when CL is present. These data challenge recent studies implicating that CL is required for Bax-mediated pore formation leading to the release of proteins from the mitochondrial intermembrane space. In contrast, they support our recently proposed two-step mechanism of cytochrome c release, which suggests that CL is required for binding cytochrome c to the inner mitochondrial membrane.     

A hypothetical model of the cytochrome c peroxidase . cytochrome c electron transfer complex

A hypothetical three-dimensional model of the cytochrome c peroxidase . tuna cytochrome c complex is presented. The model is based on known x-ray structures and supported by chemical modification and kinetic data. Cytochrome c peroxidase contains a ring of aspartate residues with a spatial distribution on the molecular surface that is complementary to the distribution of highly conserved lysines surrounding the exposed edge of the cytochrome c heme crevice, namely lysines 13, 27, 72, 86, and 87. These lysines are known to play a functional role in the reaction with cytochrome c peroxidase, cytochrome oxidase, cytochrome c1, and cytochrome b5. A hypothetical model of the complex was constructed with the aid of a computer-graphics display system by visually optimizing hydrogen bonding interactions between complementary charged groups. The two hemes in the resulting model are parallel with an edge separation of 16.5 A. In addition, a system of inter- and intramolecular pi-pi and hydrogen bonding interactions forms a bridge between the hemes and suggests a mechanism of electron transfer.     

Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.     

The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans

We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.     

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.     

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.