Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.     

Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-l-cysteine (NAC) on cell viability, hydrogen peroxide (H₂O₂) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H₂O₂ and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.     

Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.     

Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1

To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Δyca1), cytochrome c (Δcyc1,7) and both (Δcyc1,7Δyca1) were compared for AA-PCD occurrence, hydrogen peroxide (H₂O₂) production and caspase activity. AA-PCD occurs in Δcyc1,7 and Δcyc1,7Δyca1 cells slower than in wt, but similar to that in Δyca1 cells, in which no cytochrome c release occurs. Both H₂O₂ production and caspase activation occur in these cells with early and extra-activation in Δcyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.     

Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger

To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.     

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.     

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.     

Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.     

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (rho0 cells). However, unlike the case with rho+ cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic rho0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.     

Proteasome function is required for activation of programmed cell death in heat shocked tobacco Bright-Yellow 2 cells

To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.     

A functional hybrid between the cytochrome bc1 complex and its physiological membrane-anchored electron acceptor cytochrome cy in Rhodobacter capsulatus

The membrane integral ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductase (or the cyt bc1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome cy (cyt cy), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc1 complex is often correlated with a similar lack of cyt cy (Jenney, F. E., et al. (1994) Biochemistry 33, 2496-2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt cy to the cyt bc1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc1-cy fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc1-cy fusion complex has an unprecedented bis-heme cyt c1-cy subunit instead of the native mono-heme cyt c1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc1-cy fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.     

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.     

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.Key words: caspase-like activation, FRET, programmed cell death, reactive oxygen species, ultraviolet-CUltraviolet-C has been shown to be a very convenient trigger to induce PCD in plants and protoplasts.^1,2 Others have shown that UV induction of plant PCD requires light and that caspase-like proteolytic activation is induced in this process.¹ Our recent works have shown that ROS mainly localizing in mitochondria and chloroplasts are produced in a light dependent manner during the early stages of UV stress, and that ROS production and mitochondrial dysfunction play important roles during UV-induced Arabidopsis PCD (Fig. 1).² We also found that if the Arabidopsis plants, which were kept at light for 1 h after UV irradiation then were moved to the dark and kept for 60 h, showed no evident plant death phenomena (unpublished data), though burst of ROS has appeared after UV exposure and subsequent 1 h light irradiation.² In contrast, seedlings developed an obvious bleaching when kept in light for 60 h after UV treatment. These findings prompt us to carry out further investigations to dig out the role of ROS in the execution of this type of cell death, and to ask whether the produced ROS in the early stages is involved in the activation of caspase-like protease.Open in a separate windowFigure 1Hypothetical model of the signal transduction pathways in the plant programmed cell death induced by UV-C overexposure. After UV and light treatment a quick burst of ROS appear in the region of mitochondria and chloroplasts, then the mitochondria undergo functional dysfunction, which ultimately leads to cell death. Caspase-like activation and nucleus damage are also involved in the control of this type cell death. Solid line means the issues have been detected. Dotted line and question marks indicate events that have not been detected in this process. For detailed explanation, see the text.It has been reported that ROS is required for the release of cytochrome c (cyt c) and subsequent activation of caspase-like proteases during heat-shock induced plant PCD, and the addition of caspase inhibitors (zVAD-fmk or AC-DEVD-CHO) can prevent the degradation of cyt c and protect the plant cells from cell death.³ Thus these findings suggest that ROS can trigger the release of cyt c, but do not cause cell death, which requires caspase-like activation.³ Conversely, caspase inhibitors have also shown to effectively block the oxidative burst and the plant cell death induced by camptothecin incubation.⁴ These studies suggest the complex relationship between ROS production and caspase activation during execution of plant PCD event. The ROS production and the mitochondrial dysfunction during UV-induced plant PCD have been illustrated in our research. We have suggested the occurrence of MTP disruption during UV stress; however, whether cyt c is released from mitochondria has not been assessed (Fig. 1). The important roles of cyt c release and subsequent caspase activation have been suggested in various types of programmed cell death including mammal and plant cells.^3,5,6 It will be a very challenging work to detect whether cyt c is released from mitochondria and is involved in the caspase-like proteolytic activation, and to further elucidate the relationship between ROS production and caspase-like activation in UV-induced plant PCD (Fig. 1).The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD.⁷ Using synthetic fluorogenic caspase-3 substrate, DEVD cleavage activity was detected during UV or heat shock-induced apoptosis of plant cells, and caspase inhibitors were able to suppress these types of cell death.^1,3 Caspase-like activities have also been detected in plant hypersensitive response (HR) triggered by tobacco mosaic virus (TMV), or plant PCD induced by chemicals like camptothecin.^8,9 All these experiments suggest the existence of functional caspase proteolytic activity in plant cells undergoing PCD. However, most of these results are from in vitro analysis using synthetic fluorogenic substrates or synthetic peptide inhibitor to caspases, this demand us to further dig out the plant caspase encoding gene and to real-time detect the caspase-like activity in vivo.Another of our ongoing work is aiming to detect the caspase-3-like proteolytic activation in living plant cells during UV-induced plant PCD, which is achieved by using the fluorescence resonance energy transfer (FRET) technique. FRET is the phenomenon whereby a fluorescent molecule—the donor—transfers energy by a nonradiative (through space) mechanism to a neighboring chromophore - the acceptor.¹⁰ FRET as a powerful technique to monitor compartmentation and subcellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells has gained increasing importance for biotechnological applications during the last few years.¹¹ During the past few years FRET technique has been successfully used to monitor interactions and distances between molecules in living plant cells.^12–14 Presently, we have constructed a recombinant caspase substrate to monitor caspase-3-like protease activation in single living plant protoplast in real time. This recombinant is composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (ECFP-DEVD-EYFP) as the papers demonstrated. 15 Arabidopsis mesophyll protoplasts have been successfully transiently transfected with our recombinant plasmid for expression of ECFP-DEVD-EYFP fusion proteins under control of the CaMV 35S promoter according to a modified procedure (as described previously, ref. ¹⁶). Preliminary experimental results have proved the feasibility of this method to real-time detect the caspase-like activation in living plant cells during UV-induced plant PCD.Using this FRET probe, we may real-time detect the caspase-like activation during UV-induced plant PCD, and elucidate the relationship between ROS production and caspase-like activation as well as verify our hypothesis that whether ROS is necessary for the activation of caspase-like proteases during this process. So the role of ROS in the execution of this type cell death can be further investigated. These subsequent researches will certainly increase our knowledge about the signal transduction pathways in UV-induced Arabidopsis PCD.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.