Succinate-cytochrome C Reductase Research Articles

Prediction of drug-induced mitochondrial dysfunction using succinate-cytochrome c reductase activity, QSAR and molecular docking

There is increasing evidence that links mitochondrial off-target effects with organ toxicities. For this reason, predictive strategies need to be developed to identify mitochondrial dysfunction early in the drug discovery process. In this study, as a major mechanism of mitochondrial toxicity, first, the inhibitory activity of 35 compounds against succinate-cytochrome c reductase (SCR) was investigated. This in vitro study led to the generation of consistent experimental data for a diverse range of compounds, including pharmaceutical drugs and fungicides. Next, molecular docking and protein-ligand interaction fingerprinting (PLIF) analysis were used to identify significant residues and protein-ligand interactions for the Qo site of complex III and Q site of complex II. Finally, this data was used for the development of QSAR models using a regression-based approach to highlight structural and chemical features that might be responsible for SCR inhibition. The statistically validated QSAR models from this work highlighted the importance of low aqueous solubility, low ionisation, fewer 6-membered rings and shorter hydrocarbon alkane chains in the molecular structure for increased inhibition of SCR, hence mitochondrial toxicity. PLIF analysis highlighted two key residues for inhibitory activity of the Qo site of complex III: His 161 as H-bond acceptor and Pro 270 for arene interactions. Currently, there are limited structure-activity models published in the scientific literature for the prediction of mitochondrial toxicity. We believe this study helps shed light on the chemical space for the inhibition of mitochondrial electron transport chain (ETC).

Neuromodulatory effect of solvent fractions of Africa eggplant (Solanium dadyphyllum) against KCN-induced mitochondria damage, viz. NADH-succinate dehydrogenase, NADH- cytochrome c reductase, and succinate-cytochrome c reductase

BackgroundIn the past few years, there has been a tremendous increase in the number of plant-based health supplements with respect to their safety and efficacy in diseases treatment and prevention. Solanum dasyphyllum, also known as Africa eggplant is ethnomedicinally used as an antivenom, pain reliever and anticonvulsant in various part of Nigeria, however, there is no scientific data to support some of these claims.MethodsThis study evaluated the protective effect of solvent fractions of Solanum dasyphyllum, hexane fraction of S. dasyphyllum (HFSD), dichloromethane fraction of S. dasyphyllum (DFSD), ethylacetate fraction of S. dasyphyllum (EAFSD), methanolic fraction of S. dasyphyllum (MFSD) and crude fraction of S. dasyphyllum (CFSD) on cyanide-induced oxidative stress and neurotoxicity in vitro in the cerebral cortex. Neuroprotective activities were evaluated by assaying for markers of oxidative stress, neurotoxicity and electron transport system enzymes via evaluating lipid peroxidation (LPO), protein carbonyl (PC), reduced glutathione (GSH), acetylcholinesterase (AChE), NADH-succinate dehydrogenase (NSD), NADH-cytochrome c reductase (NCR), and succinate-cytochrome c reductase (SCR) in the homogenate of cerebral cortex.ResultsThe results showed that all solvent fractions of S. dasyphyllum significantly ameliorated cyanide-induced oxidative stress (P < 0.05). It inhibited the activity of acetylcholinesterase-HFSD (68.42 ± 5.37%), DFSD (36.32 ± 5.45%), EFA (20 ± 0.69%), MFSD (33.16 ± 4.8%) and CFSD (35.79 ± 2.8%), increased the activity of NSD [HFSD (94.74 ± 7.3%), EAFSD (78.95 ± 5.4%) and CFSD (60.53 ± 4.6%)], while DFSD (− 5.26 ± 1.4%) and MFSD (− 7.9 ± 0.4%) had a negative effect, increased the activity of NCR [HFSD (91.89 ± 7.1%), DFSD (90.54 ± 8.2%), EAFSD (62.16 ± 4.7%); MFSD (306.76 ± 7.2%) and CFSD (154.0 ± 8.1%)]. All the solvent fractions also significantly increased the activity of SCR [HFSD (70.59 ± 3.8%), EAFSD (58.82 ± 6.4%), MFSD (88.24 ± 9.0%) CFSD (76.47 ± 8.2%)] apart from DFSD (− 5.88 ± 1.2%) in rat cerebral cortex.ConclusionThis result shows that S. dasyphyllum has neuroprotective activities, however HFSD shows the most potent bioactivities in maintaining mitochondria integrity by preserving the electron transport system. Further work can be done on isolating and characterizing the bioactive compound in HFSD for novel natural product in the treatment of neurological disorders.

Transcriptomic profiling of mitochondrial dysfunction induced apoptosis in accelerated cardiovascular disease

IntroductionVascular calcification is a significant contributor to accelerated cardiovascular disease and mortality in patients with chronic kidney disease (CKD). We previously reported that heat shock protein 70 (HSP70) induction can inhibit human aortic smooth muscle cell (HA‐SMC) calcification in CKD. In addition, our data indicated mitochondrial complexes II, IV, NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) activities were preserved by HSP70 induction leading to reduced apoptosis. Apoptosis occurs with either receptor mediated extrinsic pathway or the mitochondrial‐dependent intrinsic pathway. The molecular mechanisms of HSP70 vasculo‐protective effects involving mitochondrial dysfunction induced apoptosis are currently unknown.HypothesisThe goal of the present study was to investigate the transcriptomic profile of genes involved in mitochondrial function and vasculo‐protective mechanisms of HSPs in apoptosis induced vascular calcification.MethodsHuman arteries were collected from healthy and CKD patients. Human aortic endothelial cells (HAECs) was treated in calcification medium (CM: 5mM β‐glycerolphosphate and 5mM CaCl2) in time‐course experiments (0–48 hours), in vitro. Total RNA from human arteries and HAECs was extracted using the Nucleospin RNA isolation kit. cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). Five million single‐ended raw reads from each sample were mapped to the human reference genome consortium GRCh38 by Burrows‐Wheeler Aligner (BWA). Gene analysis were performed by the combinations of fold‐changes on log 2 ratio, and p value &lt; 0.05.ResultsIn healthy human arteries, we found that HSPA5 (fold change, FC, 3.85), HSPA1 (FC=3.64), and HSPA9 (FC=3.85) expression was preserved and Bcl2 associated Athanogene 1 (BAG1) (FC=7.44) was upregulated. However, mitochondrial fusion regulation genes MFN1 (FC=1.83) and OPA1 (FC=2.33) were down‐regulated in CKD arteries together with downregulation of downstream anti‐apoptotic gene Bcl2 (FC=4.52) and up‐regulation of pro‐apoptotic gene BAX (FC=2.77) and P53 (FC=5.35). Lastly, caspase 3 (FC=2.6) and cytochrome c (FC=2.29) expression were significantly increased in CKD arteries compared to control. In the primary HAECs, our data indicates that BAG1 was significantly increased at 6 hours prior to upregulation of Bcl2 at 12 hours in HAECs after CM treatment. BAG1 is a co‐chaperone for heat‐shock protein 70 (HSP70) that coordinates signals for cell growth in response to stress. Stress respondent genes, DNAJB6 (HSP40) and HSPA5 (HSP70 member5) was significantly increased at 24 hours and 12 hours respectively in CM‐treated HAECs.ConclusionComplex differential gene regulation involving preservation of mitochondrial function and cell apoptosis may be involved in the pathogenesis of accelerated vascular calcification. Decreased HSPs in CKD arteries may play the key role in mitochondrial dysfunction induced endothelial cells' apoptosis, which further leading to vascular calcification. Our findings may serve to inform the rational development of therapeutic strategies for vascular calcification involving therapeutic targeting of HSPs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis of new 4-aryloxy-N-arylanilines and their inhibitory activities against succinate-cytochrome c reductase

Succinate-cytochrome c reductase (SCR) is composed of a mixture of mitochondrial complex II (succinate-ubiquinone oxidoreductase) and complex III (cytochrome bc1 complex). Meanwhile, complexes II and III are two promising targets of numerous antibiotics and fungicides. With an aim to identify new lead structures for SCR, complex II or III, a new series of 4-aryloxy-N-arylanilines were synthesized by introducing a 4-aryloxy phenyl group as one of the aryl groups in diaryl amines. With the economic Cu(OAc)2·H2O as the optimal copper promoter, a simple and facile protocol was utilized to afford 24 target products in 56–93% yields. Furthermore, extensive screening results suggested variable inhibitory activities of these compounds against SCR. Exceptionally, compounds 7k–7n showed excellent inhibition potency with their IC50 values in the nanomolar range, demonstrating higher potency than the commercial controls (penthiopyrad and azoxystrobin) by over one order of magnitude.

Alpha-mangostin from mangosteen (Garcinia mangostana Linn.) pericarp extract reduces high fat-diet induced hepatic steatosis in rats by regulating mitochondria function and apoptosis.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is caused by multiple factors including hepatic oxidative stress, lipotoxicity, and mitochondrial dysfunction. Obesity is among the risk factors for NAFLD alongside type 2 diabetes mellitus and hyperlipidemia. α- mangostin (α-MG) extracts from the pericarps of mangosteen (Garcinia mangostana Linn.) may regulate high fat diet-induced hepatic steatosis; however the underlying mechanisms remain unknown. The aim of this study was to investigate the regulatory effect of α-MG on high fat diet-induced hepatic steatosis and the underlying mechanisms related to mitochondrial functionality and apoptosis in vivo and in vitro.MethodsSprague Dawley (SD) rats were fed on either AIM 93-M control diet, a high-fat diet (HFD), or high-fat diet supplemented with 25 mg/day mangosteen pericarp extract (MGE) for 11 weeks. Thereafter, the following were determined: body weight change, plasma free fatty acids, liver triglyceride content, antioxidant enzymes (superoxide dismutase, SOD; glutathione, GSH; glutathione peroxidase, GPx; glutathione reductase GRd; catalase, CAT) and mitochondrial complex enzyme activities. In the in vitro study, primary liver cells were treated with 1 mM free fatty acid (FFA) (palmitate: oleate acid = 2:0.25) to induce steatosis. Thereafter, the effects of α-MG (10 μM, 20 μM, 30 μM) on total and mitochondria ROS (tROS, mitoROS), mitochondria bioenergetic functions, and mitochondrial pathway of apoptosis were examined in the FFA-treated primary liver cells.ResultsThe MGE group showed significantly decreased plasma free fatty acids and hepatic triglycerides (TG) and thiorbarbituric acid reactive substances (TBARS) levels; increased activities of antioxidant enzymes (SOD, GSH, GPx, GRd, CAT); and enhanced NADH-cytochrome c reductase (NCCR) and succinate-cytochrome c reductase (SCCR) activities in the liver tissue compared with HFD group. In the in vitro study, α-MG significantly increased mitochondrial membrane potential, enhanced cellular oxygen consumption rate (OCR), decreased tROS (total ROS) and mitoROS (mitochondrial ROS) levels ; reduced Ca2+ and cytochrome c (cyt c) release from mitochondria, and reduced caspases 9 and 3 activities compared with control group.ConclusionThese findings demonstrate α-MG attenuated hepatic steatosis in high fat-diet fed rats potentially through enhanced cellular antioxidant capacity and improved mitochondrial functions as well as suppressed apoptosis of hepatocytes. The findings of study represent a novel nutritional approach on the use of α-MG in the prevention and management of NAFLD.

Abstract 18849: Arterial Calcification Can Be Inhibited via Maintained Mitochondrial Homeostasis in Chronic Kidney Disease

Background: Vascular calcification (VC) is a significant contributor to cardiovascular mortality in patients with chronic kidney disease and chronic inflammatory conditions. Previous work by our group has shown that HSP72 is an inducible protein that can preserve mitochondrial complexes II, IV, NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) activities and prevent cardiomyocyte apoptosis. We recently reported that heat shock protein 72 (HSP72) could be induced in human arteries and prevent human aortic smooth muscle cell (HA-SMC) calcification. However, the molecular mechanisms involved in vasculo-protective effects of HSP72 have yet to be elucidated. In this study, we postulate that inducible HSP72 may exert vasculo-protective effects by stabilizing HA-SMCs phenotype through inhibition of mitochondrial dysfunction. Methods: In vitro long-term calcification model: HA-SMCs treated with calcification medium (CM) containing 5mM CaCl 2 and 5mM β-glycerophosphate for 21 days. Results: Our results show that inducible HSP72 and mitochondrial HSP (HSPA5) are significantly expressed following heat shock treatment in HA-SMCs. Inducible HSP72 inhibited the development of VC in our long-term calcification model, in vitro . We next showed that JC-1 complex, an indicator of mitochondrial membrane potential stability and mitochondrial respiratory chain complexes II, IV was preserved in HA-SMCs treated with calcification medium following induction of HSP72. These cellular protective effects were however abolished following HSP72 knockdown by HSP72 siRNA. Furthermore, p53, Bax and Bak were up-regulated and Bcl-2 was down-regulated in HA-SMCs treated in calcification medium and these pro-apoptotic changes could be reversed following induction of HSP72. Conclusion: HSP72’s anti-calcific effects involve stabilization of mitochondrial function and inhibition of HA-SMC apoptosis. We suggest treatment strategies involving induction of HSP72 as a new approach to inhibit VC.

Comparative Kinetics of Qi Site Inhibitors of Cytochrome bc1 Complex: Picomolar Antimycin and Micromolar Cyazofamid

Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki =0.033±0.00027nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki =12.90±0.91μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

Design, syntheses, and kinetic evaluation of 3-(phenylamino)oxazolidine-2,4-diones as potent cytochrome bc1 complex inhibitors

The cytochrome bc 1 complex (EC 1.10.2.2, bc 1) is one of the most promising targets for new drugs and agricultural fungicides. Among the existing bc 1 complex inhibitors specifically binding to the Q o site, oxazolidinedione derivatives have attracted great attention. With the aim to understand the substituent effects of oxazolidinedione derivatives on the inhibition activity against the bc 1 complex, a series of new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further inhibitory kinetics studies against porcine succinate–cytochrome c reductase (SCR) revealed that the representative compound 8d and famoxadone are both non-competitive inhibitors with respect to the substrate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH 2). In addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory activity against the porcine SCR than the porcine bc 1 complex, indicating that these two inhibitors not only inhibited the activity of the bc 1 complex, but possibly affect the interaction between the complex II and the bc 1 complex. To our knowledge, this is the first report that famoxadone and its analogs have effects on the interaction between the complex II and the bc 1 complex.

Excess no predisposes mitochondrial succinate–cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O 2 •− generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate–cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/ •OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron–H 2O 2-dependent Fenton reaction or O 2 •−-dependent Haber–Weiss mechanism. Direct EPR measurement of SCR at 77 K indicated the formation of a nonheme iron–NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce •OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.

Late phase ischemic preconditioning preserves mitochondrial oxygen metabolism and attenuates post-ischemic myocardial tissue hyperoxygenation

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism. Main methodsC57BL/6 mice were subjected to 30min coronary ligation followed by 60min or 24h reperfusion with or without LPC (3cycles of 5min I/5min R): Sham, LPC, I/R, and LPC+I/R group. Myocardial tissue Po2 and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy. Key findingsUpon reperfusion, tissue Po2 rose significantly above the pre-ischemic level in the I/R mice (23.1±2.2 vs. 12.6±1.3mmHg, p<0.01). This hyperoxygenation was attenuated by LPC in the LPC+I/R mice (11.9±2.0mmHg, p<0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC+I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC+I/R mice compared to that in the Sham control (1.24±0.01 and 1.23±0.01, p<0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268±0.0016/min) and was corrected by LPC in the LPC+I/R mice (0.0379±0.0023/min). Finally, LPC reduced the infarct size in the LPC+I/R mice (10.5±0.4% vs. 33.3±0.6%, p<0.05). SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.

Succinate cytochrome c reductase in schistosomiasis: in vitro inhibition by some schistosomicidal drugs

Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome c reductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its Vmax and Km compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection.

Mechanism of action and selectivity of a novel fungicide, pyribencarb

Pyribencarb is a novel benzylcarbamate-type fungicide, which is active against a wide range of plant pathogenic fungi. In this paper, the inhibitory effects of this fungicide on the electron transport system of fungi, plants, rat and carp were examined to elucidate its mode of action and selectivity. Pyribencarb potently inhibited succinate-cytochrome c reductase (SCR) activities of Botrytis cinerea (cucumber gray mold), Corynespora cassiicola (leaf spot) and decylubiquinol-cytochrome c reductase (UCR) activity of B. cinerea. Pyribencarb inhibited the UCR of B. cinerea in an uncompetitive manner with respect to decylubiquinol, which was the same as strobilurin fungicides, and the substrate-dependent inhibition constant was found from calculation to be 13 nM. These results suggested that the target site of pyribencarb is cytochrome b of complex III in the electron transport system of the respiratory chain. On the other hand, the inhibitory potency of pyribencarb on SCR activities of plants, rats and carp was relatively weak compared with that of strobilurin fungicides, indicating that pyribencarb is a Qo inhibitor of cytochrome b, whose properties are superior to well-known Qo inhibitor fungicides in terms of target. The binding site of pyribencarb on cytochrome b was assumed to be a little different from that of strobilurin fungicides, because pyribencarb inhibited SCRs of strobilurin fungicide-resistant strains of B. cinerea and C. cassiicola with relatively low concentrations. The binding site was also discussed through comparison of amino acid sequences of plants, rats, carp, yeast and fungi, including B. cinerea, which was elucidated in this paper.

The role of individual lysine residues of horse cytochrome c in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome-c with single or double substitutions of lysine residues near the heme cavity was prepared that provided an interaction of mitochondrial ubiquinone with cytochrome-c reductase (EC 1.10.2.2) (complex III) and cytochrome-c oxidase (EC 1.9.3.1) (complex IV). The succinate cytochrome-c reductase and cytochrome-c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome-c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol-cytochrome-c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome-c oxidase.

Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation

Diazoxide, a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener, protects the heart from ischemia-reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O(2)(-*)) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O(2)(-*) generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV-visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoK(ATP) channel and ubisemiquinone-dependent O(2)(-*) generation via inhibiting mitochondrial complex II-dependent respiration.

Dominant cold paralytic mutations on the X-chromosome of Drosophila melanogaster

Six new cold paralytic behavioural mutations on the X-chromosome of Drosophila melanogasterare reported. All mutations map to a position of 1.7 map units to the left offwhere also a previously found cold paralytic mutant has been located. On the basis of complementation tests and behavioural studies the new mutants were all considered to be mutations of the same locus as the previously found cold paralytic mutant, Ocd*, and therefore called Ocdks-2 to Ocdks-7. Only minor differences were found in the cold paralytic behaviour of Ocdks-2 to Ocdks-6 when compared to Ocdks-1. The critical temperature of Ocdks-7 was found to be 2°C lower than for the other mutants. The Arrhenius activation energy of succinate-cytochrome creductase exhibited a change at about 18°C in mitochondria from mutants Ocdks-2 to Ocdks-6. In Ocdks-7 this change had been shifted to 16°C. These data confirm the correlation between the cold paralysis and the change in activation energy which has previously been established for Ocdks-1.

Direct and Indirect Roles of Cytochrome b in the Mediation of Superoxide Generation and NO Catabolism by Mitochondrial Succinate-Cytochrome c Reductase

Mitochondrial superoxide (O2*-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2*- generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2*- generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2*- generation from SCR was detected as the spin adduct DEPMPO/*OOH. Inhibitors of the Q(o*-) site only marginally reduced (20-30%) this O2*- production, suggesting a secondary role of Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) reduction, and the reaction was mediated by the formation of an O2*- intermediate, indicating a direct role for cytochrome b in O2*- generation. In the presence of NO, DEPMPO/*OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2*-. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2*-. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2*- in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.

Isolated Mitochondrial Myopathy Associated With Muscle Coenzyme Q10 Deficiency

Primary coenzyme Q(10) (CoQ(10)) deficiency is rare. The encephalomyopathic form, described in few families, is characterized by exercise intolerance, recurrent myoglobinuria, developmental delay, ataxia, and seizures. To report a rare manifestation of CoQ(10) deficiency with isolated mitochondrial myopathy without central nervous system involvement. The patient was evaluated for progressive muscle weakness. Comprehensive clinical evaluation and muscle biopsy were performed for histopathologic analysis and mitochondrial DNA and respiratory chain enzyme studies. The patient began taking 150 mg/d of a CoQ(10) supplement. The elevated creatine kinase and lactate levels with abnormal urine organic acid and acylcarnitine profiles in this patient suggested a mitochondrial disorder. Skeletal muscle histochemical evaluation revealed ragged red fibers, and respiratory chain enzyme analyses showed partial reductions in complex I, I + III, and II + III activities with greater than 200% of normal citrate synthase activity. The CoQ(10) concentration in skeletal muscle was 46% of the normal reference mean. The in vitro addition of 50 micromol/L of coenzyme Q(1) to the succinate cytochrome-c reductase assay of the patient's skeletal muscle whole homogenate increased the succinate cytochrome-c reductase activity 8-fold compared with 2.8-fold in the normal control homogenates. Follow-up of the patient in 6 months demonstrated significant clinical improvement with normalization of creatine kinase and lactate levels. The absence of central nervous system involvement and recurrent myoglobinuria expands the clinical phenotype of this treatable mitochondrial disorder. The complete recovery of myopathy with exogenous CoQ(10) supplementation observed in this patient highlights the importance of early identification and treatment of this genetic disorder.

The operation of the alternative electron-leak pathways mediated by cytochrome c in mitochondria

Low (1 × 10 −9 M) concentrations of cytochrome c inhibit H 2O 2 production in cytochrome c-depleted mitochondria, purified succinate-cytochrome c reductase (SCR) and antimycin A inhibited cytochrome c-depleted HMP. At higher concentration (2 × 10 −6 M), cytochrome c eliminates pre-existed H 2O 2 if feeding electrons to it by succinate. Cytochrome c also decreases the OH produced by succinate-cytochrome c reductase oxidizing succinate. We conclude that the alternative electron-leak pathway mediated by cytochrome c operates very well. In the presence of antimycin A, ferrocytochrome c can suppress the generation of H 2O 2 in SCR system, but ferricytochrome c cannot. Similar results are obtained on the elimination of pre-existed H 2O 2 by cytochrome c. For hydroxyl radical, antimycin A abolishes the suppression caused by both ferrocytochrome c and ferricytochrome c. These results indicate that the reductive state of cytochrome c caused by electron-flow is necessary and sufficient for the operation of cytochrome c-mediated electron-leakage pathway.

99mTc-HMPAO distribution at SPECT is associated with succinate-cytochrome c reductase (SCR) activity in subjects with psychiatric disorders

The origin of altered 99mTc-HMPAO distribution at SPECT in psychiatric disorders is unknown. Correlations between brain 99mTc-HMPAO distribution and muscle succinate-cytochrome c reductase (SCR, complex II + III) were assessed in 20 unmedicated psychiatric patients. Significant negative correlations were found between 99mTc-HMPAO distribution in associative sensory regions and SCR activity. Sensory cortices are normally enriched in complex II activity. The production of electrons and reactive oxygen species affecting the redox state is considered to be highest from complex III, but complex II may also contribute. The negative relationship between 99mTc-HMPAO uptake and SCR activity may be due to redox state alterations influencing fixation of the radiopharmaceutical.

Heat shock pretreatment prevents cardiac mitochondrial dysfunction during sepsis.

The present study was designed to investigate the effect of previous heat shock treatment on the mitochondria function of the heart during a cecal ligation and puncture (CLP)-induced sepsis model. Rats of the heated group were heated by whole-body hyperthermia 24 h before the CLP operation. Cardiac mitochondria were freshly collected 9 and 18 h after CLP, indicating early and late sepsis, respectively. The expressions of heat shock protein 72 (Hsp72), glucose-regulated protein 75 (Grp75), and mitochondrial complexes I, II, III, and IV were evaluated by Western blot and immunochemical analysis. Enzyme activities of NADH cytochrome c reductase (NCCR), succinate cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) were measured after the reduction or oxidation of cytochrome c using a spectrophotometer. The results showed that the ATP content in the heart significantly declined during late sepsis, whereas heat shock treatment reversed this declination. The enzyme activities of NCCR, SCCR, and CCO were apparently suppressed during late stage of sepsis. The protein expressions of mitochondrial complex II and complex IV and Grp75 were also down-regulated during sepsis. Previously treated by heat shock, late-sepsis rats emerged with a high preservation of mitochondrial respiratory chain enzymes, both the protein amount and enzyme activity. Aspects of morphology were observed by electron microscopy, while heat shock treatment revealed the attenuation of cardiac mitochondrial damage induced by sepsis. In conclusion, structural deformity and the decrease of respiratory chain enzyme activity in mitochondria and its leading to a decline of ATP content are highly correlated with the deterioration of cardiac function during sepsis, and heat shock can reverse adverse effects, thus achieving a protective goal.