Respiratory Electron Transport Pathways Research Articles

Quantitative proteomics reveals the neurotoxicity of trimethyltin chloride on mitochondria in the hippocampus of mice

Trimethyltin chloride (TMT) is a potent neurotoxin widely used as a constituent of polyvinyl chloride plastic in the industrial and agricultural fields. However, the underlying mechanisms by which TMT leads to neurotoxicity remain elusive. In the present study, we constructed a dose and time dependent neurotoxic mouse model of TMT exposure to explore the molecular mechanisms involved in TMT-induced neurological damage. Based on this model, the cognitive ability of TMT exposed mice was assessed by the Morris water maze test and a passive avoidance task. The ultrastructure of hippocampus was analyzed by the transmission electron microscope. Subsequently, proteomics integrated with bioinformatics and experimental verification were employed to reveal potential mechanisms of TMT-induced neurotoxicity. Gene ontology (GO) and pathway enrichment analysis were done by using Metascape and GeneCards database respectively. Our results demonstrated that TMT-exposed mice exhibited cognitive disorder, and mitochondrial respiratory chain abnormality of the hippocampus. Proteomics data showed that a total of 7303 proteins were identified in hippocampus of mice of which 224 ones displayed a 1.5-fold increase or decrease in TMT exposed mice compared with controls. Further analysis indicated that these proteins were mainly involved in tricarboxylic acid (TCA) cycle and respiratory electron transport, proteasome degradation, and multiple metabolic pathways as well as inflammatory signaling pathways. Some proteins, including succinate-CoA ligase subunit (Suclg1), NADH dehydrogenase subunit 5 (Nd5), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (Ndufa4l2) and cytochrome c oxidase assembly factor 7 (Coa7), which were closely related to mitochondrial respiratory electron transport, showed TMT dose and time dependent changes in the hippocampus of mice. Moreover, apoptotic molecules Bax and cleaved caspase-3 were up-regulated, while anti-apoptotic Bcl-2 was down-regulated compared with controls. In conclusion, our findings suggest that impairment of mitochondrial respiratory chain transport and promotion of apoptosis are the potential mechanisms of TMT induced hippocampus toxicity in mice.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

Abstract 15870: Integrated Transcriptomic and Proteomics Analyses Identify Lipid Metabolism as a Key Regulator of RV Size and Function in a Porcine Pulmonary Artery Banded Model

Introduction: Right ventricular (RV) dysfunction is the leading cause of death in pulmonary arterial hypertension (PAH), but the molecular determinants of RV dysfunction are not well defined. In addition, most molecular evaluations of RV dysfunction use small animal models, and data from large animal models is sparse. Methods: Control and pulmonary artery banded (PAB) male piglets were subjected to cardiac MRI to quantify RV ejection fraction (EF), mass, and end systolic volume (ESV). Total RNA was isolated from RV free wall specimens and subjected to Next Gene RNAsequencing. RV mitochondrial enrichments were analyzed with quantitative mass spectrometry to define RV proteome changes. Bioinformatics analyses integrated transcriptomics, proteomics, and MRI data to identified pathways associated with RV dysfunction, hypertrophy, and volume. Results: Both transcriptomic and proteomic profiling revealed significantly different molecular signatures when comparing control to PAB piglets. Both analyses identified metabolism of lipids, lysosome, and mitochondrial fatty acid beta-oxidation pathways were associated with RVEF, while dilated cardiomyopathy, and arrhythmogenic RV cardiomyopathy pathways correlated with RV mass, and metabolism, metabolic pathways, citric acid cycle, and respiratory electron transport pathways were associated with RVESV. Fatty acid metabolism was identified on our analyses, and ferroptosis, a nonapoptotic form of cell death initiated by lipid peroxidation, was associated with RVESV and RV mass. Finally, in addition to coding genes we identified 72 lncRNAs that were strongly (r>|0.85|) associated with RVEF. Conclusions: A combined proteomics and transcriptomics analysis identified multiple lncRNAs and lipid metabolism pathways associated with pathological RV remodeling and RV function. Future studies are needed to define how modulation of these pathways could impact modulate RV size and function in large animal models.

Nitric oxide alleviates mitochondrial oxidative damage and maintains mitochondrial functions in peach fruit during cold storage

Mitochondrial oxidative damage induces aging and apoptosis of organisms, so improving the antioxidant capacity is vital to maintain mitochondrial functions. Peaches were treated with water (control), 15 μmol L − 1 nitric oxide (NO), and 5 μmol L − 1 c-PTIO (NO scavenger), and stored at 0 °C. Mitochondrial reactive oxygen species (ROS) content, mitochondrial swelling, mitochondrial membrane potential, mitochondrial membrane fluidity, and the activities of two mitochondrial respiratory electron transport pathways were measured. Results indicated that NO could extenuate mitochondrial swelling, maintain mitochondrial membrane potential and membrane fluidity, and delay the decrease in activities of the cytochrome pathway and cyanide-insensitive pathway of the mitochondrial respiratory chain. Simultaneously, NO maintained the lower mitochondrial oxygen consumption and cytochrome c content. In summary, NO could efficiently sustain the mitochondrial structure and functions by alleviating the mitochondrial oxidative damage of peaches during cold storage.

Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

Ractopamine-Induced Changes in the Mitochondrial Proteome of Postmortem Beef Longissimus Lumborum

ObjectivesRactopamine is a β-adrenergic agonist approved as growth promotant in beef cattle, and it increases muscle deposition while limiting fat deposition. Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem beef skeletal muscles is influenced by fiber type. Therefore, dietary ractopamine may potentially affect mitochondrial functionality. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from beef cattle.Materials and MethodsPen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 d prior to the harvest. The animals were harvested, and carcasses were chilled for 24 h. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. Mitochondrial proteome was analyzed using two-dimensional electrophoresis, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P < 0.10) between RAC and CON were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsSeven differentially abundant proteins were identified in the mitochondrial proteome. Three proteins were over-abundant (P < 0.10) in RAC, whereas four spots were over-abundant in CON. The proteins over-abundant in RAC mitochondrial proteome was complement component 1 Q subcomponent-binding protein, very long-chain specific acyl-CoA dehydrogenase, and aconitate hydratase. On the other hand, ATP synthase subunit β, prohibitin, Cytochrome b-c1 complex subunit, and thioredoxin-dependent peroxide reductase were over-abundant in CON samples. The differentially abundant proteins belong to four functional groups; i.e., energy metabolism (ATP synthase subunit β, Cytochrome b-c1 complex subunit 1, and aconitate hydratase), chaperone activity (complement component 1 Q subcomponent-binding protein and prohibitin), antioxidant activity (thioredoxin-dependent peroxide reductase), and lipid metabolism (very long-chain specific acyl-CoA dehydrogenase).ConclusionDietary ractopamine impacts mitochondrial proteome in postmortem beef LL muscle and influences the abundance of proteins involved in cellular metabolism and protective mechanisms. The increased protein synthesis and leanness previously reported in ractopamine-fed cattle may be attributed to the decreased expression of enzymes involved in respiratory electron transport pathways and the increased expression of enzymes involved in lipolysis.

Ractopamine-Induced Changes in the Mitochondrial Proteome of Postmortem Beef Longissimus Lumborum

ObjectivesRactopamine is a β-adrenergic agonist approved as growth promotant in beef cattle, and it increases muscle deposition while limiting fat deposition. Dietary ractopamine causes a muscle fiber shift in cattle, and the biochemistry of mitochondria in postmortem beef skeletal muscles is influenced by fiber type. Therefore, dietary ractopamine may potentially affect mitochondrial functionality. Nonetheless, the influence of ractopamine on beef skeletal muscle mitochondrial proteome has not been evaluated. Therefore, the objective of this study was to examine the effects of ractopamine on mitochondrial proteome of postmortem longissimus lumborum (LL) from beef cattle.Materials and MethodsPen-housed crossbred steers were fed either a corn-based basal diet (CON) or a diet top-dressed with Optaflexx 45 (Elanco Animal Health) to provide 400 mg of ractopamine hydrochloride/steer per day (RAC). Ractopamine was fed the last 28 d prior to the harvest. The animals were harvested, and carcasses were chilled for 24 h. The LL muscle samples were obtained from nine (n = 9) RAC and nine (n = 9) CON carcasses. Mitochondrial proteome was analyzed using two-dimensional electrophoresis, and the digital gel images were analyzed. The protein spots exhibiting more than 1.5-fold intensity differences (P < 0.10) between RAC and CON were subjected to in-gel tryptic digestion and were identified by tandem mass spectrometry.ResultsSeven differentially abundant proteins were identified in the mitochondrial proteome. Three proteins were over-abundant (P < 0.10) in RAC, whereas four spots were over-abundant in CON. The proteins over-abundant in RAC mitochondrial proteome was complement component 1 Q subcomponent-binding protein, very long-chain specific acyl-CoA dehydrogenase, and aconitate hydratase. On the other hand, ATP synthase subunit β, prohibitin, Cytochrome b-c1 complex subunit, and thioredoxin-dependent peroxide reductase were over-abundant in CON samples. The differentially abundant proteins belong to four functional groups; i.e., energy metabolism (ATP synthase subunit β, Cytochrome b-c1 complex subunit 1, and aconitate hydratase), chaperone activity (complement component 1 Q subcomponent-binding protein and prohibitin), antioxidant activity (thioredoxin-dependent peroxide reductase), and lipid metabolism (very long-chain specific acyl-CoA dehydrogenase).ConclusionDietary ractopamine impacts mitochondrial proteome in postmortem beef LL muscle and influences the abundance of proteins involved in cellular metabolism and protective mechanisms. The increased protein synthesis and leanness previously reported in ractopamine-fed cattle may be attributed to the decreased expression of enzymes involved in respiratory electron transport pathways and the increased expression of enzymes involved in lipolysis.

Targets identification and mechanism analysis for macrophage activation of low molecular weight saccharides from Cistanche deserticola based on molecular affinity chromatography

This study aims to investigate the targets and targets-involved mechanism for the macrophage activation of low molecular weight saccharides from Cistanche deserticola (LMSC). The phagocytic activity and NO release of RAW264.7 cells were detected, and results showed that LMSC exerts immune activation effect by significantly increasing the phagocytic activity and NO release. LMSC-conjugated epoxy-activated sepharose beads were prepared as affinity reagent to capture the target proteins. Twenty-four proteins such as Eef2 were identified by LC-MS/MS analysis. Pathway enrichment analysis showed LMSC activated RAW264.7 cells by regulating Fcgamma receptor dependent phagocytosis, TNF-alpha NF-κB signaling pathway, glycolysis/gluconeogenesis and the citric acid cycle and respiratory electron transport pathway.

Alternative oxidase impacts ganoderic acid biosynthesis by regulating intracellular ROS levels in Ganoderma lucidum.

The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

Targeting mitochondrial translation by inhibiting DDX3: a novel radiosensitization strategy for cancer treatment.

DDX3 is a DEAD box RNA helicase with oncogenic properties. RK-33 is developed as a small molecule inhibitor of DDX3 and showed potent radiosensitizing activity in preclinical tumor models. This study aimed to assess DDX3 as a target in breast cancer and to elucidate how RK-33 exerts its anti-neoplastic effects. High DDX3 expression was present in 35% of breast cancer patient samples and correlated with markers of aggressiveness and shorter survival. With a quantitative proteomics approach, we identified proteins involved in the mitochondrial translation and respiratory electron transport pathways to be significantly downregulated after RK-33 or DDX3 knockdown. DDX3 localized to the mitochondria and DDX3 inhibition with RK-33 reduced mitochondrial translation. As a consequence, oxygen consumption rates and intracellular ATP concentrations decreased and reactive oxygen species (ROS) increased. RK-33 antagonized the increase in oxygen consumption and ATP production observed after exposure to ionizing radiation and reduced DNA repair. Overall, we conclude that DDX3 inhibition with RK-33 causes radiosensitization in breast cancer through inhibition of mitochondrial translation, which results in reduced oxidative phosphorylation capacity and increased ROS levels, culminating in a bioenergetic catastrophe.

Photosynthetic, respiratory and extracellular electron transport pathways in cyanobacteria

Cyanobacteria have evolved elaborate electron transport pathways to carry out photosynthesis and respiration, and to dissipate excess energy in order to limit cellular damage. Our understanding of the complexity of these systems and their role in allowing cyanobacteria to cope with varying environmental conditions is rapidly improving, but many questions remain. We summarize current knowledge of cyanobacterial electron transport pathways, including the possible roles of alternative pathways in photoprotection. We describe extracellular electron transport, which is as yet poorly understood. Biological photovoltaic devices, which measure electron output from cells, and which have been proposed as possible means of renewable energy generation, may be valuable tools in understanding cyanobacterial electron transfer pathways, and enhanced understanding of electron transfer may allow improvements in the efficiency of power output. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Delocalised electron transport and chemiosmosis in Escherichia coli

The membrane organisation of electron transport and chemiosmosis remains a topic of intense debate, with current models in many bioenergetic membranes favouring the assembly of multiple chemiosmotic components into supercomplexes that could control the pathways of electron flow and utilisation of the proton-motive force. We set out to investigate the distribution and dynamics of OXPHOS components in the plasma membrane of Escherichia coli using a combination of fluorescent protein tagging and fluorescence microscopy with dynamic tracking and single-particle analysis. Complexes investigated included NDH-1, SDH, Cytochrome bd-I and the proton-translocating ATPase, which could all be tagged with a variety of fluorescent proteins, with minimal loss of function [1,2]. Fluorescence microscopy in vivo showed that all complexes tested are concentrated in mobile domains in the membrane, with dimensions of about 100–200 nm and containing 10 s to 100 s of the tagged complex. Simultaneous visualisation of pairs of tagged complexes showed that different complexes are concentrated in separate domains, with no significant co-localisation and therefore no supercomplexes [2]. Since the pairs of complexes tested include the two complexes involved in one of the major respiratory electron transport pathways, and a major source and sink for the proton-motive force, it follows that both electron transport and the proton motive force are largely delocalised over the entire membrane area in E. coli. Consistent with this model, we observed rapid long-range diffusion of a fluorescent quinone analogue. We suggest that long-range quinone diffusion serves to carry electrons between islands of distinct electron transport complexes in the membrane.

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.

Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat

Germination of plant embryo is a dynamic phase-changing process that is driven by a rapid increase in mitochondrial respiration. We studied the development of respiratory electron transport pathways and the profiles of their transcript and protein components during this critical period using wheat embryos. Oxygen consumption through both the cytochrome and alternative pathways increased rapidly upon imbibition. Quantitative RT-PCR analysis using specific primers and western blot analysis using specific antibodies suggested that this respiratory burst was supported both by the stored mRNA and protein components and ones synthesized de novo at least in the cytochrome pathway. Dry embryos also contained transcript and protein of alternative oxidase (AOX), but their levels remained constant during the studied period. By contrast, the alternative pathway capacity showed a marked increase when the cytochrome pathway was inhibited by antimycin A and this increase was associated with increased levels of AOX transcript and protein. Our results suggest that mitochondrial biogenesis is accompanied by sequential and differential gene expression and protein accumulation, and that AOX allows the complex I to continue to conserve energy thus to support embryo germination and initial seedling growth in wheat when the cytochrome pathway is restricted.

Remote control of photosynthetic genes by the mitochondrial respiratory chain

In this paper, we study whether mitochondrial respiration has an impact on the biogenesis of photosynthetic apparatus in the unicellular alga, Chlamydomonas reinhardtii. When respiration was activated by acetate in the dark, mRNAs of nuclear-encoded photosynthetic genes were induced. This induction did not occur in the cells treated with respiration inhibitors or in respiration mutants. An uncoupler of oxidative phosphorylation did not inhibit this mRNA induction; rather, it enhanced it in response to the increase in respiratory electron transport (RET). Plant and algal mitochondria have two RET pathways: the cytochrome pathway and the alternative pathway. Inhibitors of the former pathway inhibited mRNA induction, but inhibitors of the latter enhanced it. Taken together, these indicate that photosynthetic gene mRNAs are induced in response to activation of the cytochrome pathway. This RET-responsive induction is analogous to the photosynthetic electron transport (PET)-responsive induction of photosynthetic gene mRNAs (Matsuo and Obokata, Plant Cell Physiol. 43, 1189). PET-responsive induction occurred in photo-autotrophic and mixotrophic conditions, while RET-responsive induction occurred in mixotrophic and dark heterotrophic conditions. These results indicate that the regulatory system of photosynthetic genes changes between chloroplastic PET-dependent type and mitochondrial RET-dependent type in response to shifts in the dominant energy source between photosynthesis and respiration.

Role of Reactive Oxygen Species in Abiotic and Biotic Stresses in Plants

Biotic and abiotic stresses induce increased formation of reactive oxygen species (ROS) through distinct pathways: pathogen infections activate specific ROS-producing enzymes (i.e. NADPH oxidase, cell wall peroxidases), which results in accumulation of cellular or intercellular ROS, such as superoxide or hydrogen peroxide. Abiotic stresses, on the other hand, cause elevated ROS production principally through an impairment of photosynthetic and respiratory electron transport pathways. Also, these two types of stresses have diverse effects on the antioxidant system of the plant. Results of experiments studying the interaction of abiotic and biotic stresses largely depend on the degree of the applied abiotic stress treatment, the compatible or incompatible host-pathogen interaction and the timing of inoculation in relation to the timing of a preceding abiotic stress treatment.

Inhibitory Effects of Nitric Oxide on Oxidative Phosphorylation in Plant Mitochondria

Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89–92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways—the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O2 consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Δψ) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Δψ to the uninhibited level. Consistent with its inhibition of Δψ, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Nitric oxide suppresses the energy transduction of plant mitochondria

Plant mitochondria are known to possess two respiratory electron transport pathways, i.e. the cytochrome and alternative pathways. The physiological function(s) of the alternative pathway are not fully understood. It has been proposed that the alternative pathway may play an important role in preventing oxidative damage induced by nitric oxide (NO). We have recently revealed that plant nitrate reductase (NR) is capable of converting nitrite to NO. By using the NR-catalyzed NO production system, we investigated in vitro effects of NO on the energy transduction system of mitochondria. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O2 consumption driven by the cytochrome pathway. In contrast, the alternative pathway in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in recovery of the membrane potential to the uninhibited level. Consistent with its inhibition of the membrane potential, NO produced by NR strongly suppressed ATP synthesis in mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO. We suggest that the alternative pathway could be required in photoinhibiting conditions where NR potentially overproduces NO.

INFLUENCE OF CYTOSKELETAL AGENTS ON THE RESPIRATORY ELECTRON TRANSPORT PATHWAYS IN THE CELLS OF WINTER WHEAT LEAVES

The effects of actin and tubulin polymerization inhibitors on the respiratory electron transport pathway activities were investigated using abscisic acid (ABA)- and cold-treated winter wheat seedling leaves. In unstressed control plants, cytochalasin B (15μm) decreased the capacity of the cytochrome pathway, but stimulated the cyanide-resistant pathway, whereas oryzalin (15μm) produced the opposite effects. Cold hardening (3°C for 7 days) and ABA treatment 30μm changed the respiratory pattern in a similar manner to cytochalasin B but to lesser effects. This points to cold- and ABA-induced reduction in microfilament sensitivity to these drugs and hence stabilization of actin-dependent processes. In contrast, oryzalin had only weak effects on control samples and its effects were strengthened in the presence of the cytoskeleton-modifying factors. The data suggest that the potential targets for the agent either increase and/or the degree of involvement of microtubules in the respiratory chain regulation, and therefore that the cytoskeleton can modify the functioning of the respiratory electron transport pathways in winter wheat cells.