Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (Mr 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (Em = -224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

The effect of pyruvate transport inhibitors on the regulation of pyruvate dehydrogenase in the perfused rat heart.

The effect of the mitochondrial pyruvate transport inhibitors, α-cyanocinnamate and α-cyano-4-hydroxycinnamate, on the regulation of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated perfused rat heart. Metabolic flux through pyruvate dehydrogenase was monitored by measuring ¹⁴CO₂ production from [1-¹⁴C]pyruvate infused into the heart. A stepwise increase in the concentration of the inhibitor in the influent perfusate effected a stepwise reduction of the flux through the enzyme complex at all pyruvate concentrations tested. However, the magnitude of the α-cyanocinnamate-insensitive flux through pyruvate dehydrogenase increased markedly as the infused pyruvate concentration was elevated. The inhibition of pyruvate decarboxylation in the heart was nearly completely reversed following cessation of the inhibitor infusion. α-Cyanocinnamate was nearly 10 times more potent than α-cyano-4-hydroxycinnamate as an inhibitor of the flux through pyruvate dehydrogenase. Maximally inhibiting levels of α-cyano-4-hydroxycinnamate caused an increase in the ratio of the active form of pyruvate dehydrogenase to the total extractable enzyme complex from a value of 0.5 at 1 mm infused pyruvate (in the absence of the inhibitor) to a value of near unity. This result indicated that the intramitochondrial pyruvate concentration was severely depleted by the infusion of the inhibitor and that the enzyme complex was interconverted to its active form under these conditions. Removal of the inhibitor from the perfusion medium again lowered the ratio of the active/total pyruvate dehydrogenase to near its original level of 0.5 and restored the original flux through the enzyme complex indicating that mitochondrial pyruvate transport has been restored. The results of this study indicate that α-cyanocinnamate and its derivatives are effective inhibitors of pyruvate transport in the perfused heart and that carrier-mediated pyruvate transport can be an important parameter in the regulation of the activation state and the metabolic flux through the pyruvate dehydrogenase multienzyme complex in the heart.     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (M_r 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (E_m = ?224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

Regulation of oxidative decarboxylation of branched-chain 2-oxo acids in rat liver mitochondria

At 0.1 mM 2-oxo[1-14C]isocaproate or 2-oxo[1-14C]isovalerate plots of the reciprocal of the rate of 14CO2 formation by branched-chain 2-oxo acid dehydrogenase complex in mitochondria vs alpha-cyanocinamate concentration were linear up to high inhibitor concentrations, indicating that the monocarboxylate carrier-mediated transport was the rate-limiting step. At low (0.025 mM) concentration of 2-oxo[1-14C]isocaproate or 2-oxo[1-14C]isovalerate the 1/v vs I plots became nonlinear indicating that the branched-chain 2-oxo acid dehydrogenase activity determined the rate of 14CO2 formation. Inhibition of branched-chain 2-oxo acid dehydrogenase complex by clofibric acid or arsenite showed that at 0.1 mM 2-oxoisovalerate the activity of the complex became the rate-limiting step of the pathway. The availability of the 2-oxoisocaproate or 2-oxoisovalerate seems to affect the phosphorylation and the activity of the branched-chain 2-oxo acid dehydrogenase complex only at low, physiological concentrations of these substrates (less than 0.025 mM).     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Selective inhibition of NADH-CoQ oxidoreductase (complex I) of rat brain mitochondria by arachidonic acid

The effects of arachidonic acid on the enzyme complexes in the electron transport system were investigated using submitochondrial particles from rat brain. Arachidonic acid irreversibly inhibited NADH-CoQ oxidoreductase (complex I) activity, but had no effect on the activities of succinate-CoQ oxidoreductase (complex II), CoQH2-cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), ATPase (complex V), glutamate dehydrogenase, and malate dehydrogenase up to 50 microM. The inhibition was dose-dependent with an IC50 value of 110 nmol/mg protein. The Lineweaver-Burk plot revealed that the inhibition by arachidonic acid was noncompetitive against CoQ with a Ki value of 33 microM and uncompetitive against NADH with a Ki value of 22 microM.     

Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters respiratory properties.

Chloroplasts and cyanobacteria contain genes encoding polypeptides homologous to some subunits of the mitochondrial respiratory NADH-ubiquinol oxidoreductase complex (NADH dehydrogenase). Nothing is known of the role of the NADH dehydrogenase complex in photosynthesis, respiration, or other functions in chloroplasts, and little is known about the specific roles of the perhaps 42 subunits of this complex in the mitochondrion. Inactivation of a gene for subunit 4 (ndhD-2, ndh4) of this complex in the cyanobacterium Synechocystis 6803 has no effect on photosynthesis, judging from the rate of photoautotrophic growth of mutant cells, but the mutant's respiratory rate is about 6 times greater than that of wild-type cells. Respiratory electron transport activity in cyanobacteria is associated both with photosynthetic thylakoid membranes and with the outer cytoplasmic membrane of the cell. Cytoplasmic membranes of mutant cells have much greater NADH-dependent cytochrome reductase activity than preparations from wild-type cells; this activity remains at wild-type levels in isolated thylakoid membranes. It is suggested that the 56.6-kD product of ndhD-2 is not essential for the activity of a cytoplasmic membrane-bound NADH dehydrogenase but that it regulates the rate of electron flow through the complex, establishing a link between this ndh gene and respiration. The activity of the molecularly distinct thylakoid-bound NADH dehydrogenase is apparently unaffected by the loss of ndhD-2.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.     

Cyanobacterial NADPH dehydrogenase complexes

Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO₂ uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO₂ uptake. Other than CO₂ uptake, chloroplastic NDH-1 complex has a similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described for comparison.     

The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.     

还原型二(三)磷酸吡啶核苷酸[NAD(P)H]脱氢酶复合体及叶绿体呼吸研究进展

除了经过光系统II和光系统I的非循环电子传递以外,围绕光系统I的循环电子传递对维持高效率的光合作用也是不可缺少的,其中叶绿体还原型二(三)磷酸吡啶核苷酸[NAD(P)H]脱氢酶复合体(NDH复合体)介导的循环电子传递是目前研究的热点。随着质体末端氧化酶(PTOX)的发现,NDH参与的循环电子传递与叶绿体呼吸在补充光合作用所需能量以及抵御光氧化胁迫过程中的作用正日渐引起研究者的重视。文章根据近年的研究进展就叶绿体NDH复合体及其介导的循环电子传递与叶绿体呼吸的生理功能做了综述。     

Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion

Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.     

Identification of a novel protein, CRR7, required for the stabilization of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

An Arabidopsis thaliana mutant, crr7 (chlororespiratory reduction), was isolated using chlorophyll fluorescence imaging to detect reduced activity in NAD(P)H dehydrogenase (NDH). The chloroplast NDH complex is considered to have originated from cyanobacteria in which the NDH complex is involved in respiration, photosystem I (PSI) cyclic electron transport and CO2 uptake. In higher plants the NDH complex functions in PSI cyclic electron transport within the chloroplast. Despite exhaustive biochemical approaches, the entire subunit composition of the NDH complex is unclear in both cyanobacteria and chloroplasts. In crr7 accumulation of the NDH complex was specifically impaired. In vivo analysis of electron transport supported the specific loss of the NDH complex in crr7. CRR7 (At5g39210) encodes a protein of 156 amino acids, including a putative plastid target signal, and does not contain any known motifs. In contrast to CRR2 and CRR4, involved in the expression of chloroplast ndh genes, CRR7 is conserved in cyanobacterial genomes. Although CRR7 did not contain any transmembrane domains, it localized to the membrane fraction of the chloroplast. CRR7 was unstable in the crr2-2 mutant background, in which the expression of ndhB was impaired. These results strongly suggest that CRR7 is a novel subunit of the chloroplast NDH complex.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria—case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.     

Chlororespiratory reduction 6 is a novel factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I cyclic electron transport and chlororespiration in higher plants. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction 6 (crr6) mutant lacking NDH activity was identified by means of chlorophyll fluorescence imaging. Accumulation of the NDH complex was impaired in crr6. Physiological characterization of photosynthetic electron transport indicated the specific defect of the NDH complex in crr6. In contrast to the CRR7 protein that was recently identified as a potential novel subunit of the NDH complex by means of the same screening, the CRR6 protein was stable under the crr2 mutant background in which the NDH complex does not accumulate. The CRR6 gene (At2g47910) encodes a novel protein without any known motif. Although CRR6 does not have any transmembrane domains, it is localized in the thylakoid membrane fraction of the chloroplast. CRR6 is conserved in phototrophs, including cyanobacteria, from which the chloroplast NDH complex has evolutionally originated, but not in Chlamydomonas reinhardtii, in which the NDH complex is absent. We believe that CRR6 is a novel specific factor for the assembly or stabilization of the NDH complex.