NdhM Subunit Is Required for the Stability and the Function of NAD(P)H Dehydrogenase Complexes Involved in CO2 Uptake in Synechocystis sp. Strain PCC 6803

The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO₂ uptake, and cyclic electron transport around photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS′, are involved in the CO₂ uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO₂ uptake, resulting in a lethal phenotype under air CO₂ condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS′ as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the ΔndhM mutant but not in the NdhI-deleted mutant ΔndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS′ involved in CO₂ uptake in Synechocystis sp. strain PCC 6803.     

Expression and activity of type 1 NAD(P)H dehydrogenase at different growth phases of the cyanobacterium, Synechocystis PCC 6803

The expression and activity of type 1 NAD(P)H dehydrogenase (NDH-1) were investigated in Synechocystis PCC 6803 cells during different growth phases (i.e. lag, logarithmic, stationary and decline phases). The relative amount of NDH-1, estimated by Western blot analysis using antibodies against NdhH, NdhI and NdhK, increased more than two-fold during growth from the lag to the logarithmic phase and then decreased after the logarithmic phase to reach lowest levels after 15 days (decline phase). The activity of light-dependent NADPH oxidation and cyclic electron flow around photosystem I (PSI) changed nearly in parallel with the amount of NdhH, NdhI and NdhK in cells across the growth phases. In contrast, the activity of photosynthetic O₂ evolution and respiratory O₂ uptake was not significantly different across phases of growth; the fluctuation of the activity at different phases was within 40%. These results suggested that the activity of light-dependent NADPH oxidation and PSI-cyclic electron flow are restricted by the amount of NDH-1 and that other factor(s) are limiting the rates of photosynthesis and respiration.     

Identification of novel Ssl0352 protein (NdhS), essential for efficient operation of cyclic electron transport around photosystem I, in NADPH:plastoquinone oxidoreductase (NDH-1) complexes of Synechocystis sp. PCC 6803

Cyanobacterial NADPH:plastoquinone oxidoreductase, or type I NAD(P)H dehydrogenase, or the NDH-1 complex is involved in plastoquinone reduction and cyclic electron transfer (CET) around photosystem I. CET, in turn, produces extra ATP for cell metabolism particularly under stressful conditions. Despite significant achievements in the study of cyanobacterial NDH-1 complexes during the past few years, the entire subunit composition still remains elusive. To identify missing subunits, we screened a transposon-tagged library of Synechocystis 6803 cells grown under high light. Two NDH-1-mediated CET (NDH-CET)-defective mutants were tagged in the same ssl0352 gene encoding a short unknown protein. To clarify the function of Ssl0352, the ssl0352 deletion mutant and another mutant with Ssl0352 fused to yellow fluorescent protein (YFP) and the His(6) tag were constructed. Immunoblotting, mass spectrometry, and confocal microscopy analyses revealed that the Ssl0352 protein resides in the thylakoid membrane and associates with the NDH-1L and NDH-1M complexes. We conclude that Ssl0352 is a novel subunit of cyanobacterial NDH-1 complexes and designate it NdhS. Deletion of the ssl0352 gene considerably impaired the NDH-CET activity and also retarded cell growth under high light conditions, indicating that NdhS is essential for efficient operation of NDH-CET. However, the assembly of the NDH-1L and NDH-1M complexes and their content in the cells were not affected in the mutant. NdhS contains a Src homology 3-like domain and might be involved in interaction of the NDH-1 complex with an electron donor.     

Identification of a cyanobacterial CRR6 protein,Slr1097, required for efficient assembly of NDH‐1 complexes in Synechocystis sp. PCC 6803

Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH‐1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH‐1 complexes are poorly understood. By transformation of wild‐type cells with a transposon‐tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH‐1‐mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH‐1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH‐1 complexes using a yeast two‐hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ?slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH‐1M complex, whereas almost half the wild‐type levels of these subunits were present in NDH‐1L complex; similar results were observed in the NdhI‐less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH‐1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH‐1L complex.     

Effects of low CO2 on NAD(P)H dehydrogenase,a mediator of cyclic electron transport around photosystem I in the cyanobacterium synechocystis PCC6803

The expression and activity of type-1 NAD(P)H dehydrogenase (NDH-1) was compared between cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO(2) conditions. Western analysis indicated that L-cells contain higher amounts of the NDH-1 subunits, NdhH, NdhI and NdhK. An NADPH-specific subcomplex of NDH-1 showed higher NADPH-nitroblue tetrazolium oxidoreductase activity in L-cells. The activities of both NADPH-menadione oxidoreductase and light-dependent NADPH oxidation driven by photosystem I were much higher in L-cells than in H-cells. The initial rate of re-reduction of P700(+) following actinic light illumination in the presence of DCMU under background far-red light was enhanced in L-cells. In addition, rotenone, a specific inhibitor of NDH-1, suppressed the relative rate of post-illumination increase in Chl fluorescence of L-cells more than that of H-cells, suggesting that the involvement of NDH-1 in cyclic electron flow around photosystem I was enhanced by low CO(2). Taken together, these results suggest that NDH-1 complex and NDH-1-mediated cyclic electron transport are stimulated by low CO(2) and function in the acclimation of cyanobacteria to low CO(2).     

Identification of NdhL and Ssl1690 (NdhO) in NDH-1L and NDH-1M complexes of Synechocystis sp. PCC 6803

The subunit compositions of two types of NAD(P)H dehydrogenase complexes of Synechocystis sp. PCC 6803, NDH-1L and NDH-1M, were studied by two-dimensional blue-native/SDS-PAGE followed by electrospray tandem mass spectrometry. Fifteen proteins were observed in NDH-1L including hydrophilic subunits (NdhH, -K, -I, -J, -M, and -N) and hydrophobic subunits (NdhA, -B, -E, -G, -D1, and -F1). In addition, NdhL and a novel subunit, Ssl1690 (designated NdhO), were shown to be components of this complex. All subunits mentioned above were present in the NDH-1M complex except NdhD1 and NdhF1. NdhL and Ssl1690 (NdhO) were homologous to hypothetical proteins encoded by genomic DNA in higher plants, suggesting that chloroplast NDH-1 complexes contain related subunits. Diagnostic sequence motifs were found for both NdhL and NdhO homologous proteins. Analysis of ndhL deletion mutant (M9) revealed the presence of assembled NDH-1L and NDH-1M complexes, but these complexes appear to be functionally impaired in the absence of NdhL. Both NDH-1 complexes were absent in the ndhB deletion mutant (M55).     

NdhP Is an Exclusive Subunit of Large Complex of NADPH Dehydrogenase Essential to Stabilize the Complex in Synechocystis sp. Strain PCC 6803

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO₂ acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl β-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His₆. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His₆ was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His₆ and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex.     

集胞藻6803NdhO蛋白多克隆抗体制备及其初步应用

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸.迄今为止,人们在蓝藻细胞中已鉴定出17种NDH-1复合体亚基(NdhA-NdhQ).最近,人们还获得了NdhO亚基的缺失突变株.然而,人们对NdhO亚基的研究还不充份,至今仍不清楚它的功能角色.通过PC...     

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO₂ conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO₂ growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

利用同源重组构建蓝藻集胞藻6803ndhO基因突变株及其分子鉴定

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。迄今为止,人们在蓝藻细胞中已鉴定出15种NDH-1复合体亚基(NdhA-NdhO)。然而,人们对NdhO亚基的研究尚不够,至今未见有反向遗传学等方面的研究。在通过构建同源重组载体、自然转化和多次继代筛选后,对转化子进行了PCR和蛋白免疫印迹鉴定。结果表明,卡那霉素基因已成功地插入到ndhO基因的保守区域,并完全破坏了ndhO基因的蛋白表达,从而获得了ndhO基因缺失的突变株,为进一步研究NdhO亚基对NDH-1复合体的稳定性和生理功能等奠定了实验基础。     

Response of NAD(P)H dehydrogenase complex to the alteration of CO2 concentration in the cyanobacterium Synechocystis PCC6803

An NADPH-specific NDH-1 sub-complex was separated by native-polyacrylamide gel electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis PCC6803. Low CO2 caused an increase in the activity of this sub-complex quickly, accompanied by an evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that can reflect the activity of NDH-1-mediated cyclic electron transport. During incubation with high CO2, the activities of NDH-1 sub-complex and PSI-driven NADPH oxidation as well as the protein level of NdhK slightly increased at the beginning, but decreased evidently in various degrees along with incubation time. These results suggest that CO2 concentration in vitro as a signal can control the activity of NDH-1 complex, and NDH-1 complex may in turn function in the regulation of CO2 uptake.     

Interaction of purified NDH-1 from Escherichia coli with ubiquinone analogues

The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K_m for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.     

Features of Subunit NuoM (ND4) in Escherichia coli NDH-1: TOPOLOGY AND IMPLICATION OF CONSERVED GLU144 FOR COUPLING SITE 1*

The bacterial H⁺-pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA–N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I–IX and XII–XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu¹⁴⁴ of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914–36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu¹⁴⁴ for proton translocation has been discussed.     

PGR5 and NDH-1 systems do not function as protective electron acceptors but mitigate the consequences of PSI inhibition

Avoidance of photoinhibition at photosystem (PS)I is based on synchronized function of PSII, PSI, Cytochrome b₆f and stromal electron acceptors. Here, we used a special light regime, PSI photoinhibition treatment (PIT), in order to specifically inhibit PSI by accumulating excess electrons at the photosystem (Tikkanen and Grebe, 2018). In the analysis, Arabidopsis thaliana WT was compared to the pgr5 and ndho mutants, deficient in one of the two main cyclic electron transfer pathways described to function as protective alternative electron acceptors of PSI. The aim was to investigate whether the PGR5 (pgr5) and the type I NADH dehydrogenase (NDH-1) (ndho) systems protect PSI from excess electron stress and whether they help plants to cope with the consequences of PSI photoinhibition. First, our data reveals that neither PGR5 nor NDH-1 system protects PSI from a sudden burst of electrons. This strongly suggests that these systems in Arabidopsis thaliana do not function as direct acceptors of electrons delivered from PSII to PSI – contrasting with the flavodiiron proteins that were found to make Physcomitrella patens PSI resistant to the PIT. Second, it is demonstrated that under light-limiting conditions, the electron transfer rate at PSII is linearly dependent on the amount of functional PSI in all genotypes, while under excess light, the PGR5-dependent control of electron flow at the Cytochrome b₆f complex overrides the effect of PSI inhibition. Finally, the PIT is shown to increase the amount of PGR5 and NDH-1 as well as of PTOX, suggesting that they mitigate further damage to PSI after photoinhibition rather than protect against it.     

Multistep assembly of chloroplast NADH dehydrogenase-like subcomplex A requires several nucleus-encoded proteins, including CRR41 and CRR42, in Arabidopsis

Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60β4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.     

Subunit Q Is Required to Stabilize the Large Complex of NADPH Dehydrogenase in Synechocystis sp. Strain PCC 6803

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1, NdhF1, and NdhP, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, NDH-1-dependent cyclic electron transport around photosystem I, and CO₂ uptake. Two mutants sensitive to high light for growth and impaired in cyclic electron transport around photosystem I were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in an open reading frame encoding a product highly homologous to NdhQ, a single-transmembrane small subunit of the NDH-1L complex, identified in Thermosynechococcus elongatus by proteomics strategy. Deletion of ndhQ disassembled about one-half of the NDH-1L to NDH-1M and consequently impaired respiration, but not CO₂ uptake. During prolonged incubation of the thylakoid membrane with n-dodecyl-β-d-maltoside at room temperature, the rest of the NDH-1L in ΔndhQ was disassembled completely to NDH-1M and was much faster than in the wild type. In the ndhP-deletion mutant (ΔndhP) background, absence of NdhQ almost completely disassembled the NDH-1L to NDH-1M, similar to the results observed in the ΔndhD1/ΔndhD2 mutant. We therefore conclude that both NdhQ and NdhP are essential to stabilize the NDH-1L complex.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011a) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around PSI, and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain, regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past few years, significant achievements have been made in resolving the subunit compositions and functions of the multiple NDH-1 complexes in several cyanobacterial strains (for review, see Battchikova and Aro, 2007; Ogawa and Mi, 2007; Ma, 2009; Battchikova et al., 2011b; Ma and Ogawa, 2015). Four types of NDH-1 have been identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter, Synechocystis 6803), and all four types of NDH-1 are involved in NDH-1-dependent cyclic electron transport (CET) around PSI (NDH-CET; Bernát et al., 2011). The NDH-CET plays an important role in coping with various environmental stresses, regardless of its elusive mechanism. For example, this function can greatly alleviate high light-sensitive growth phenotypes (Endo et al., 1999; Battchikova et al., 2011a; Dai et al., 2013; Zhang et al., 2014; Zhao et al., 2014). Therefore, high light strategy can help in identifying the proteins essential to NDH-CET.Proteomics studies revealed the presence of three major NDH-1 complexes in cyanobacteria: a large complex (NDH-1L), a medium size complex (NDH-1M), and a small complex (NDH-1S) with molecular masses of about 460, 350, and 200 kD, respectively (Herranen et al., 2004). NDH-1M consists of 14 subunits (i.e. NdhA–NdhC, NdhE, NdhG–NdhO, and NdhS). In addition to these subunits, the NDH-1L complex contains NdhD1, NdhF1, NdhP, and NdhQ (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Zhang et al., 2005, 2014; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Ma and Ogawa, 2015) and is involved in respiration (Zhang et al., 2004). NDH-1S is composed of NdhD3, NdhF3, CO₂ uptake A (CupA), and CupS (Ogawa and Mi, 2007) and is considered to be associated with NDH-1M in the cells as a functional complex NDH-1MS (Zhang et al., 2004, 2005) participating in CO₂ uptake. Among the several copies of ndhD and ndhF genes found in cyanobacterial genomes, ndhD1 and ndhF1 show the highest homology to chloroplast ndhD and ndhF genes, respectively, and CupA and CupS subunits of the cyanobacteria have no counterparts in higher plants. These facts suggest that the structure and composition of NDH-1L, but not the NDH-1MS complex, are similar to those of the chloroplast NDH-1 complex (Battchikova and Aro, 2007; Ogawa and Mi, 2007; Shikanai, 2007; Ma, 2009; Suorsa et al., 2009; Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a; Ma and Ogawa, 2015). Despite their similarity, a large number of subunits that constitute the chloroplast NDH-1 complex, including ferredoxin-binding subcomplex subunits NdhT and NdhU and all the subunits of subcomplex B and lumen subcomplex, are absent in the cyanobacterial NDH-1L complex (Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a). This implies that the stabilization strategies for the cyanobacterial NDH-1L complex and chloroplastic NDH-1 complex might be significantly different.Recently, a new oxygenic photosynthesis-specific small subunit NdhQ was identified in the NDH-1L complex purified by Ni²⁺ affinity chromatography from Thermosynechococcus elongatus (Nowaczyk et al., 2011). NdhQ is extensively present in cyanobacteria, but its homolog is absent in higher plants (Nowaczyk et al., 2011). In this study, we demonstrate that deletion of NdhQ disassembled the NDH-1L into NDH-1M, but not NDH-1MS, in Synechocystis 6803 and consequently impaired respiration, but not CO₂ uptake. NdhQ and NdhP stabilize the NDH-1L complex. Thus, the stabilization strategy of cyanobacterial NDH-1L is distinctly different from that of the chloroplastic NDH-1 complex.     

Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations.Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K_m value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.     

Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study

Cyanobacterial NDH-1 is a multisubunit complex involved in proton translocation, cyclic electron flow around photosystem I and CO₂ uptake. The function and location of several of its small subunits are unknown. In this work, the location of the small subunits NdhL, -M, -N, -O and CupS of Synechocystis 6803 NDH-1 was established by electron microscopy (EM) and single particle analysis. To perform this, the subunits were enlarged by fusion with the YFP protein. After classification of projections, the position of the YFP tag was revealed; all five subunits are integrated in the membrane domain. The results on NDH-1 demonstrate that a GFP tag can be revealed after data processing of EM data sets of moderate size, thus showing that this way of labeling is a fast and reliable way for subunit mapping in multisubunit complexes after partial purification.     

The 5 kDa Protein NdhP Is Essential for Stable NDH-1L Assembly in Thermosynechococcus elongatus

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO₂ concentration, and not detectable in the NDH-1MS and NDH-1MS'' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.     

Molecular dynamics and structural models of the cyanobacterial NDH-1 complex

NDH-1 is a gigantic redox-driven proton pump linked with respiration and cyclic electron flow in cyanobacterial cells. Based on experimentally resolved X-ray and cryo-EM structures of the respiratory complex I, we derive here molecular models of two isoforms of the cyanobacterial NDH-1 complex involved in redox-driven proton pumping (NDH-1L) and CO₂-fixation (NDH-1MS). Our models show distinct structural and dynamic similarities to the core architecture of the bacterial and mammalian respiratory complex I. We identify putative plastoquinone-binding sites that are coupled by an electrostatic wire to the proton pumping elements in the membrane domain of the enzyme. Molecular simulations suggest that the NDH-1L isoform undergoes large-scale hydration changes that support proton-pumping within antiporter-like subunits, whereas the terminal subunit of the NDH-1MS isoform lacks such structural motifs. Our work provides a putative molecular blueprint for the complex I-analogue in the photosynthetic energy transduction machinery and demonstrates that general mechanistic features of the long-range proton-pumping machinery are evolutionary conserved in the complex I-superfamily.