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.     

Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803

To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DeltaNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the DeltaNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (DeltaNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in DeltaNdhD1/D2 and M55. In contrast with the wild type and DeltaNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the DeltaNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3- transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.     

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 CO(2) 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-CO(2) 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.     

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.     

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.     

Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43' protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.     

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.     

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).     

Cyanobacterial NDH-1 complexes: multiplicity in function and subunit composition

In cyanobacteria, the NAD(P)H:quinone oxidoreductase (NDH-1) is involved in a variety of functions like respiration, cyclic electron flow around PSI and CO₂ uptake. Several types of NDH-1 complexes, which differ in structure and are responsible for these functions, exist in cyanobacterial membranes. This minireview is based on data obtained by reverse genetics and proteomics studies and focuses on the structural and functional differences of the two types of cyanobacterial NDH-1 complexes: NDH-1L, important for respiration and PSI cyclic electron flow, and NDH-1MS, the low-CO₂ inducible complex participating in CO₂ uptake. The NDH-1 complexes in cyanobacteria share a common NDH-1M 'core' complex and differ in the composition of the distal membrane domain composed of specific NdhD and NdhF proteins, which in complexes involved in CO₂ uptake is further associated with the hydrophilic carbon uptake (CUP) domain. At present, however, very important questions concerning the nature of catalytically active subunits that constitute the electron input device (like NADH dehydrogenase module of the eubacterial 'model' NDH-1 analogs), the substrate specificity and reaction mechanisms of cyanobacterial complexes remain unanswered and are shortly discussed here.     

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.     

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.     

Identification, regulation and physiological functions of multiple NADPH dehydrogenase complexes in cyanobacteria

Cyanobacteria possess multiple, functionally distinct NADPH dehydrogenase (NDH-1) complexes. In this mini-review, we describe the cyanobacterial NDH-1 complexes by focusing on their identification, regulatory properties, and multiple functions. The multiple functions can be divided into basic and extending functions, and the basic functions are compared with those in chloroplasts. Many questions related to cyanobacterial NDH-1 complexes remain unanswered and are briefly summarized here.     

几种蓝藻光合NAD（P）H脱氢酶复合体研究的新进展

蓝藻NAD（P）H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。就几种蓝藻NDH-1复合体的鉴定、结构、生理功能等研究的新进展进行了综述与分析,并对今后NDH-1复合体的研究作了展望。     

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。     

The mitochondrial respiratory chain of Ustilago maydis

Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca²⁺, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation.The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool.     

Synthesis and structural characterization of the mono- and diphosphine-containing diiron propanedithiolate complexes related to [FeFe]-hydrogenases. Biomimetic H2 evolution catalyzed by (mu-PDT)Fe2(CO)4[(Ph2P)2N(n-Pr)

As the new H-cluster models, six diiron propanedithiolate (PDT) complexes with mono- and diphosphine ligands have been prepared and structurally characterized. The monophosphine model complex (μ-PDT)Fe₂(CO)₅[Ph₂PNH(t-Bu)] (1) was prepared by reaction of parent complex (μ-PDT)Fe₂(CO)₆ (A) with 1 equiv of Ph₂PNH(t-Bu) in refluxing xylene, whereas A reacted with 1 equiv of Me₃NO · 2H₂O in MeCN at room temperature followed by 1 equiv of Ph₂PH to give the corresponding monophosphine model complex (μ-PDT)Fe₂(CO)₅(Ph₂PH) (2). Further treatment of 2 with 1 equiv of n-BuLi in THF at −78 °C followed by 1 equiv of CpFe(CO)₂I from −78 °C to room temperature afforded monophosphine model complex (μ-PDT)Fe₂(CO)₅[Ph₂PFe(CO)₂Cp] (3), whereas the diphosphine model complexes (μ-PDT)Fe₂(CO)₄(Ph₂PC₂H₄PPh₂) (4), (μ-PDT)Fe₂(CO)₄[(Ph₂P)₂N(n-Pr)] (5) and (μ-PDT)Fe₂(CO)₄[(Ph₂P)₂N(n-Bu)] (6) were obtained by reactions of A with ca.1 equiv of the corresponding diphosphines in refluxing xylene. All the new model complexes were characterized by elemental analysis, spectroscopy and particularly for 1 and 3-6 by X-ray crystallography. On the basis of electrochemical and spectroelectrochemical studies, model 5 was found to be a catalyst for HOAc proton reduction to H₂, and for this electrocatalytic reaction an ECCE mechanism was proposed.     

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.     

Reduction of Photosystem I Reaction Center in DrgA Mutant of the Cyanobacterium Synechocystis sp. PCC 6803 Lacking Soluble NAD(P)H:Quinone Oxidoreductase

Photoautotrophically grown cells of the cyanobacterium Synechocystis sp. PCC 6803 wild type and the Ins2 mutant carrying an insertion in the drgA gene encoding soluble NAD(P)H:quinone oxidoreductase (NQR) did not differ in the rate of light-induced oxygen evolution and Photosystem I reaction center (P700+) reduction after its oxidation with a white light pulse. In the presence of DCMU, the rate of P700+ reduction was lower in mutant cells than in wild type cells. Depletion of respiratory substrates after 24 h dark-starvation caused more potent decrease in the rate of P700+ reduction in DrgA mutant cells than in wild type cells. The reduction of P700+ by electrons derived from exogenous glucose was slower in photoautotrophically grown DrgA mutant than in wild type cells. The mutation in the drgA gene did not impair the ability of Synechocystis sp. PCC 6803 cells to oxidize glucose under heterotrophic conditions and did not impair the NDH-1-dependent, rotenone-inhibited electron transfer from NADPH to P700+ in thylakoid membranes of the cyanobacterium. Under photoautotrophic growth conditions, NADPH-dehydrogenase activity in DrgA mutant cells was less than 30% from the level observed in wild type cells. The results suggest that NQR, encoded by the drgA gene, might participate in the regulation of cytoplasmic NADPH oxidation, supplying NADP+ for glucose oxidation in the pentose phosphate cycle of cyanobacteria.