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.     

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.     

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.     

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.     

Structure of the NDH-2 – HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ₁) interactions in the yeast Ndi1–UQ₁ complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Synthetic DNA system for structure-function studies of the high affinity CO2 uptake NDH-13 protein complex in cyanobacteria

The CO₂-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO₂ around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO₂-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO₂ to bicarbonate. This CO₂-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO₂ hydration to NDH-1 proton pumping. The development of the first molecular genetic system to probe structure-function relationships of this important enzyme system is described. A CO₂-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO₂-hydration system. This was tested by introducing a construct to an ectopic location that gives constitutive expression, rather than native inducible expression, of the ndhF3-ndhD3-cupA-cupS, (cupA operon) encoding high affinity CO₂-hydration complex, NDH-1₃. Uptake assays show the restoration of high affinity for CO₂ uptake, but demonstrate that the CupA complex can drive only modest uptake fluxes, underlining the importance of its tandem operation with the CupB-containing complex NDH-1₄, the complementary high flux, low affinity CO₂ hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-1₃ complex is strongly inhibited, yet the remaining NDH-1₄ activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO₂–hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO₂ uptake systems.     

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.     

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.     

NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

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.     

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.     

Apoptosis-inducing Factor (AIF) and Its Family Member Protein,AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.     

The molecular mechanism of menadione resistance in the <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700⁺) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700⁺ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.     

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.     

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 Neuronal Kv4 Channel Complex

Kv4 channel complexes mediate the neuronal somatodendritic A-type K⁺ current (I_SA), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated α-subunits (Shal/Kv4) and at least two classes of auxiliary β-subunits: KChIPs (K ⁺-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary β-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn²⁺ site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of I_SA function and regulation. Special issue article in honor of Dr. Ricardo Tapia.     

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).     

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.