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 CO 2 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 DNdhD1/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 þ rereduction in darkness in the DNdhD1/D2 mutant grown at low CO 2 were similar to those in the wild type, whereas in the M55 mutant (DNdhB), lacking both NDH-1L and NDH-1M, the rate of P700 þ 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 CO 2 in the wild type as well as in DNdhD1/D2 and M55. In contrast with the wild type and DNdhD1/D2, which show normal CO 2 uptake, M55 is unable to take up CO 2 even when the NDH-1S complex is present. Conversely, the DNdhD3/D4 mutant, also unable to take up CO 2 , lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO 2 . These results demonstrate that both NDH-1S and NDH-1M are essential for CO 2 uptake and that NDH-1M is a functional complex. We also show that the Na þ /HCO 3 ÿ transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO 2 .
Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner-Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/ night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO 2 via the Calvin-Benson cycle. T he breakdown of glucose is central for energy and biosynthetic metabolism throughout all domains of life. The Embden-Meyerhof-Parnas (EMP) pathway (glycolysis) and the oxidative pentose phosphate (OPP) pathway are the backbones of eukaryotic carbon and energy metabolism (1, 2). They generate ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. Prokaryotes, in contrast, exhibit a broad diversity in sugar oxidation pathways (3-5). These routes differ in ATP yield, in the enzymes and cofactors involved, and in the chemical intermediates of the pathways. The most common glycolytic routes in prokaryotes are the EMP, ED, and OPP pathways (Fig. 1). The key enzyme unique to the ED pathway is 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase (Eda), whereas phosphofructokinase (PFK) is unique to the EMP pathway in the catabolic direction (3, 6). KDPG as a metabolite is exclusively found in the ED pathway (Fig. 1). The first two steps of the OPP pathway are catalyzed by glucose 6-phosphate-dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd). As the pentose phosphate pathway can either run in its oxidative mode (OPP pathway) to oxidize carbohydrates or in its reductive mode ...
The activity of the bidirectional hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was found not to be regulated in parallel to respiration but to photosynthesis. A mutant with a deletion in the large hydrogenase subunit gene (hoxH), which contains the active site, was impaired in the oxidation of photosystem I (PSI) when illuminated with light, which excites either PSI alone or both photosystems. The fluorescence of photosystem II (PSII) of this mutant was higher than that of wild-type cells. The transcript level of the photosynthetic genes psbA, psaA and petB was found to be different in the hydrogenase-free mutant cells compared to wild-type cells, which indicates that the hydrogenase has an effect on the regulation of these genes. Collectively, these results suggest that the bidirectional hydrogenase functions as a valve for low-potential electrons generated during the light reaction of photosynthesis, thus preventing a slowing down of electron transport. This conclusion is supported by growth curves demonstrating that the mutant cells need more time to adapt to changing light intensities. Investigations of the wild-type and deltahoxH strains strongly suggest that Synechocystis contains only the bidirectional hydrogenase, which seems to be essentially insensitive to oxygen.
Background: Cyanobacterial hydrogenases are claimed to produce hydrogen via NAD(P)H, which contradicts thermodynamic considerations; the physiological function of these hydrogenases is unresolved. Results: Flavodoxin/ferredoxin reduce cyanobacterial hydrogenases, which are essential under mixotrophic, nitrate-limiting conditions. Conclusion: Cyanobacterial bidirectional hydrogenases are electron sinks for reduced flavodoxin/ferredoxin. Significance: This study provides a basis for a target-oriented enhancement of hydrogen production and explains the aquatic distribution of cyanobacterial hydrogenases.
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