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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 .

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BackgroundFlavodiiron proteins (FDPs) comprise a group of modular enzymes that function in oxygen and nitric oxide detoxification in Bacteria and Archaea. The FDPs in cyanobacteria have an extra domain as compared to major prokaryotic enzymes. The physiological role of cyanobacteria FDPs is mostly unknown. Of the four putative flavodiiron proteins (Flv1–4) in the cyanobacterium Synechocystis sp. PCC 6803, a physiological function in Mehler reaction has been suggested for Flv1 and Flv3.Principal FindingsWe demonstrate a novel and crucial function for Flv2 and Flv4 in photoprotection of photosystem II (PSII) in Synechocystis. It is shown that the expression of Flv2 and Flv4 is high under air level of CO2 and negligible at elevated CO2. Moreover, the rate of accumulation of flv2 and flv4 transcripts upon shift of cells from high to low CO2 is strongly dependent on light intensity. Characterization of FDP inactivation mutants of Synechocystis revealed a specific decline in PSII centers and impaired translation of the D1 protein in Δflv2 and Δflv4 when grown at air level CO2 whereas at high CO2 the Flvs were dispensable. Δflv2 and Δflv4 were also more susceptible to high light induced inhibition of PSII than WT or Δflv1 and Δflv3.SignificanceAnalysis of published sequences revealed the presence of cyanobacteria-like FDPs also in some oxygenic photosynthetic eukaryotes like green algae, mosses and lycophytes. Our data provide evidence that Flv2 and Flv4 have an important role in photoprotection of water-splitting PSII against oxidative stress when the cells are acclimated to air level CO2. It is conceivable that the function of FDPs has changed during evolution from protection against oxygen in anaerobic microbes to protection against reactive oxygen species thus making the sustainable function of oxygen evolving PSII possible. Higher plants lack FDPs and distinctly different mechanisms have evolved for photoprotection of PSII.

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