cKMT1 is a New Lysine Methyltransferase That Methylates the Ferredoxin-NADP(+) Oxidoreductase and Regulates Energy Transfer in Cyanobacteria.

Lysine methylation is a conserved and dynamic regulatory posttranslational modification performed by lysine methyltransferases (KMTs). KMTs catalyze the transfer of mono-, di-, or tri-methyl groups to substrate proteins and play a critical regulatory role in all domains of life. To date, only one KMT has been identified in cyanobacteria. Here, we tested all of the predicted KMTs in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and we biochemically characterized sll1526 that we termed cKMT1 (cyanobacterial lysine methyltransferase 1) and determined that it can catalyze lysine methylation both in vivo and in vitro. Loss of cKMT1 alters photosynthetic electron transfer in Synechocystis. We analyzed cKMT1-regulated methylation sites in Synechocystis using a timsTOF Pro instrument. We identified 305 class I lysine methylation sites within 232 proteins, and of these, 80 methylation sites in 58 proteins were hypomethylated in ΔcKMT1 cells. We further demonstrated that cKMT1 could methylate ferredoxin-NADP(+) oxidoreductase (FNR) and its potential sites of action on FNR were identified. Amino acid residues H118 and Y219 were identified as key residues in the putative active site of cKMT1 as indicated by structure simulation, site-directed mutagenesis, and KMT activity measurement. Using mutations that mimic the unmethylated forms of FNR, we demonstrated that the inability to methylate K139 residues results in a decrease in the redox activity of FNR and affects energy transfer in Synechocystis. Together, our study identified a new KMT in Synechocystis and elucidated a methylation-mediated molecular mechanism catalyzed by cKMT1 for the regulation of energy transfer in cyanobacteria.

Redox Properties of Bacillus subtilis Ferredoxin:NADP+ Oxidoreductase: Potentiometric Characteristics and Reactions with Pro-Oxidant Xenobiotics.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

Root-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses.

In Arabidopsis, two leaf-type ferredoxin-NADP+ oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP+ , while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using ß-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.

Reactions of Plasmodium falciparum Ferredoxin:NADP+ Oxidoreductase with Redox Cycling Xenobiotics: A Mechanistic Study.

Ferredoxin:NADP+ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E17). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar "intrinsic" reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

Arabidopsis cryptochrome 1 functions in nitrogen regulation of flowering.

The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP(+)-oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-of-function mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP(+) and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process.

Antiplasmodial Activity of Nitroaromatic Compounds: Correlation with Their Reduction Potential and Inhibitory Action on Plasmodium falciparum Glutathione Reductase.

With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP+ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E17). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E17 values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.

Detailed characterization of Synechocystis PCC 6803 ferredoxin:NADP+ oxidoreductase interaction with model membranes.

Direct interaction of ferredoxin:NADP+ oxidoreductase (FNR) with thylakoid membranes was postulated as a part of the cyclic electron flow mechanism. In vitro binding of FNR to digalactosyldiacylglycerol and monogalactosyldiacylglycerol membranes was also shown. In this paper we deal with the latter interaction in more detail describing the effect for two FNR forms of Synechocystis PCC 6803. The so-called short FNR (sFNR) is homologous to FNR from higher plant chloroplasts. The long FNR (lFNR) form contains an additional domain, responsible for the interaction with phycobilisomes. We compare the binding of both sFNR and lFNR forms to native and non-native lipids. We also include factors which could modulate this process: pH change, temperature change, presence of ferredoxin, NADP+ and NADPH and heavy metals. For the lFNR, we also include phycobilisomes as a modulating factor. The membrane binding is generally faster at lower pH. The sFNR was binding faster than lFNR. Ferredoxin isoforms with higher midpoint potential, as well as NADPH and NADP+, weakened the binding. Charged lipids and high phosphate promoted the binding. Heavy metal ions decreased the rate of membrane binding only when FNR was preincubated with them before injection beneath the monolayer. FNR binding was limited to surface lipid groups and did not influence hydrophobic chain packing. Taken together, FNR interaction with lipids appears to be non-specific, with an electrostatic component. This suggests that the direct FNR interaction with lipids is most likely not a factor in directing electron transfer, but should be taken into account during in vitro studies.

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen.

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 µmolH2 min-1 mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.

Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii.

Ferredoxins (FDX) and the FDX:NADP+ oxidoreductase (FNR) represent a key junction of electron transport downstream of photosystem I (PSI). Dynamic recruitment of FNR to the thylakoid membrane has been considered as a potential mechanism to define the fate of photosynthetically derived electrons. In this study, we investigated the functional importance of the association of FNR with the photosynthetic apparatus in Chlamydomonas reinhardtii. In vitro assays based on NADP+ photoreduction measurements as well as NMR chemical shift perturbation analyses showed that FNR preferentially interacts with FDX1 compared to FDX2. Notably, binding of FNR to a PSI supercomplex further enhanced this preference for FDX1 over FDX2, suggesting that FNR is potentially capable of channelling electrons towards distinct routes. NADP+ photoreduction assays and immunoblotting revealed that the association of FNR with the thylakoid membrane including the PSI supercomplex is impaired in the absence of Proton Gradient Regulation 5 (PGR5) and/or Proton Gradient Regulation 5-Like photosynthetic phenotype 1 (PGRL1), implying that both proteins, directly or indirectly, contribute to the recruitment of FNR to the thylakoid membrane. As assessed via in vivo absorption spectroscopy and immunoblotting, PSI was the primary target of photodamage in response to high-light stress in the absence of PGR5 and/or PGRL1. Anoxia preserved the activity of PSI, pointing to enhanced electron donation to O2 as the source of the observed PSI inactivation and degradation. These findings establish another perspective on PGR5/PGRL1 knockout-related phenotypes and potentially interconnect FNR with the regulation of photosynthetic electron transport and PSI photoprotection in C. reinhardtii.

Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure.

NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD(+). Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg(187) is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH(•)/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed.

Electron-transfer kinetics in cyanobacterial cells: methyl viologen is a poor inhibitor of linear electron flow.

The inhibitor methyl viologen (MV) has been widely used in photosynthesis to study oxidative stress. Its effects on electron transfer kinetics in Synechocystis sp. PCC6803 cells were studied to characterize its electron-accepting properties. For the first hundreds of flashes following MV addition at submillimolar concentrations, the kinetics of NADPH formation were hardly modified (less than 15% decrease in signal amplitude) with a significant signal decrease only observed after more flashes or continuous illumination. The dependence of the P700 photooxidation kinetics on the MV concentration exhibited a saturation effect at 0.3 mM MV, a concentration which inhibits the recombination reactions in photosystem I. The kinetics of NADPH formation and decay under continuous light with MV at 0.3 mM showed that MV induces the oxidation of the NADP pool in darkness and that the yield of linear electron transfer decreased by only 50% after 1.5-2 photosystem-I turnovers. The unexpectedly poor efficiency of MV in inhibiting NADPH formation was corroborated by in vitro flash-induced absorption experiments with purified photosystem-I, ferredoxin and ferredoxin-NADP(+)-oxidoreductase. These experiments showed that the second-order rate constants of MV reduction are 20 to 40-fold smaller than the competing rate constants involved in reduction of ferredoxin and ferredoxin-NADP(+)-oxidoreductase. The present study shows that MV, which accepts electrons in vivo both at the level of photosystem-I and ferredoxin, can be used at submillimolar concentrations to inhibit recombination reactions in photosystem-I with only a moderate decrease in the efficiency of fast reactions involved in linear electron transfer and possibly cyclic electron transfer.

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: a versatile probe for in vivo measurements of rates, yields and pools.

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP(+) to NADPH by ferredoxin-NADP(+)-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP(+)+NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin-Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP(+) oxidoreductase.

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.

Thioredoxin Reductase-Type Ferredoxin: NADP+ Oxidoreductase of Rhodopseudomonas palustris: Potentiometric Characteristics and Reactions with Nonphysiological Oxidants.

Rhodopseudomonas palustris ferredoxin:NADP+ oxidoreductase (RpFNR) belongs to a novel group of thioredoxin reductase-type FNRs with partly characterized redox properties. Based on the reactions of RpFNR with the 3-acetylpyridine adenine dinucleotide phosphate redox couple, we estimated the two-electron reduction midpoint potential of the FAD cofactor to be -0.285 V. 5-Deaza-FMN-sensitized photoreduction revealed -0.017 V separation of the redox potentials between the first and second electron transfer events. We examined the mechanism of oxidation of RpFNR by several different groups of nonphysiological electron acceptors. The kcat/Km values of quinones and aromatic N-oxides toward RpFNR increase with their single-electron reduction midpoint potential. The lower reactivity, mirroring their lower electron self-exchange rate, is also seen to have a similar trend for nitroaromatic compounds. A mixed single- and two-electron reduction was characteristic of quinones, with single-electron reduction accounting for 54% of the electron flux, whereas nitroaromatics were reduced exclusively via single-electron reduction. It is highly possible that the FADH· to FAD oxidation reaction is the rate-limiting step during the reoxidation of reduced FAD. The calculated electron transfer distances in the reaction with quinones and nitroaromatics were close to those of Anabaena and Plasmodium falciparum FNRs, thus demonstrating their similar "intrinsic" reactivity.

Roles of Ferredoxin-NADP+ Oxidoreductase and Flavodoxin in NAD(P)H-Dependent Electron Transfer Systems.

Distinct isoforms of FAD-containing ferredoxin-NADP+ oxidoreductase (FNR) and ferredoxin (Fd) are involved in photosynthetic and non-photosynthetic electron transfer systems. The FNR (FAD)-Fd [2Fe-2S] redox pair complex switches between one- and two-electron transfer reactions in steps involving FAD semiquinone intermediates. In cyanobacteria and some algae, one-electron carrier Fd serves as a substitute for low-potential FMN-containing flavodoxin (Fld) during growth under low-iron conditions. This complex evolves into the covalent FNR (FAD)-Fld (FMN) pair, which participates in a wide variety of NAD(P)H-dependent metabolic pathways as an electron donor, including bacterial sulfite reductase, cytochrome P450 BM3, plant or mammalian cytochrome P450 reductase and nitric oxide synthase isoforms. These electron transfer systems share the conserved Ser-Glu/Asp pair in the active site of the FAD module. In addition to physiological electron acceptors, the NAD(P)H-dependent diflavin reductase family catalyzes a one-electron reduction of artificial electron acceptors such as quinone-containing anticancer drugs. Conversely, NAD(P)H: quinone oxidoreductase (NQO1), which shares a Fld-like active site, functions as a typical two-electron transfer antioxidant enzyme, and the NQO1 and UDP-glucuronosyltransfease/sulfotransferase pairs function as an antioxidant detoxification system. In this review, the roles of the plant FNR-Fd and FNR-Fld complex pairs were compared to those of the diflavin reductase (FAD-FMN) family. In the final section, evolutionary aspects of NAD(P)H-dependent multi-domain electron transfer systems are discussed.

The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana.

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP+ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP+ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH.

Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non­sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s-1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.

Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water.

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP+ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: "Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production". Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells - for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.

Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

NADPH-generating systems in bacteria and archaea.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.