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.     

Tethering of ferredoxin:NADP+ oxidoreductase to thylakoid membranes is mediated by novel chloroplast protein TROL

Working in tandem, two photosystems in the chloroplast thylakoid membranes produce a linear electron flow from H₂O to NADP⁺. Final electron transfer from ferredoxin to NADP⁺ is accomplished by a flavoenzyme ferredoxin:NADP⁺ oxidoreductase (FNR). Here we describe TROL (t hylakoid r ho danese‐l ike protein), a nuclear‐encoded component of thylakoid membranes that is required for tethering of FNR and sustaining efficient linear electron flow (LEF) in vascular plants. TROL consists of two distinct modules; a centrally positioned rhodanese‐like domain and a C‐terminal hydrophobic FNR binding region. Analysis of Arabidopsis mutant lines indicates that, in the absence of TROL, relative electron transport rates at high‐light intensities are severely lowered accompanied with significant increase in non‐photochemical quenching (NPQ). Thus, TROL might represent a missing thylakoid membrane docking site for a complex between FNR, ferredoxin and NADP⁺. Such association might be necessary for maintaining photosynthetic redox poise and enhancement of the NPQ.     

Homology of the N-terminal domain of the petH gene product from Anabaena sp. PCC 7119 to the CpcD phycobilisome linker polypeptide

The complete nucleotide sequence of the petH gene encoding ferredoxin-NADP⁺ reductase from the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7119 has been determined. The encoded polypeptide is 136 amino acids longer than the enzyme obtained after purification to homogeneity. The extended N-terminal domain consists of 80 amino acids which shows homology to the CpcD phycobilisome linker polypeptide, through which FNR might be anchored to the thylakoid-bound phycobilisomes. A 56 amino acid interdomain fragment is found which could be a target for proteolysis.     

The complete purification and characterization of three forms of ferredoxin-NADP(+) oxidoreductase from a thermophilic cyanobacterium Synechococcus elongatus

The petH gene, encoding ferredoxin-NADP(+) oxidoreductase (FNR), was isolated from a thermophilic cyanobacterium, Synechococcus elongatus (the same strain as Thermosynechococcus elongatus). The petH gene of S. elongatus was a single copy gene, and the N-terminal region of PetH showed a sequence similarity to the CpcD-phycobilisome linker polypeptide. The amino acid sequence of the catalytic domains of PetH was markedly similar to those from mesophilic cyanobacterial PetH and higher plant FNR. The enzymatically active FNR protein was purified to homogeneity from S. elongatus as three forms corresponding to the 45-kDa form retaining the CpcD-like domain, the 34-kDa form lacking the CpcD-like domain, and the 78-kDa complex with phycocyanin. The FNR in the 78-kDa complex was tolerant to proteolytic cleavage. However, the dissociation of phycocyanin from the 78-kDa complex induced to specific proteolysis between the CpcD-like domain and the FAD-binding domain to give rise to the 34-kDa form of FNR. The enzymatic activity of the 45-kDa form was thermotolerant, but the 45-kDa form readily aggregated under the storage at -30 degrees C. These results suggest that the association with phycocyanin via CpcD-like domain gives remarkable stability to S. elongatus FNR.     

PetH is rate-controlling in the interaction between PetH, a component of the supramolecular complex with photosystem II, and PetF, a light-dependent electron transfer protein

Cyanobacterial PetH is similar to ferredoxin-NADP⁺ oxidoreductase (FNR) of higher plants and comprises 2 components, CpcD-like rod linker and FNR proteins. Here, I show that PetH controls the rate of the interaction with PetF (ferredoxin [Fd1]). Purified recombinant PetH protein, which cut off a CpcD-like rod linker domain, and Fd1 were used in detailed surface plasmon resonance analyses. The interaction between FNR and Fd1 chiefly involved extremely fast binding and dissociation reactions and the FNR affinity for Fd1 was stronger than the Fd1 affinity for FNR. The dissociation constant values were determined as approximately 93.65 μM (FNR) for Fd1 and 1.469 mM (Fd1) for FNR.     

Two isoforms of ferredoxin:NADP+ oxidoreductase from wheat leaves: purification and initial biochemical characterization

Ferredoxin:NADP⁺ oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP⁺ is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.     

Activation of Ferredoxin-NADP+Oxidoreductase in Vicia fabaLeaves Induced by a Short-Term Increase in Irradiance

The effect of a short-term increase in growth irradiance (I) by 1.5–5 times on the rate of the photosynthetic electron transport and the activity of ferredoxin-NADP⁺oxidoreductase (FNR) in the leaves of broadbean (Vicia fabaL.) plants grown under an irradiance of 8 W/m²was studied. NADPH-diaphorase and cytochrome creductase activities of FNR were determined in isolated chloroplasts and leaf homogenates. The duration of the plant exposure to a higher I varied from 1–30 min to 2 or 24 h. The rate of noncyclic electron transport from water to NADP⁺and the NADPH-diaphorase activity of FNR increased significantly 15 min after a twofold increase in the I. FNR activation was also found after a short-term (1 min) increase in growth I by 1.5 times. The degree of light-induced activation of FNR was dependent on the light intensity, the duration of plant exposure, and the leaf age. The activation of FNR induced by a short-term increase in the I was reversible. However, inactivation of FNR proceeded more slowly than its light-induced activation. Thus, a relatively small change in the I was sufficient to induce the adaptive response of the photosynthetic apparatus at the level of the electron-transport chain. The results obtained confirm a conclusion made previously that a rapid activation of FNR induced by an increase in the I occurs in the absence of de novoprotein synthesis.     

Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria

The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c ₅₅₂, b ₆ and b ₅₅₉ were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.Abbreviations CD circular dichroism - CP chlorophyll-protein complex - LDS lithium dodecyl sulfate - NF norflurazon     

Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ana A. Arteni and Lu-Ning Liu equally contributed to the work.     

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP⁺ oxidoreductase (FNR) is known to provide NADPH for CO₂ assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR_S) similar to the one found in plant plastids and a large one (FNR_L) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR_L exhibits a higher NADP⁺/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR_L, named FNR_L-PC. The enzymatic activities of FNR_L-PC were compared with those of FNR_S. During NADPH oxidation, FNR_L-PC exhibits a 30% decrease in the Michaelis constant K_m(NADPH), and a 70% increase in K_{m(ferredoxin)}, which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP⁺ reduction, the FNR_L-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP⁺. The increase in K_{m(ferredoxin)} and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR_L-PC. Both isoforms are capable of catalyzing the NADP⁺ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.     

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme

Two isoforms of ferredoxin-NADP⁺ reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure–function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP⁺ with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k_cat/K_m), as an NADPH-dependent diaphorase, higher than those of both the root (～2-fold) and the leaf enzyme (～5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.     

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP⁺ reductase (FNR) was previously inferred to bind to the cytochrome b₆f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b₆f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b₆f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b₆f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b₆f interaction is explained by FNR interaction with micelles of the undecyl β-D maltoside (UDM) detergent micelles used to purify b₆f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP⁺-binding domains of FNR caused by detergent. The enthalpy, ΔH^o, and the entropy, ΔS^o, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b₆f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.     

Depletion of leaf-type ferredoxin-NADP(+) oxidoreductase results in the permanent induction of photoprotective mechanisms in Arabidopsis chloroplasts

Arabidopsis thaliana contains two photosynthetically competent chloroplast‐targeted ferredoxin‐NADP⁺ oxidoreductase (FNR) isoforms that are largely redundant in their function. Nevertheless, the FNR isoforms also display distinct molecular phenotypes, as only the FNR1 is able to directly bind to the thylakoid membrane. We report the consequences of depletion of FNR in the F₁ (fnr1 × fnr2) and F₂ (fnr1 fnr2) generation plants of the fnr1 and fnr2 single mutant crossings. The fnr1 × fnr2 plants, with a decreased total content of FNR, showed a small and pale green phenotype, accompanied with a marked downregulation of photosynthetic pigment‐protein complexes. Specifically, when compared with the wild type (WT), the quantum yield of photosystem II (PSII) electron transport was lower, non‐photochemical quenching (NPQ) was higher and the rate of P700⁺ re‐reduction was faster in the mutant plants. The slight over‐reduction of the plastoquinone pool detected in the mutants resulted in the adjustment of the reactive oxygen species (ROS) scavenging systems, as both the content and de‐epoxidation state of xanthophylls, as well as the content of α‐tocopherol, were higher in the leaves of the mutant plants when compared with the WT. The fnr1 fnr2 double mutant plants, which had no detectable FNR and possessed an extremely downregulated photosynthetic machinery, survived only when grown heterotrophically in the presence of sucrose. Intriguingly, the fnr1 fnr2 plants were still capable of sustaining the biogenesis of a few malformed chloroplasts.     

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP⁺ reductase (FNR) and ferredoxin, the proteins that reduce NADP⁺ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel β-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753–3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP⁺ arises primarily from substitutions in FNR that favor interactions with the 2′ phosphate of NADP⁺. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe–2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

The presence of multidomain linkers determines the bundle-shape structure of the phycobilisome of the cyanobacterium Gloeobacter violaceus PCC 7421

The complete genome sequence of Gloeobacter violaceus [Nakamura et al. (2003a, b) DNA Res 10:37–45, 181–201] allows us to understand better the structure of the phycobilisomes (PBS) of this cyanobacterium. Genomic analysis revealed peculiarities in these PBS: the presence of genes for two multidomain linker proteins, a core membrane linker with four repetitive sequences (REP domains), the absence of rod core linkers, two sets of phycocyanin (PC) α and β subunits, two copies of a rod PC associated linker (CpcC), and two rod cap associated linkers (CpcD). Also, there is one ferredoxin–NADP⁺ oxidoreductase with only two domains. The PBS proteins were investigated by gel electrophoresis, amino acid sequencing and peptide mass fingerprinting (PMF). The two unique multidomain linkers contain three REP domains with high similarity and these were found to be in tandem and were separated by dissimilar Arms. One of these, with a mass of 81 kDa, is found in heavy PBS fragments rich in PC. We propose that it links six PC hexamers in two parallel rows in the rods. The other unique linker has a mass of 91 kDa and is easily released from the heavy fragments of PBS. We propose that this links the rods to the core. The presence of these multidomain linkers could explain the bundle shaped rods of the PBS. The presence of 4 REP domains in the core membrane linker protein (129 kDa) was established by PMF. This core linker may hold together 16 AP trimers of the pentacylindrical core, or alternatively, a tetracylindrical core of the PBS of G. violaceus.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

The first eukaryotic cells — Acid hot-spring algae

The Cyanidiophyceae members (PreRhodophyta) may serve as a transitional algal group bridging the cyanobacteria and the unicellular Rhodophyta. This thermoacidic algal group is composed of three genera containing several species. We suggested placing these algae in progressively evolutionary steps: (Cyanidioschyzon Cyanidium Galdieria). This evolutional ladder is based upon various areas of research like biochemistry, amount of nuclear genome and shape of chloroplast nucleoid, ultrastructure and ecological aspects. The first alga —Cyanidioschyzon — is the cornerstone of this succession; it shows mixed features between cyanobacterium and archaebacteria(Thermoplasma-like cell). It demonstrates simple eukaryotic cellular features and has the smallest amount of nuclear and chloroplast DNA. The intermediate alga in this line,Cyanidium, is also a simple cell, but shows more progressive characterizations than theCyanidioschyzon. The third taxon,Galdieria, is already very close to the unicellular rhodophytes (red algae) and indicates typical advanced eukaryotic characterization. We propose thatCyanidioschyzon (considered to be the simplest eukaryote) may have evolved from an association betweenThermoplasma-like archaebacterium and a thermophilic cyanobacterium. Autogenous (non-symbiotic) compartmental steps may have taken place fromCyanidioschyzon toCyanidium and then toGaldieria, and from this alga (group) towards the other unicellular red algae.Dedicated toDr. Jerome F. Fredrick, an enthusiast of our favorite algaCyanidium, on his retirement from directorship of Dodge Chemical Laboratories in Bronx, NYC.