Molecular structure and regulation of phototropin kinase by blue light

The ferredoxin:NADP+ oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.     

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.     

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.     

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.     

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.     

The root form of ferredoxin-NADP oxidoreductase is expressed in rice coleoptiles for the assimilation of nitrate

Two ferredoxin-dependent proteins, nitrite reductase and glutamate synthase, play a role in nitrate assimilation during the anaerobic germination of rice (Oryza sativa L.). This paper reports the expression of the root form of ferredoxin-NADP⁺ oxidoreductase (FNR), the protein responsible for providing reduced ferredoxin in rice coleoptiles. Using an antibody against FNR, a protein with the expected molecular mass for root FNR (35 kDa) was recognized by Western blot analysis in extracts from aerobic and anaerobic coleoptiles. The enzyme is synthesized de novo, as shown by immunoprecipitation of the radiolabeled 35-kDa protein from anaerobic seedlings grown in the presence of [³⁵S]methionine. Northern blot analysis with specific probes for root and leaf FNR showed the presence of mRNA for the root form but not for the leaf form, in both aerobic and anaerobic rice coleoptiles. The inductive effect of exogenous nitrate on the expression of FNR is further evidence for the presence of the root type of FNR in rice coleoptiles. The importance of the expression of root FNR during the anaerobic development of rice seedlings is discussed. Received: 7 October 1996 / Accepted: 22 January 1997     

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.     

Subcellular localization of ferredoxin-NADP+ oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms

Ferredoxin-NADP⁺ oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP⁺-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP⁺-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.     

Protection of photosystem I during sudden light stress depends on ferredoxin:NADP(H) reductase abundance and interactions

Plant tolerance to high light and oxidative stress is increased by overexpression of the photosynthetic enzyme Ferredoxin:NADP(H) reductase (FNR), but the specific mechanism of FNR-mediated protection remains enigmatic. It has also been reported that the localization of this enzyme within the chloroplast is related to its role in stress tolerance. Here, we dissected the impact of FNR content and location on photoinactivation of photosystem I (PSI) and photosystem II (PSII) during high light stress of Arabidopsis (Arabidopsis thaliana). The reaction center of PSII is efficiently turned over during light stress, while damage to PSI takes much longer to repair. Our results indicate a PSI sepcific effect, where efficient oxidation of the PSI primary donor (P700) upon transition from darkness to light, depends on FNR recruitment to the thylakoid membrane tether proteins: thylakoid rhodanase-like protein (TROL) and translocon at the inner envelope of chloroplasts 62 (Tic62). When these interactions were disrupted, PSI photoinactivation occurred. In contrast, there was a moderate delay in the onset of PSII damage. Based on measurements of ΔpH formation and cyclic electron flow, we propose that FNR location influences the speed at which photosynthetic control is induced, resulting in specific impact on PSI damage. Membrane tethering of FNR therefore plays a role in alleviating high light stress, by regulating electron distribution during short-term responses to light.

Altered location of a key enzyme involved in the post-photosystem I electron transport chain ameliorates damage to photosystem I during increasing light intensity.     

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.     

Ferredoxin-NADP+ reductase uses the same site for the interaction with ferredoxin and flavodoxin

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.     

Ferredoxin:NADP+ oxidoreductase as a target of Cd2+ inhibitory action--biochemical studies

The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b₆f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre - ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K_i value calculated for research condition was 1.72 mM.FNR molecule can bind a large number of cadmium ions, as shown by the application of cadmium-selective electrode, but just one ion remains bound after dialysis. The effect of cadmium binding is significant disturbance in the electron transfer process from flavin adenine dinucleotide (FAD) to dibromothymoqinone, but less interference with the reduction of ferricyanide. However, it caused a strong inhibition of Fd reduction, indicating that Cd-induced changes in the FNR structure disrupt Fd binding. Additionally, the protonation of the thiol groups is shown to be of great importance in the inhibition process. A mechanism for cadmium-caused inhibition is proposed and discussed with respect to the in vitro and in vivo situation.     

Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275-4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.     

Effect of Ferredoxin on the Diaphorase Activity of Cyanobacterial Ferredoxin-NADP Reductase

The interaction of ferredoxin-NADP reductase (FNR) and ferredoxin (Fd) results in an enhanced rate of reaction and a shift of the pH optimum for the FNR-mediated diaphorase reaction. Low concentrations of NaCl (<100 millimolar), favorable for formation of the FNR:Fd complex, further magnify the alteration of the diaphorase reaction; the activity is enhanced 3-fold and pH optimum is shifted from 9.5 to 7.8. The Fd-stimulated diaphorase activity of FNR may result either from a conformational change of the enzyme and/or from a transition from a two electron to a one electron reaction.     

Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates.

The role of the negative charge of the E139 side-chain of Anabaena Ferredoxin-NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side-chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge-reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side-chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild-type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.     

Highly nonproductive complexes with Anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin:NADP+ reductase

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena function in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is extremely impaired relative to the reaction with wt FNR, and the dependency of k(obs) on E139K concentration shows strong upward curvature at protein concentrations > or = 10 microM. However, at values of mu > or = 200 mM, reaction rates approach those of wild-type FNR, and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR concentrations and low mu values, the dependency of k(obs) on E139Q concentration shows a smaller degree of upward curvature at mu = 12 and 100 mM and shows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 microM at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are explained by a mechanism in which the mutual orientation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increased E139K concentrations, the added mutant FNR reacts via a collisional interaction with the reduced Fd present in the unreactive complex. The et reactivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a negative charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.     

Effects of Amino and Thiol Group Reagents on the Ferredoxin:NADP+ Oxidoreductase Catalysed Reduction of Dibromothymoquinone

Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP⁺ oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 M Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.     

Structural analysis of interactions for complex formation between Ferredoxin-NADP+ reductase and its protein partners

The three-dimensional structures of K72E, K75R, K75S, K75Q, and K75E Anabaena Ferredoxin-NADP+ reductase (FNR) mutants have been solved, and particular structural details of these mutants have been used to assess the role played by residues 72 and 75 in optimal complex formation and electron transfer (ET) between FNR and its protein redox partners Ferredoxin (Fd) and Flavodoxin (Fld). Additionally, because there is no structural information available on the interaction between FNR and Fld, a model for the FNR:Fld complex has also been produced based on the previously reported crystal structures and on that of the rat Cytochrome P450 reductase (CPR), onto which FNR and Fld have been structurally aligned, and those reported for the Anabaena and maize FNR:Fd complexes. The model suggests putative electrostatic and hydrophobic interactions between residues on the FNR and Fld surfaces at the complex interface and provides an adequate orientation and distance between the FAD and FMN redox centers for efficient ET without the presence of any other molecule as electron carrier. Thus, the models now available for the FNR:Fd and FNR:Fld interactions and the structures presented here for the mutants at K72 and K75 in Anabaena FNR have been evaluated in light of previous biochemical data. These structures confirm the key participation of residue K75 and K72 in complex formation with both Fd and Fld. The drastic effect in FNR activity produced by replacement of K75 by Glu in the K75E FNR variant is explained not only by the observed changes in the charge distribution on the surface of the K75E FNR mutant, but also by the formation of a salt bridge interaction between E75 and K72 that simultaneously "neutralizes" two essential positive charged side chains for Fld/Fd recognition.