No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.     

Flavodoxin cofactor binding induces structural changes that are required for protein–protein interactions with NADP+ oxidoreductase and pyruvate formate-lyase activating enzyme

Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV–vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP⁺-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.     

Cofactor‐induced reversible folding of Flavodoxin‐4 from Lactobacillus acidophilus

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN‐binding mechanism to flavodoxins‐4 were obtained from the NMR structures of the apo‐protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO ) and exhibits the characteristic flavodoxin fold, with a central five‐stranded parallel β–sheet and five α‐helices forming an α/β‐sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β‐sheet and covers the FMN‐binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.     

Electron transfer by diflavin reductases

Diflavin reductases are enzymes which emerged as a gene fusion of ferredoxin (flavodoxin) reductase and flavodoxin. The enzymes of this family tightly bind two flavin cofactors, FAD and FMN, and catalyze transfer of the reducing equivalents from the two-electron donor NADPH to a variety of one-electron acceptors. Cytochrome P450 reductase (P450R), a flavoprotein subunit of sulfite reductase (SiR), and flavoprotein domains of naturally occurring flavocytochrome fusion enzymes like nitric oxide synthases (NOS) and the fatty acid hydroxylase from Bacillus megaterium are some of the enzymes of this family. In this review the results of the last decade of research are summarized, and some earlier results are reevaluated as well. The kinetic mechanism of cytochrome c reduction is analyzed in light of other results on flavoprotein interactions with nucleotides and cytochromes. The roles of the binding sites of the isoalloxazine rings of the flavin cofactors and conformational changes of the protein in electron transfer are discussed. It is proposed that minor conformational changes during catalysis can potentiate properties of the redox centers during the catalytic turnover. A function of the aromatic residue that shields the isoalloxazine ring of the FAD is also proposed.     

Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities

Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.     

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.     

Flavodoxin from the blue-green alga Nostoc strain MAC

A flavodoxin was isolated from the blue-green alga Nostoc strain MAC grown photoautotrophically or chemoheterotrophically in iron-deficient medium. In vitro, the flavodoxin would support NADP⁺ photoreduction by photosynthetic membranes, pyruvate oxidation by the phosphoroclastic system of Clostridium pasteurianum, and electron transfer to Cl. pasteurianum hydrogenase. In its oxidized form, the flavodoxin had absorbance maxima at 274 sh283 sh293, 376 sh432 and 466 sh488 nm. Reduction by dithionite proceeded via a neutral, blue semiquinone radical. The flavodoxin contained 1 mol of FMN per mol of protein and the amino acid composition showed a predominance of acidic residues; cysteine was apparently absent. A minimum MW of ca 22 000 derived from these data was confirmed by electrophoresis on SDS-polyacrylamide gels and by ultracentrifugal analysis. This flavodoxin thus belongs to the higher MW group of these low potential electron transfer proteins.     

黄素氧化还原蛋白研究进展

黄素氧化还原蛋白（flavodoxin,Fld）是一类通过非共价键与黄素单核苷酸（flavinmononucleotide,FMN）紧密结合的小黄素蛋白,在原核生物及低等真核生物中作为重要的低电势电子传递载体参与了众多的生化反应。在Fld的结构和分类、FMN与脱辅基Fld结合途径以及Fld结构与功能的关系等方面综述了近年来的相关研究进展。     

Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH*/FMNH2 couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.     

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: implications for their interactions with photosystem I and ferredoxin-NADP reductase

The two proteins ferredoxin and flavodoxin can replace each other in the photosynthetic electron transfer chain of cyanobacteria and algae. However, structure, size, and composition of ferredoxin and flavodoxin are completely different. Ferredoxin is a small iron-sulfur protein (approximately 100 amino acids), whereas flavodoxin is a flavin-containing protein (approximately 170 amino acids). The crystal structure of both proteins from the cyanobacteria Anabeana PCC 7120 is known. We used these two protein structures to investigate the structural basis of their functional equivalence. We apply the Hodgkin index to quantify the similarity of their electrostatic potentials. The technique has been applied successfully in indirect drug design for the alignment of small molecule and bioisosterism elucidation. It requires no predefined atom-atom correspondences. As is known from experiments, electrostatic interactions are most important for the association of ferredoxin and flavodoxin with their reaction partners photosystem I and ferredoxin-NADP reductase. Therefore, use of electrostatic potentials for the structural alignment is well justified. Our extensive search of the alignment space reveals two alignments with a high degree of similarity in the electrostatic potential. In both alignments, ferredoxin overlaps completely with flavodoxin. The active sites of ferredoxin and flavodoxin rather than their centers of mass coincide in both alignments. This is in agreement with electron microscopy investigations on photosystem I cross-linked to ferredoxin or flavodoxin. We identify residues that may have the same function in both proteins and relate our results to previous experimental data.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

Fatty acid metabolism, conformational change, and electron transfer in cytochrome P-450(BM-3)

Crystal structure-based mutagenesis studies on cytochrome P-450(BM-3) have confirmed the importance of R47, Y51, and F87 in substrate binding. Replacing F87 has profound effects on regioselectivity. In contrast, changing either R47 or Y51 alone to other residues results in limited impact on substrate binding affinity. Mutating both, however, leads to large changes. Substrate-induced protein conformational changes not only lead to specific substrate binding in the heme domain, but also affect interactions with the FMN domain. Unlike the microsomal P-450 reductase, the FMN semiquinone is the active electron donor to the heme iron in P-450(BM-3). The crystal structure of P-450(BM-3) heme/FMN bidomain provides important insights into why the FMN semiquinone is the preferred electron donor to the heme as well as how substrate-induced structural changes possibly affect the FMN and heme domain-domain interaction.     

Conformational and thermodynamic control of electron transfer in neuronal nitric oxide synthase

Multiple solution-state techniques have been employed in investigating the nature and control of electron transfer in the context of the proposed "domain shuffle hypothesis" for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. NADPH analogues and fragments have been used to map those regions of this substrate that are important in eliciting a conformational change, observed in both the fluorescence emission of the flavin cofactors of the enzyme and the EPR spectra of the FMN flavosemiquinone state. EPR and UV-visible potentiometric methods have demonstrated a substantial calmodulin-dependent perturbation in the midpoint reduction potentials of the redox couples of both flavin cofactors, in contrast to a previous report [Noble, M. A., et al. (1999) Biochemistry 38, 16413-16418]. These studies support a model in which FMN domain mobility, triggered by Ca2+-calmodulin binding and antagonized by substrate binding, facilitates electron transfer in nitric oxide synthase through conformational change and effects a major change in the midpoint reduction potentials of the flavin redox couples. These results are discussed in light of the recent crystal structure of the NADPH-locked reductase domain.     

Temperature-jump studies of Desulfovibrio vulgaris flavodoxin: Kinetics of FMN binding and of reduction of semiquinone by methyl viologen

Temperature-jump relaxation spectrometry has been used with the flavodoxin from Desulfovibrio vulgaris to investigate the kinetics of FMN binding to apoprotein and of reduction of half-reduced to fully-reduced protein by methyl viologen. FMN binding was characterized by a single, concentration dependent, exponential relaxation which corresponded to a diminution of flavin fluorescence intensity. This implies the absence of detectable intermediates during coenzyme binding and a positive enthalpy of binding. The latter was confirmed by microcalorimetry and fluorometric titration studies at two temperatures. The reduction of the half-reduced holoprotein also displayed simple exponential kinetics, again implying the absence of intermediates. The results of these studies are compared with earlier work with other flavodoxins.     

1H, 13C and 15N resonance assignments of the apo and holo states of flavodoxin YqcA from Escherichia coli

Flavodoxins are a family of FMN binding proteins widely distributed in prokaryotes. They involve in various electron transfer reactions using the non-covalently bound FMN cofactor as the redox center. The Escherichia coli yqcA gene was identified to encode a short-chain favodoxin based on sequence information. However, the structure of YqcA protein is unknown and its exact biological function in cell is yet to be investigated. Herein, we report the resonance assignments of ¹H, ¹³C and ¹⁵N atoms of E. coli YqcA in both the apo and holo states.     

Refined structures of oxidized flavodoxin from Anacystis nidulans

Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the first member of the flavodoxin family to be characterized, and is the structural prototype for the "long-chain" flavodoxins that have molecular masses of approximately 20 kDa. Crystal structure analyses and refinements of three orthorhombic forms of oxidized A. nidulans flavodoxin are reported, and salient features of the fold and the FMN binding site are compared with other flavodoxins. The structure of form I (wild-type: P212121, a=57.08 A, b=69.24 A, c=45.55 A), determined initially by multiple isomorphous replacement, has been refined to R=0.183 and R(free)=0.211 for data from 10.0 to 1.7 A resolution. Structures of form II (wild-type: P212121, a=60.05 A, b=65.85 A, c=51.36 A) and form III (Asn58Gly: P212121, a=51.30 A, b=59.15 A, c=94.44 A) have been determined by molecular replacement and refined versus data to 2.0 A and 1.85 A, respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermolecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper.     

A Bridging Interaction Allows Calmodulin to Activate NO Synthase through a Bi-modal Mechanism

Calmodulin (CaM) activates the nitric-oxide synthases (NOS) by a mechanism that is not completely understood. A recent crystal structure showed that bound CaM engages in a bridging interaction with the NOS FMN subdomain. We investigated its importance in neuronal NOS (nNOS) by mutating the two residues that primarily create the bridging interaction (Arg⁷⁵² in the FMN subdomain and Glu⁴⁷ in CaM). Mutations designed to completely destroy the bridging interaction prevented bound CaM from increasing electron flux through the FMN subdomain and diminished the FMN-to-heme electron transfer by 90%, whereas mutations that partly preserve the interaction had intermediate effects. The bridging interaction appeared to control FMN subdomain interactions with both its electron donor (NADPH-FAD subdomain) and electron acceptor (heme domain) partner subdomains in nNOS. We conclude that the Arg⁷⁵²–Glu⁴⁷ bridging interaction is the main feature that enables CaM to activate nNOS. The mechanism is bi-modal and links a single structural aspect of CaM binding to specific changes in nNOS protein conformational and electron transfer properties that are essential for catalysis.     

Role of reductase domain cluster 1 acidic residues in neuronal nitric-oxide synthase. Characterization of the FMN-FREE enzyme.

The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp(918) and Glu(919)) and one conserved aromatic residue (Phe(892)), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steady-state and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.     

Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp. PCC 7119.

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.