Báo cáo khoa học: Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family pdf

Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family Naoki Shibata1, Yasufumi Ueda1, Daisuke Takeuchi2, Yoshihiro Haruyama2, Shuichi Kojima3, Junichi Sato4, Youichi Niimura4, Masaya Kitamura2 and Yoshiki Higuchi1 Department of Life Science, University of Hyogo, Japan Department of Applied Chemistry and Bioengineering, Osaka City University, Japan Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan Department of Bioscience, Tokyo University of Agriculture, Japan Keywords crystal structure; electron transfer; flavin mononucleotide; flavin reductase family; sulfate-reducing bacterium Correspondence M Kitamura, Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Fax: +81 666 05 2769 Tel: +81 666 05 3091 E-mail: kitamura@bioa.eng.osaka-cu.ac.jp Y Higuchi, Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan Fax: +81 791 58 0177 Tel: +81 791 58 0179 E-mail: hig@sci.u-hyogo.ac.jp Database The coordinates and structure factor data have been deposited in the PDB, under the accession number 2D5M The nucleotide and amino acid sequence data may be found in the DDBJ, EMBL and GenBank sequence databases under the accession numbers AB214904 and BAD99043, respectively The crystal structure of flavoredoxin from Desulfovibrio vulgaris Miyazaki ˚ F was determined at 1.05 A resolution and its ferric reductase activity was examined The aim was to elucidate whether flavoredoxin has structural similarity to ferric reductase and ferric reductase activity, based on the sequence similarity to ferric reductase from Archaeoglobus fulgidus As expected, flavoredoxin shared a common overall structure with A fulgidus ferric reductase and displayed weak ferric reductase and flavin reductase activities; however, flavoredoxin contains two FMN molecules per dimer, unlike A fulgidus ferric reductase, which has only one FMN molecule per dimer Compared with A fulgidus ferric reductase, flavoredoxin forms three additional hydrogen bonds and has a significantly smaller solvent-accessible surface area These observations explain the higher affinity of flavoredoxin for FMN Unexpectedly, an electron-density map indicated the presence of a Mes molecule on the re-side of the isoalloxazine ring of FMN, and that two zinc ions are bound to the two cysteine residues, Cys39 and Cys40, adjacent to FMN These two cysteine residues are close to one of the putative ferric ion binding sites of ferric reductase Based on their structural similarities, we conclude that the corresponding site of ferric reductase is the most plausible site for ferric ion binding Comparing the structures with related flavin proteins revealed key structural features regarding the discrimination of function (ferric ion or flavin reduction) and a unique electron transport system (Received 30 March 2009, revised 28 May 2009, accepted 29 June 2009) doi:10.1111/j.1742-4658.2009.07184.x Abbreviations DvMF, Desulfovibrio vulgaris Miyazaki F; FeR, ferric reductase; Fre, flavin reductase; PDB, Protein Data Bank 4840 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al Structure of flavoredoxin Introduction Flavins play major roles as cofactors for a wide variety of redox proteins and enzymes; these reactions depend on the redox ability of the flavin species Although the basic redox reactions are identical or similar, it is of interest to understand the molecular bases for the different reactivities displayed by flavins in different protein contexts Flavoredoxin is an electron-transfer protein that has one FMN molecule per subunit or monomer [1] The only flavoredoxins characterized to date are from the sulfate-reducing bacterium Desulfovibrio gigas [1–3] and an Archaeon Methanosarcina acetivorans [4] Deletion and mutation analyses of this bacterium have indicated that flavoredoxin is involved in the thiosulfate reduction process [3] It has been proposed that flavoredoxin receives an electron, which was originally generated by a hydrogenase, from flavodoxin or ferredoxin, and transfers it to a sulfite reductase, desulfoviridin [3] The D gigas flavoredoxin has an apparent sequence identity of 22% with Archaeoglobus fulgidus ferric reductase (FeR) [1] FeR catalyzes the reduction of Fe(III) chelates, such as Fe(III)–EDTA, in a NAD(P)Hdependent manner [5,6] The crystal structure of FeR has been determined with and without NADP+ [6] These authors were unsuccessful in their attempts to solve the structure in the Fe(III) ion-bound state The catalytic mechanism of ferric ion reduction by this enzyme has been proposed based on biochemical [5] and structural studies of FeR [6], although the key residues for ferric ion binding need to be identified to elucidate the complete reaction mechanism It was surprising that, as pointed out by Chiu et al [6], FeR revealed a common overall fold with the FMN-binding protein from Desulfovibrio vulgaris Miyazaki F (DvMF), whose crystal structure was determined by our group [7] However, FeR and the FNM-binding protein from have relatively low sequence identity (12%) FeR also has structural similarity to the flavin reductase (Fre, NADH : flavin oxidoreductase) family, which includes the Fre component of the two-component flavin-diffusible monooxygenase [5,6] The Fre component reduces a flavin with NADH or NADPH to provide a reduced flavin, which is used to activate molecular oxygen for the oxygenase reaction [8] In this family, neither component of the enzyme binds flavin tightly as a cofactor, but rather utilizes it as another substrate [8] Considering the amino acid sequence similarities between flavoredoxin and related flavin proteins, the question arises as to whether flavoredoxin possesses ferric ion or flavin reductase activity Which structures determine the unique functions of these flavin proteins? In this study, we present the crystal structure of DvMF flavoredoxin and discuss the key residues for ligand binding and metal ion binding, based on the crystal structures Results Cloning and sequencing of the flavoredoxin gene We determined the nucleotide sequence of the entire flavoredoxin gene (accession number AB214904 in the DDBJ, EMBL and GenBank nucleotide databases) The ORF that encodes flavoredoxin comprises 190 amino acid residues A potential ribosome-binding site (GAGG, nucleotides 737–740 in the PstI–KpnI fragment) is present upstream of the initiation codon (ATG), and there are potential promoter regions at nucleotides 643–648 (TTGCCG) and 666–671 (CAAACT) in the PstI–KpnI fragment Nucleotides 1339– 1371 comprise the putative transcriptional terminator, forming a stem-and-loop structure The results of a BLAST homology search indicate that the product of this ORF is highly homologous to flavoredoxins from other bacteria, especially that of D vulgaris (Hildenborough), with an identity of 71%; therefore, we confirmed this ORF to be the flavoredoxin gene Recombinant flavoredoxin purification We used an Escherichia coli expression system to express the flavoredoxin gene Recombinant flavoredoxin was detected in transformed E coli crude cell extracts by SDS ⁄ PAGE Through chromatographic steps using DE52 and Superdex 75, a large amount of flavoredoxin was purified to homogeneity by SDS ⁄ PAGE (Fig S1) The molecular mass of the expressed flavoredoxin was estimated to be  23 000 Da by SDS ⁄ PAGE, which is different from the value calculated based on the amino acid sequence (20 800 Da) We also estimated the molecular mass in the native state to be  37 000 Da using a Superdex 75 gel-filtration column This value is about twice that calculated from the amino acid sequence, indicating that the native form of flavoredoxin is a dimer Amino acid sequence analysis of flavoredoxin The N-terminal amino acid sequence of flavoredoxin from DvMF was determined to be Met–Lys–Lys–Ser– Leu–Gly–Ala, and the Met was formylated When the flavoredoxin amino acid sequences of DvMF and other FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4841 Structure of flavoredoxin N Shibata et al organisms were compared, they were found to be highly conserved The three characteristic co-ordination motifs (36TSKP–62FGVSVL–124GTHTL) of the FeR from A fulgidus, which is linked to FMN or NAD binding [5], were also found in DvMF flavoredoxin (40CSQP–66FTISIP–128GLHTQ) These co-ordination motifs are not homologous to those of flavodoxin or FMN-binding protein Identification of the prosthetic group To identify the prosthetic group bound to the recombinant flavoredoxin, UV-visible spectra of the purified holoprotein were recorded (Fig S2) In the visible region, absorption maxima were observed at 381 and 452 nm, which are characteristic of proteins that bind to flavin derivatives The recombinant flavoredoxin was subjected to reverse-phase HPLC on a C8 column, and the retention time of the obtained prosthetic group was compared with those of flavin derivatives The retention time of the prosthetic group bound to recombinant flavoredoxin was identical to that of FMN (Fig S3) The A448 : A268 ratio of the holoprotein was 0.267, suggesting that the flavoredoxin expressed in E coli as a holoprotein binds to FMN as a prosthetic group at a molar ratio of Overall structure of DvMF flavoredoxin DvMF flavoredoxin was crystallized in the P3121 space group with one molecule in the asymmetric unit The structure was refined to a crystallographic R factor of ˚ 0.135 and Rfree of 0.162 at 1.05 A resolution (Table 1) Residues 128–130 and 187–190 (four C-terminal residues) were excluded from the structural model because of poor electron densities in these regions DvMF flavoredoxin contains four a helices (a1–4), two 310 helices (3101–2) and 12 b strands (b1–12) as secondary structural elements; it also has a Greek key motif with seven anti-parallel b strands (Figs and 2A), which is also found in DvMF FMN-binding protein [7] and A fulgidus FeR [6] Flavoredoxin contains two FMN molecules per dimer, unlike FeR, which has only one FMN molecule per dimer (Fig 2B) The FMN molecule is located in the hollow, encompassed mainly by a1, a2 and b3 A structural homology search was carried out using the DALI server [9] Among the proteins of known function, the M acetivorans flavoredoxin exhibited ˚ the lowest rmsd (1.5 A) and the highest Z score (25.5), as expected from the highest sequence identity (30%) of the known structures A fulgidus FeR ˚ showed the second lowest rmsd (1.9 A) and the 4842 Table Summary of x-ray data collection, phasing and refinement statistics Native Methylmercuric chloride derivative Data collection Beamline BL41XU, SPring-8 BL44XU, SPring-8 Space group P3121 P3121 Unit-cell a = b = 53.35, a = b = 53.5, ˚ parameters (A) c = 116.22 c = 116.2 ˚ Wavelength (A) 0.7100 1.0000 ˚ Resolution range (A) 50–1.05 (1.09–1.05) 50–1.71 (1.77–1.71) Measured reflections 950,955 205,263 Unique reflections 89,930 21,327 Completeness (%) 99.6 (97.0) 99.6 (97.6) Rmerge 0.088 (0.573) 0.087 (0.158) Multiplicity 10.6 (8.7) 9.6 (7.2) I ⁄ r(I) 42.1 (3.7) 59.8 (14.0) SAD phasing for methylmercuric chloride derivative Figure of merit, 0.165 ⁄ 0.609 centric ⁄ acentric Phasing power 5.126 Refinement ˚ Resolution range (A) 10–1.05 (1.09–1.05) Rwork 0.135 (0.208) Rfree 0.162 R.m.s deviations from ideal values ˚ Bond lengths (A) ⁄ 0.016 ⁄ 0.031 angle distances (°) Ramachandran plot Most-favored 136 Additionally allowed 16 Generously allowed Disallowed (Val167) - second highest Z score (18.8), although the sequence identity between DvMF flavoredoxin and A fulgidus FeR is low (17%) Among the flavin-containing electron-transfer proteins of Desulfovibrio species, the structures of the FMN-binding protein and flavodoxin were determined by X-ray crystallography; the structure of DvMF flavoredoxin resembles ˚ the former (rmsd = 2.5 A) rather than the latter ˚ ) (rmsd = 3.4 A As deduced by gel-filtration chromatography, DvMF flavoredoxin forms a dimer, as evidenced by the crystallographic two-fold axis in the crystal When the dimeric structure of DvMF flavoredoxin was compared with those of FeR and FMN-binding protein, the flavoredoxin dimer was superimposed on the former (Fig 2B) but not on the latter (Fig 2C) In flavoredoxin, the twofold axis associated with the dimer passes through the vicinity of the side chains of Pro13, Ile126, Gln133 and Ile163 The corresponding FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al Structure of flavoredoxin Fig Amino acid sequence alignment of DvMF flavoredoxin, Methanosarcina acetivorans flavoredoxin, ferric reductase and HpaC component of Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase Secondary structure elements of flavoredoxin are shown on the lines of residue numbers Residues involved in binding of FMN are shown in red Residues shown in bold are aligned based on crystal structures Residues shown in regular characters indicate that structural information is unavailable or that structurally equivalent residues are not present Alignment for HpaC was performed with CLUSTAL W [48] A B Fig Structures of flavoredoxin and other related proteins (A) Overall structure of the flavoredoxin dimer Each subunit is shown in green–cyan and violet–red models FMN molecules are depicted as ball-and-stick models (B) Superimposed Ca-traces of flavoredoxin (green and violet) and FeR (light gray) (C) Superimposed Ca-traces of flavoredoxin (green and violet) and FMN-binding protein (light gray) C FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4843 Structure of flavoredoxin N Shibata et al residues in the FMN-binding protein are exposed to the solvent In terms of dimer interactions, both N- and C-terminal loops (Met1–Pro13 and Val174–Lys186, respectively) appear to play important roles The six N-terminal residues (Met1–Gly6) are extended in the opposite direction along the b10 of the other monomer, forming an anti-parallel intermolecular b sheet (Fig 2A) The subsequent residues of the loop (Ala7– Pro13) turn into the interior of the dimer interface, and Leu10 and Tyr12 form a hydrophobic core with Ile70, Met116, Val141, Pro154, Ile156 and Pro161 of the other monomer Similarly, Gly175–Ala181 forms an anti-parallel intermolecular b sheet with the b12 of the other monomer, and towards the C-terminus, the subsequent residues, Phe182–Lys186, pass through the a2 vicinity of the other monomer For the FeR dimer, the N-terminal loop is replaced by an a helix, and side chain-to-side chain interactions play a major role in dimer interactions through this region By contrast, a similar intermolecular b sheet through the C-terminal loop is conserved Structure of the FMN-binding region The hydrogen bonds and salt bridge that encompass the ribitol moiety and the phosphate group of FeR and M acetivorans flavoredoxin are moderately and completely conserved in DvMF flavoredoxin (Fig 3A,B and Fig S4A), respectively In the case of FeR, Ser84 replaces Asn29 of DvMF flavoredoxin, which forms a hydrogen bond to the O3P atom of FMN (Fig 3A,B) DvMF flavoredoxin forms three additional hydrogen bonds between the NH2 moiety of Arg51 and three atoms of FMN (N1, O2 and O3’) In both FeR and M acetivorans flavoredoxin, the corresponding residue is asparagine (Asn47 and Asn52, respectively) to which only the O2 atom of FMN forms a hydrogen bond (Figs 3A,B and Fig S4A) The isoalloxazine ring of FMN is surrounded by hydrophobic residues, Leu16, Trp35, Ile84, Phe164, Tyr171 and Phe182, the first five residues of which correspond to Leu13, Thr31, Phe81, Tyr147 and Tyr150 in FeR (Fig 3A,B), and Val18, Trp36, Leu85, Leu162, Tyr169 and Leu180 in M acetivorans flavoredoxin (Fig S4A) It should be noted that a positively charged residue, Lys92, is involved in the binding of the phosphate group(s) of FMN or FAD Lys92 forms a salt bridge with the O3P of FMN A salt bridge that involves flavin species has not been reported in the structures of the other electron-transfer flavoproteins; however, a salt bridge between the lysine ⁄ arginine residue and 4844 FMN ⁄ FAD is found frequently in flavin-dependent enzymes To date, from the 130 FMN protein PDB entries 22 have at least one FMN–lysine and 62 have at least one FMN–arginine interaction For FAD proteins, from the 210 entries have at least one FAD–lysine and 55 have at least one FAD–arginine interaction One of these proteins, FeR, has a Lys89 residue that interacts with the phosphate group of FMN As pointed out by Chiu et al [6], the structure of FeR resembles the flavin-binding domain of ferredoxin : NADP+ reductase [10] The lysine residue is not conserved; instead, an arginine residue interacts with the FAD molecule of the enzyme In the case of FMN-binding protein, Lys53 is adjacent to FMN, forming a hydrogen bond with the phosphate group of FMN through its main-chain N atom, whereas the side ˚ chain amino group is A from the FMN molecule The surface charge models of these proteins indicate that the level of positive charge at the phosphate group binding site is considerably higher in both flavoredoxin and FeR than in the FMN-binding protein (Fig S5A–C) The accessible surface areas of FMN in flavoredoxin ˚ and FeR have been calculated to be 57 and 95 A2, respectively Three residues, Trp35, Arg51 and Phe182, are responsible for this difference (Fig 3A,B) Both Trp35 and Arg51 of flavoredoxin have larger volumes than the corresponding residues (Thr31 and Asn47) of FeR No residue in FeR corresponds to Phe182 (Fig 3A,B) The surface model of flavoredoxin indicates that the re-side of the isoalloxazine ring is partially covered by these residues (Fig S5A) By contrast, the re-side of the isoalloxazine ring of FeR is completely exposed to the solvent (Fig S5B) Resolution of the structure of NADP+-bound FeR revealed that the nicotinamide moiety of NADP+ faces the re-side of the isoalloxazine ring, and that the 2¢-P-AMP moiety is held in the groove between the 310 helix and the third a helix [6] Unexpectedly, in flavoredoxin, this site is occupied by Mes, which was added to crystallization buffer solution The Mes molecule is held in place through a salt bridge with Arg169, hydrogen bonds with Thr9 and Val167, and hydrophobic interactions with Trp35 and FMN (Fig 3C) Structure of the metal ion binding site The electron-density map displayed three isolated spheres with significantly greater density than normal water oxygen atoms Two of these are close to the FMN binding site, and the other is on the opposite surface of the protein An anomalous-difference map calculated from the native dataset showed significant FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al Structure of flavoredoxin A B C Fig FMN binding site (A,B) Superimposed models of FMN binding sites of flavoredoxin and FeR, showing residues that interact with the FMN molecule Hydrogen bonds are shown as green dotted lines Flavoredoxin is displayed by atom color, with the exception that the carbon atoms of FMN are in yellow FeR is shown as a transparent model The Mes molecule is omitted for clarity The view in (B) is rotated 180° about the vertical axis Residue labels are shown as flavoredoxin ⁄ FeR (C) Residues of flavoredoxin involved in Mes binding NADP+ derived from the superimposed model of FeR is also shown as a transparent model (D) Zinc ion-binding site Zinc ions are depicted as green spheres The remaining color codes are the same as in (A) and (B) D peaks at each of these sites These densities were assigned as zinc ions derived from the crystallization solution as an additive The zinc ion closest to FMN, Zn201, is coordinated by the Sc atom of one of the two conformers of Cys40 and two water molecules ˚ (Fig 3D) Zn203, which is 5.1 A from Zn201, is also FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4845 Structure of flavoredoxin N Shibata et al coordinated by Cys39 and the other conformer of Cys40 This zinc ion is bound to His131, which corresponds to the histidine residue that is completely conserved among the FeR homologs [5,6] Interestingly, the residues corresponding to metal ion binding are totally different in both FeR and M acetivorans flavoredoxin These cysteine residues are replaced by threonine and leucine in FeR and asparagine and valine in M acetivorans flavoredoxin (Figs and 3D; Fig S4C) In the case of FeR, however, Cys45 is adjacent to this site instead (Fig 3D) observed increase in absorption around 340 nm is caused by the addition of NADH or NADPH Based on the amino acid comparison (Fig 1) and the crystal structure of flavoredoxin, we propose that flavoredoxin has a NAD(P)H binding site similar to that of A fulgidus FeR, and therefore we expect that NAD(P)H is bound to this site and reduces the FMN of flavoredoxin; however, no decrease in absorption in the visible region because of flavoredoxin reduction was observed, even when NADH or NADPH was added (data not shown); therefore, we conclude that flavoredoxin has no oxidase activity Redox potential of recombinant flavoredoxin Figure shows the results of linear regression analysis of the logarithms for the redox ratio of the mediator versus that of recombinant flavoredoxin The redox potential of oxidized flavoredoxin ⁄ reduced flavoredoxin (Eflr) was calculated as )343 mV at pH 7.0, determined using Neutral Red (Em,7 = )325 mV, n = 2) [11] or benzyl viologen (Em,7 = )359 mV, n = 1) [12] An n value of was used in these experiments, which fit the experimental data closely Although recombinant flavoredoxin was fully reduced by sodium dithionite, no semiquinone intermediate was found Flavoredoxin reduction by NAD(P)H Reduction experiments were performed under anaerobic conditions substituted by oxygen-free argon, and NADH or NADPH was used as the reductant The Ferric reductase and flavin reductase activities of recombinant flavoredoxin and related proteins Flavoredoxin uses both NADH and NADPH as electron donors to reduce Fe3+–EDTA and FMN; however, we found that both FeR and Fre activities using NADH were lower than those using NADPH (data not shown) FeR and Fre activities of flavoredoxin were 2.58 · 10)3 and 2.70 · 10)3 unitsỈmgỈprotein)1, respectively, whereas those of DrgA from Synechocystis sp PCC6803, which was used as a positive control, were 8.66 · 10)2 and 3.22 unitsỈmgỈprotein)1 under aerobic conditions, respectively [13] Even though both activities of flavoredoxin could be detected, FeR and Fre activities of flavoredoxin were 33.6-fold and 1200fold lower than those of DrgA, respectively However, FMN-binding protein [14] showed neither FeR nor Fre activity Discussion Fig Linear regression analysis of the logarithms of concentration ratios for oxidized and reduced forms of a mediator versus that of recombinant flavoredoxin (°) Neutral Red used as the mediator; (D) Benzyl viologen is used as the mediator 4846 Based on the high structural similarity between DvMF flavoredoxin and A fulgidus FeR, we expected that the flavoredoxin would have FeR and Fre activities, both of which confer reduction of a flavin by receiving electrons from NADH or NADPH; however, these activities of flavoredoxin were much lower than those of A fulgidus FeR The FeR activity of A fulgidus, which is also a sulfate reducer, was reported to be 3503 unitsỈmgỈprotein)1 at 85 °C using NADPH and FMN [5] This value is  1.36 · 106-fold higher than that of DvMF flavoredoxin DvMF FMN-binding protein, which also shares structural similarity with A fulgidus FeR, did not exhibit any detectable FeR activity It should be noted that the FeR activity of the A fulgidus enzyme is at least 1000-fold higher than all other bacterial enzymes [5] In addition, the FeR activity of A fulgidus enzyme was  1070-fold higher than DrgA, which we used as a positive control, even under anaerobic conditions (3.28 unitsỈmgỈprotein)1) [13] FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al Thus, it appears that only A fulgidus FeR possesses extremely high FeR activity, compared with other FeRs The FeR activity of flavoredoxin is 102–104 times lower than that of other bacterial FeR-active enzymes Taking this into account, we postulate that DvMF flavoredoxin reacts similarly to FeR, although its activity is not comparable with that of A fulgidus FeR The same may be true of flavin reductase activity Two explanations can be proposed for the large differences noted in ferric ion and flavin reductase activities between DvMF flavoredoxin and A fulgidas FeR The first possibility is that NAD(P)H binds to flavoredoxin, but hardly transfers an electron to FMN The fact that DvMF flavoredoxin has a lower redox potential than NADH ()343 versus )320 mV) supports this explanation The second possibility is that the binding of NAD(P)H is sterically hindered by the surrounding residues Regarding this second possibility, when the flavoredoxin–Mes complex is superimposed on the FeR–NADP+ complex, steric hindrances occur between flavoredoxin and NADP+ at three different sites (Fig 3C) First, the adenine ring of NADP+ has severe steric contacts with Pro166, Val167 and Ser168, which comprise the loop between b11 and b12 Second, the phosphate and ribose groups overlap with the phenyl group of Phe182 Third, the nicotinamide moiety is immediately adjacent to Trp35 At the first site, the adenine ring has to move away from these overlapping residues to bind to the corresponding site of flavoredoxin At the second site, such steric repulsions could be relieved by displacement of the residues and ⁄ or a modified configuration of NADP+, because most Phe182 is exposed to solvent and the side chain rotates freely about its Ca–Cb and Cb–Cc bonds The steric contacts of Trp35 at the third site are not severe Chiu et al [6] have suggested that the dimethylbenzene moiety is a candidate for ferric ion binding If this is the case, flavoredoxin cannot bind a ferric ion at this site unless Phe182 moves away from the site Then, because NAD(P)H was not the most suitable electron donor for DvMF flavoredoxin, the FeR and flavin reductase activities of DvMF flavoredoxin might have been underestimated, and we cannot ignore that the physiologic function of flavoredoxin is to transfer an electron from reduced ferredoxin or flavodoxin to a sulfite reductase, i.e desulfoviridin in the thiosulfate reduction process [3] It has been reported that A fulgidus FeR activity decreases during purification because of the loss of a flavin molecule, and is restored by adding FMN or FAD, which suggests that the affinities for flavin species are not sufficiently high In D gigas flavoredoxin, however, FMN binds tightly with a dissociation Structure of flavoredoxin constant of 0.12 nm [1], i.e 2500-fold lower than the Km value (0.3 lm) for FMN of FeR [5] This large difference can be explained in part by the difference in the exposed surface areas of FMN, as described above Among the NADH : flavin oxidoreductase family, one structure, the HpaC component of Sulfolobus tokodaii 4-hydroxyphenylacetate 3-monooxygenase, which is a member of the two-component flavin-diffusible monooxygenases, in the FMN-bound form has been reported [15] The Km of S tokodaii HpaC for FMN is unknown, but that of E coli HpaC is reported to be 2.1 lm [16], which is comparable with the values obtained for other NADH : flavin oxidoreductases [16–23] The accessible surface area of FMN of ˚ S tokodaii HpaC was calculated to be 152 A2 As expected, this area is significantly larger than those of ˚ ˚ flavoredoxin (57 A2) and FeR (95 A2) The large accessible surface area for FMN of HpaC is quite reasonable considering that HpaC releases the FMN molecule immediately upon reduction, with which the oxygenase component catalyzes the oxygenation reaction A fulgidus FeR is reported to be able to use FMN, but not riboflavin, as the electron acceptor, although most FeR molecules can use both For example, the Km and kcat values of E coli Fre for riboflavin are reported as 2.5 lm and 52.4 s)1 with NADPH as the electron donor and 1.3 lm and 30.6 s)1 with NADH [13] For FMN, Km and kcat values of E coli Fre were calculated as 2.67 lm and 0.724 s)1 with NADPH as the electron donor and 0.67 lm and 1.84 s)1 with NADH (our unpublished data) These differences may influence FeR and Fre activities Chiu et al [6] claimed that His126 of A fulgidus FeR is one of the candidates for the ferric ion-binding residue based on the structure of the mercury-bound derivative, although a ferric ion was not found at the site of either the native or NADP+-bound form; instead this residue is bound to the nicotinamide moiety of NADP+ in the NADP+-bound form These authors also proposed that Cys45 of FeR, which corresponds to Ser49 of DvMF flavoredoxin, is another candidate [6] The Oc atom of Ser49 does not bind to ˚ a zinc ion but is only 5.68 and 5.50 A from Zn201 and Zn203, respectively (Fig 3D) In FeR, Cys39 and Cys40 are replaced by leucine and threonine, and Cys45 rather than threonine would be the ligand for a ferric ion, because the hydroxyl group of threonine has a lower affinity for ferric ions than the thiol group of cysteine DrgA, which was used as a positive control in activity measurements, has a cysteine (Cys147) and three histidine (His15, His20 and His45) residues, which could be candidates for ferric ion-binding FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4847 Structure of flavoredoxin N Shibata et al residues However, DrgA seems to have a different folding from flavoredoxin and FeR, as the secondary structure prediction by PSIPRED [24] suggests that DrgA has a helix-rich structure (data not shown), unlike the b-rich structures of flavoredoxin and FeR Structural analysis needs to be carried out to elucidate the ferric ion-binding site of DrgA Suharti et al [4] have recently shown that M acetivorans flavoredoxin does not transfer an electron to ferric and chelated ferric ion, and does not have a cystein residue at the metal-binding site The corresponding region is formed by Val40, Asn41, Gly50 and Phe127 (Fig S4C), which are unlikely to have high affinity to ferric ions It should be noted that E coli HpaC displayed FeR activity [16] Sequence alignment indicates that it has a cysteine residue at the position that corresponds to Cys45 (Fig 1); therefore, flavin proteins, which have similar folding and ferric reductase activity, have at least one cysteine residue at or around the site corresponding to the zinc-binding site of flavoredoxin These lines of evidence raise questions as to why a zinc ion binds robustly to DvMF flavoredoxin and why no metal ions were found in the A fulgidus FeR structure even in a ferric ion atmosphere FeR may have low affinity for ferric ions in the oxidized state As Chiu et al [6] have suggested, the solvent-exposed dimethylbenzene moiety is one candidate for binding the ferric ion In this case, the ferric ion would bind only weakly to FeR, because none of the residues that could act as a ligand for the ferric ion are found at this position A zinc ion is preferentially trapped at the metal ion binding site of DvMF flavoredoxin Indeed, the ferric reductases of Azotobacter vinelandii [25] and Legionella pneumophila [26] are inhibited in the presence of zinc ions This is probably because a zinc ion is bound tightly at the ferric ion binding site, although the detailed inhibition mechanism remains unknown In the case of A vinelandii ferric reductase, the enzyme was purified as a mixture of two proteins or a heterodimer with molecular masses of 44 600 and 69 000 Da [25] Although none of the amino acid sequences of A vinelandii is recorded as ferric reductase, an NCBI entry, ‘Oxidoreductase FAD ⁄ NAD(P)-binding: Oxidoreductase FAD-binding region’ (ZP_00418100), might be the amino acid sequence of the smaller component of A vinelandii ferric reductase This entry contains 443 residues with a calculated molecular mass of 49 180 Da and has apparent identity with the FAD and NADH domains of BenC, which is the reductase component of benzoate dioxygenase reductase The crystal structure of BenC [27] shows that Cys307, ˚ which corresponds to Cys412 of the entry, is only A from the isoalloxazine ring of FAD, which is compara4848 ble with flavoredoxin and FeR If the entry corresponds to the smaller component of the A vinelandii ferric reductase, Cys412 could be the ferric ion binding site Experimental procedures Cloning and sequencing of the flavoredoxin gene E coli JM109 (recA1, supE44, endA1, hsdR17, gryA96, relA1, thi, D(lac-proAB), F’[traD36, proAB+, lacIq, lacZDM15]) was used for cloning and expression of the flavoredoxin gene DvMF was grown [28] and used for genomic DNA preparation Restriction and modification enzymes were purchased from New England BioLabs (Pickering, Ontario, Canada), Nippon Gene (Tokyo, Japan) and Toyobo (Osaka, Japan) The [32P]ATP[cP] (185 TBqỈmmol)1) was obtained from MP Biomedicals (Irvine, CA, USA) All other chemicals were of analytic grade for biochemical use Genomic DNA isolated from DvMF was prepared by the method of Saito & Miura [29] To amplify the flavoredoxin gene, we searched for the published amino acid sequences of flavoredoxin from other bacteria, because the DvMF flavoredoxin amino acid sequence was unknown, but we could not find the PCR conditions; however, we noted that the ABC transporter gene, the amino acid sequence of which is highly conserved across species, was in the complementary strand upstream of the flavoredoxin gene in sulfate-reducing bacteria Assuming that gene mapping is similar among sulfate-reducing bacteria, we designed two primers according to the conserved regions of the amino acid sequence of the ABC transporter gene from D vulgaris (Hildenborough), and amplified part of the ABC transporter gene using PCR with DvMF genomic DNA as a template The PCR primer sequences were as follows: ABC01, 5¢-TGGATAGCCGCCAAGATGGG GTT-3¢ (23-mer corresponding to the amino acid sequence 58 W–I–A–A–K–M–G–F); and ABC02, 5¢-GCGAGCGG GGCCGAATCGTAGAA-3¢ (23-mer complementary sequence to the corresponding amino acid sequence 163 F–Y–D–S–A–P–L–A) The PCR products were separated by agarose gel electrophoresis and a fragment of  340 bp was extracted using the MinElute Gel Extraction Kit (Qiagen, Venlo, Netherlands) The nucleotide sequence of this fragment revealed a putative amino acid sequence that was similar to the amino acid sequence of another ABC transporter from sulfate-reducing bacteria We then synthesized five primers, which were used to determine the sequence upstream of the ABC transporter gene using genomic DNA as the template The primer sequences were as follows: ABC04, 5¢-CCAGCTTCACCTTGCCCTTC-3¢ (20-mer); ABC05, 5¢-CTTGTCCACGTAGGCGAAGG-3¢ (20-mer); Flr05, 5¢-TCTCGTGGGCACATACGACC-3¢ (20-mer); Flr06, 5¢-TCAACAAGGTGGATCCGGTG-3¢ (20-mer); and Flr07, FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ê 2009 FEBS N Shibata et al 5Â-ACGTGAAGGTGGACGAATCC-3Â (20-mer) We identified the flavoredoxin gene in the complementary strand upstream of the ABC transporter, and then designed a 30-mer probe DNA (5¢-TCGGAGGTACCGCGCTGCAC GCCCAGCTTC-3¢), which is a complementary sequence corresponding the amino acid sequence 133V–K–L–G–V–Q– R–G–T–S–E We carried out Southern hybridization with this labeled oligonucleotide at 65 °C and detected a band that hybridized to an  4.3-kb PstI–KpnI fragment using a Bioimaging Analyzer (BAS1000; Fuji, Tokyo, Japan) Then, we digested the genomic DNA with PstI and KpnI, and fractionated the products on an agarose gel The separated fragments were ligated into the corresponding sites in pUC18, and the resultant ligation mixture was transformed into E coli JM109 One such transformant, isolated by the colony hybridization method, was found to harbor a plasmid that carried the  4.3-kbp PstI–KpnI fragment of DvMF DNA, and was designated pABCFLR The nucleotide sequence of the inserted fragment was determined by sequencing the restriction fragment that was cloned into pUC18 using the dideoxy chain termination method [30] with a DNA sequencer Expression and purification of recombinant flavoredoxin For high-level expression in E coli, we used pMK2 [31], which is an expression vector that carries the tac promoter The coding region of the flavoredoxin gene was amplified by PCR with KOD-Plus- DNA polymerase (Toyobo) DvMF genomic DNA was used as the PCR template The PCR primer sequences were as follows: flr09, 5¢CGACCCGGGTCATGAAGAAATCCCTGG-3¢ (27-mer); and flr10, 5¢-TTTGTCGACTGATCAGGAGCGCAGGC C-3¢ (27-mer) PCR was carried out at 94 °C for min, followed by 35 cycles of 94 °C for 15 s, 55 °C for 30 s, and 68 °C for The PCR products were digested with SmaI and SalI and ligated into similarly digested pUC18 The cloned fragment was confirmed by sequencing, digested with SmaI and HindIII, and then ligated into pMK2, which was initially digested with EcoRI, blunt-ended with the Klenow fragment and then digested with HindIII to give the expression vector pMKFLR9-10 E coli was transformed with pMKFLR9-10, and transformants were grown in 1.7 mL LB medium containing 100 lgỈmL)1 ampicillin for h at 37 °C Twelve flasks containing 167 mL of the same medium were inoculated with 1.7 mL culture and incubated overnight with shaking at 37 °C Cells were harvested by centrifugation at 4000 g for 10 at °C The cell pellet was resuspended in 10 mm Tris ⁄ HCl buffer (pH 8.0), sonicated using a Model 201M sonicator (Kubota, Tokyo, Japan) at 9000 Hz and 200 W for 10 min, then ultracentrifuged at 100 000 g for h at °C The supernatant was then dialyzed against distilled water overnight at °C Structure of flavoredoxin For flavoredoxin purification, the dialysate was loaded onto a DEAE-cellulose column (DE52, 2.2 · 15 cm) equilibrated with 10 mm Tris ⁄ HCl (pH 8.0) The column was washed with 150 mL of 100 mm NaCl and 10 mm Tris ⁄ HCl (pH 8.0) Flavoredoxin was eluted with 200 mL of 300 mm NaCl and 10 mm Tris ⁄ HCl (pH 8.0) The colored eluent was dialyzed against distilled water overnight at °C, and then reloaded onto a DE52 column equilibrated with 10 mm Tris ⁄ HCl (pH 8.0) Flavoredoxin was eluted with a linear gradient of 100–300 mm NaCl in 10 mm Tris ⁄ HCl (pH 8.0) in a total volume of 300 mL The flavoredoxincontaining fractions were identified based on absorbance at 448 nm The colored fractions were collected and dialyzed against distilled water, and then lyophilized or concentrated using Vivaspin (MW 5000 cut-off; Sartorius AG, Gottină gen, Germany) Gel filtration on a Superdex 75 HR10 ⁄ 30 column was carried out using the Pharmacia FPLC system (Uppsala, Sweden) in 200 mm NaCl, 10 mm Tris ⁄ HCl (pH 8.0) at a flow rate of 0.5 mLỈmin)1, and the purified recombinant flavoredoxin was eluted after 23 SDS ⁄ PAGE (15%) was carried out according to the method of Laemmli [32] For amino acid sequence analysis, further purification was performed The purified recombinant flavoredoxin was loaded onto a TSK-GEL TMS-250 (Tosoh, Tokyo, Japan) The column was washed with 0.1% trifluoroacetic acid, and then developed with a gradient of 0–80% acetonitrile in 0.1% trifluoroacetic acid The flow rate was 0.8 mLỈmin)1 Purified apo-flavoredoxin was separated by SDS ⁄ PAGE and electroblotted For deformylation, a sample was treated with 0.6 m HCl overnight at room temperature The total protein concentration was determined using the BCA protein assay kit (Thermo Scientific Pierce, Waltham, MA, USA) or calculated from the absorbance value at 448 nm Identification of the prosthetic group To identify the prosthetic group, purified recombinant flavoredoxin and a mixture of riboflavin, FAD and FMN were loaded onto an HPLC C8 column (NUCLEOSIL 10 C8; Shinwa Chemical Industries Ltd., Kyoto, Japan) The column was washed with 0.1% trifluoroacetic acid, and then developed with a gradient of 0–20% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.8 mLỈmin)1 Crystallization, data collection and processing The details of crystallization and data collection have been reported previously [33] The purified protein solution was concentrated to 25 mgỈmL)1 by centrifugation using a Vivaspin (Mr 5000 cut-off; Sartorius) Flavoredoxin was crystallized using the sitting-drop vapor diffusion method Protein droplets were prepared by mixing lL protein solution with lL reservoir solution and equilibrated against 100 lL reservoir solution containing 10% (w ⁄ v) poly(ethylene gycol)8000, 0.2 m zinc acetate and 100 mm Mes (pH FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4849 Structure of flavoredoxin N Shibata et al 6.0), at 283 K A methylmercuric chloride derivative crystal was prepared by soaking the crystals for 24 h in a reservoir solution that contained mm methylmercuric chloride Before data collection, the crystals were passed quickly through a cryoprotectant solution that contained 30% (v ⁄ v) glycerol in addition to well solution components The crystals were flash-cooled to 100 K in a stream of nitrogen ˚ gas The native dataset extending to 1.05 A resolution and ˚ the methylmercuric chloride derivative extending to 1.7 A resolution were collected at 100 K on an ADSC Q315 charge-coupled device detector at the BL41XU beamline and on a DIP2040 image-plate detector at the BL44XU beamline (SPring-8, Hyogo, Japan) Data processing, scaling, and reduction were performed using the HKL-2000 system [34] Structure determination and refinement The structure was solved by single-wavelength anomalous diffraction using the methylmercuric chloride derivative Two mercury sites, located by the program snb [35], were used for phasing with sharp [36] The initial model was built automatically with arp ⁄ warp [37], followed by manual building with xfit [38] for the unbuilt regions The initial crystallographic refinement was performed using cns [39] The initial model was first subjected to cycles of energy minimization and individual B-factor refinement After the refinement step, a SIGMAA-weighted 2Fo–Fc map was calculated and used for manual model rebuilding of the molecule Next, water molecules were added using the waterpick protocol of cns After several cycles of refinement, the structure was further refined using shelxl [40] In the later stages, anisotropic B-factor refinement was performed and several double conformations were applied In the final stage of refinement, protons were included in the riding-on mode Standard restraints were applied throughout the refinement Most parts of the electron- density maps were well-defined, although residues 128–130 and 187–190 could not be built because of poor electron densities in these regions The Ramachandran plot indicated that all residues were in the most-favored and allowed regions, except for Val167 (Table 1) located at the Mes-binding site, which was well-defined by the electron- density map Structural comparison and analysis Structures were compared using the DALI server [9] Root mean square deviation and rotation-translation matrices for superimposing structures were calculated with ssm [41] Sol˚ vent-accessible surface areas were calculated with a 1.4 A probe using cns [39] Electrostatic potentials were calculated with the delphi program [42], which uses finite difference methods to solve the linearized Poisson–Boltzmann equation Interior and exterior dielectric constants of and ˚ 80, respectively, and a probe radius of 1.4 A were used 4850 The atomic charge parameters for the molecules were taken from the default library The model figures were generated with molscript [43] ⁄ raster3d [43,44] for Figs and and with chimera [45] for Fig S4 Determination of oxidation ⁄ reduction potential The redox potential for oxidized ⁄ reduced flavoredoxin (Eflr) was determined by means of equilibrium reactions and spectrophotometric measurements [46] with mixtures of flavoredoxin and a mediator, which was Neutral Red or benzyl viologen The redox potential, Eh, for the system at equilibrium was calculated with the Nernst equation, Eh ¼ Em;7 dyeị ỵ RT ẵoxidized dye ln nF ẵreduced dye where R denotes the gas constant, T is the absolute temperature, F is the Faraday constant and n is the number of electrochemical equivalents A solution of flavoredoxin and a dye in 100 mm potassium phosphate buffer (pH 7) in a closed all-glass cuvette was made anaerobic by repeated cycles of evacuation and flushing with oxygen-free argon When Neutral Red was used as the mediator, absorbances at 452 and 540 nm were monitored Neutral Red in the reduced state and flavoredoxin not absorb at 540 nm, and flavoredoxin in the fully reduced state does not absorb at 452 nm When benzyl viologen was used as the mediator, absorbances at 452 and 400 nm were monitored Benzyl viologen in the oxidized state does not absorb at either 452 or 400 nm To determine Eflr, sodium dithionite solution was added to the dye along with the flavoredoxin in the oxidized state Redox potentials were calculated by linear regression analysis of the logarithms of the concentration ratios for oxidized and reduced forms of the mediator versus that of recombinant flavoredoxin Reduction titration of flavoredoxin by NAD(P)H The reduction of flavoredoxin by NAD(P)H was observed at 25 °C by spectral change in the visible region, using a U-3000IR spectrophotometer (Hitachi, Tokyo, Japan) A solution of flavoredoxin in 50 mm Tris ⁄ HCl (pH 7.5) in a closed all-glass cuvette was made anaerobic by repeated cycles of evacuation and flushing with oxygen-free argon For the observation of flavoredoxin reduction, mm NADH in 50 mm Tris ⁄ HCl (pH 7.5) was added to flavoredoxin in the oxidized state Measurements of ferric reductase and flavin reductase activities FeR and Fre activity were examined using flavoredoxin, DrgA [13] and FMN-binding protein [14] DrgA from Synechocystis sp PCC6803 and FMN-binding protein from FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al DvMF were prepared according to the methods of Takeda et al [13] and Kitamura et al [14], respectively On the basis of structural homologies, both flavoredoxin and FMN-binding protein are considered to have FeR motifs, and DrgA was used as a positive control Both activities were measured under aerobic conditions, because purified flavoredoxin and FMN-binding protein not react with oxygen The assay was performed using NADPH as a substrate To measure FeR activity, the reaction was initiated by the addition of 100 lm Fe3+–EDTA solution to 41 lm flavoredoxin or 97 lm FMN-binding protein or 0.44 lm DrgA and 150 lm of NADPH in 50 mm sodium phosphate buffer (pH 7.0) in the presence or absence of 15 lm FMN in quartz cuvettes The total volume was mL at 30 °C The reaction was monitored at 340 nm in a spectrophotometer [47] Activity was determined by measuring the difference in NADPH consumption at 340 nm in the presence and absence of Fe3+–EDTA The absorbance coefficient used for NADPH was 6.20 mm)1Ỉcm)1 Flavin reductase activity was measured using the same reaction mixture as for FeR, but using a final concentration of 100 mm FMN solution and omitting Fe3+–EDTA One unit of activity is defined as lmol NADPH oxidized per minute References Broco M, Soares CM, Oliveira S, Mayhew SG & Rodrigues-Pousada C (2007) Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin FEBS Lett 581, 4397–4402 Agostinho M, Oliveira S, Broco M, Liu MY, LeGall J & Rodrigues-Pousada C (2000) Molecular cloning of the gene encoding flavoredoxin A flavoprotein from Desulfovibrio gigas Biochem Biophys Res Commun 272, 653–656 Broco M, Rousset M, Oliveira S & Rodrigues-Pousada C (2005) Deletion of flavoredoxin gene in Desulfovibrio gigas reveals its participation in thiosulfate reduction FEBS Lett 579, 4803–4807 Suharti S, Murakami KS, de Vries S & Ferry JG (2008) Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans Biochemistry 47, 11528–11535 Vadas A, Monbouquette HG, Johnson E & Schroder I (1999) Identification and characterization of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus J Biol Chem 274, 36715–36721 Chiu HJ, Johnson E, Schroder I & Rees DC (2001) Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+ Structure 9, 311–319 Suto K, Kawagoe K, Shibata N, Morimoto Y, Higuchi Y, Kitamura M, Nakaya T & Yasuoka N (2000) How the X-ray structure and the NMR structure of Structure of flavoredoxin 10 11 12 13 14 15 16 17 18 19 FMN-binding protein differ? Acta Crystallogr D Biol Crystallogr 56, 368–371 Fontecave M, Coves J & Pierre JL (1994) Ferric reductases or flavin reductases? Biometals 7, 3–8 Holm L & Sander C (1993) Protein structure comparison by alignment of distance matrices J Mol Biol 233, 123–138 Bruns CM & Karplus PA (1995) Refined crystal struc˚ ture of spinach ferredoxin reductase at 1.7 A resolution: oxidized reduced and 2¢-phospho-5¢-AMP bound states J Mol Biol 247, 125–145 Park DH, Laivenieks M, Guettler MV, Jain MK & Zeikus JG (1999) Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production Appl Environ Microbiol 65, 2912–2917 Blaut M, Whittaker K, Valdovinos A, Ackrell BA, Gunsalus RP & Cecchini G (1989) Fumarate reductase mutants of Escherichia coli that lack covalently bound flavin J Biol Chem 264, 13599–13604 Takeda K, Iizuka M, Watanabe T, Nakagawa J, Kawasaki S & Niimura Y (2007) Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction FEBS J 274, 1318–1327 Kitamura M, Kojima S, Ogasawara K, Nakaya T, Sagara T, Niki K, Miura K, Akutsu H & Kumagai I (1994) Novel FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) Cloning and expression of its gene in Escherichia coli J Biol Chem 269, 5566– 5573 Okai M, Kudo N, Lee WC, Kamo M, Nagata K & Tanokura M (2006) Crystal structures of the shortchain flavin reductase HpaC from Sulfolobus tokodaii strain in its three states: NAD(P)(+)(-)free NAD(+)(-)bound, and NADP(+)(-)bound Biochemistry 45, 5103–5110 Galan B, Diaz E, Prieto MA & Garcia JL (2000) Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new flavin:NAD(P)H reductase subfamily J Bacteriol 182, 627–636 Uetz T, Schneider R, Snozzi M & Egli T (1992) Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from ‘Chelatobacter’ strain ATCC 29600 J Bacteriol 174, 1179–1188 Parry RJ & Li W (1997) An NADPH:FAD oxidoreductase from the valanimycin producer Streptomyces viridifaciens Cloning, analysis, and overexpression J Biol Chem 272, 23303–23311 Filisetti L, Fontecave M & Niviere V (2003) Mechanism and substrate specificity of the flavin reductase ActVB from Streptomyces coelicolor J Biol Chem 278, 296– 303 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4851 Structure of flavoredoxin N Shibata et al 20 Witschel M, Nagel S & Egli T (1997) Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103 J Bacteriol 179, 6937–6943 21 Kirchner U, Westphal AH, Muller R & van Berkel WJ (2003) Phenol hydroxylase from Bacillus thermoglucosidasius A7 A two-protein component monooxygenase with a dual role for FAD J Biol Chem 278, 47545– 47553 22 Thibaut D, Ratet N, Bisch D, Faucher D, Debussche L & Blanche F (1995) Purification of the two-enzyme system catalyzing the oxidation of the d-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis J Bacteriol 177, 5199–5205 23 Kendrew SG, Harding SE, Hopwood DA & Marsh EN (1995) Identification of a flavin:NADH oxidoreductase involved in the biosynthesis of actinorhodin Purification and characterization of the recombinant enzyme J Biol Chem 270, 17339–17343 24 Bryson K, McGuffin LJ, Marsden RL, Ward JJ, Sodhi JS & Jones DT (2005) Protein structure prediction servers at University College London Nucleic Acids Res 33, W36–W38 25 Huyer M & Page WJ (1989) Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+ J Bacteriol 171, 4031–4037 26 Poch MT & Johnson W (1993) Ferric reductases of Legionella pneumophila Biometals 6, 107–114 27 Karlsson A, Beharry ZM, Matthew Eby D, Coulter ED, Neidle EL, Kurtz DM Jr, Eklund H & Ramaswamy S (2002) X-Ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp strain ADP1 J Mol Biol 318, 261–272 28 Postgate JR (1984) The Sulphate-reducing Bacteria, 2nd edn, pp 30–50 Cambridge University Press, Cambridge, UK 29 Saito H & Miura KI (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment Biochim Biophys Acta 72, 619–629 30 Sanger F, Nicklen S & Coulson AR (1992) DNA sequencing with chain-terminating inhibitors Biotechnology 24, 104–108 31 Hibino T, Misawa S, Wakiyama M, Maeda S, Yazaki K, Kumagai I, Ooi T & Miura K (1994) High-level expression of porcine muscle adenylate kinase in Escherichia coli: effects of the copy number of the gene and the translational initiation signals J Biotechnol 32, 139–148 32 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 33 Ueda Y, Shibata N, Takeuchi D, Kitamura M & Higuchi Y (2008) Crystallization and preliminary X-ray crystallographic study of flavoredoxin from Desulfovibrio vulgaris Miyazaki F Acta Crystallogr Sect F Struct Biol Cryst Commun 64, 851–853 4852 34 Otwinowski Z & Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326 35 Smith GD, Nagar B, Rini JM, Hauptman HA & Blessing RH (1998) The use of SnB to determine an anomalous scattering substructure Acta Crystallogr D Biol Crystallogr 54, 799–804 36 de La Fortelle E & Bricogne G (1997) Maximum-likelihood heavy-atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods Methods Enzymol 276, 472–494 37 Morris RJ, Perrakis A & Lamzin VS (2003) ARP ⁄ wARP and automatic interpretation of protein electrondensity maps Methods Enzymol 374, 229–244 38 Duncan EM (1993) Practical Protein Crystallography Academic Press, San Diego, CA 39 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al (1998) Crystallography and NMR system: a new software suite for macromolecular structure determination Acta Crystallogr D Biol Crystallogr 54, 905–921 40 Sheldrick GM & Schneider TR (1997) SHELXL: high resolution refinement Methods Enzymol 277, 319–343 41 Krissinel E & Henrick K (2004) Secondary-structure matching (SSM) A new tool for fast protein structure alignment in three dimensions Acta Crystallogr D Biol Crystallogr 60, 2256–2268 42 Nicholls A & Honig B (1991) A rapid finite difference alogrithm, utililizing successive over-relaxation to solve the Poisson–Boltzman equation J Comput Chem 12, 435–445 43 Kraulis J (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallogr 24, 946–950 44 Merritt EA & Bacon DJ (1997) Raster3D: photorealistic molecular graphics Methods Enzymol 277, 505–524 45 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC & Ferrin TE (2004) UCSF Chimera – a visualization system for exploratory research and analysis J Comput Chem 25, 1605–1612 46 Curley GP, Carr MC, Mayhew SG & Voordouw G (1991) Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough) Eur J Biochem 202, 1091–1100 47 Niviere V, Fieschi F, Decout JL & Fontecave M (1996) Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin–NADP+ reductase family? Evidence for the catalytic role of serine 49 residue J Biol Chem 271, 16656–16661 48 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al (2007) Clustal W and Clustal X version 2.0 Bioinformatics 23, 2947–2948 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS N Shibata et al Supporting information The following supplementary material is available: Fig S1 SDS ⁄ PAGE of purified recombinant flavoredoxin Fig S2 Ultraviolet and visible-light spectra of purified flavoredoxin expressed in Escherichia coli transformed with pMKFLR9-10 Fig S3 Identification of the prosthetic group by HPLC Fig S4 Superimposed models of the vicinity of FMN binding sites of DvMF and Methanosarcina acetivorans flavoredoxins as in Fig Structure of flavoredoxin Fig S5 Surface charge models viewed from the same perspective as in Fig This supplementary material can be found in the online article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4853 ... in DvMF flavoredoxin (Fig 3A,B and Fig S4A), respectively In the case of FeR, Ser84 replaces Asn29 of DvMF flavoredoxin, which forms a hydrogen bond to the O3P atom of FMN (Fig 3A,B) DvMF flavoredoxin. .. isoalloxazine ring of FeR is completely exposed to the solvent (Fig S5B) Resolution of the structure of NADP+-bound FeR revealed that the nicotinamide moiety of NADP+ faces the re-side of the isoalloxazine... potential of recombinant flavoredoxin Figure shows the results of linear regression analysis of the logarithms for the redox ratio of the mediator versus that of recombinant flavoredoxin The redox