doi:10.1016/j.jmb.2010.06.024 J Mol Biol (2010) 401, 403–414 Available online at www.sciencedirect.com Asymmetric Dimeric Structure of Ferredoxin-NAD(P) + Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP + Norifumi Muraki 1,2 †, Daisuke Seo ⁎†, Tomoo Shiba , Takeshi Sakurai and Genji Kurisu 2,4 ⁎ Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received 26 December 2009; received in revised form June 2010; accepted 11 June 2010 Available online 18 June 2010 Edited by M Guss Ferredoxin-NAD(P)+ oxidoreductase (FNR) catalyzes the reduction of NAD(P)+ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to planttype FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR) Here, we report the crystal structure of C tepidum FNR to 2.4 Å resolution, which reveals a unique structure– function relationship C tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C tepidum FNR © 2010 Elsevier Ltd All rights reserved Keywords: ferredoxin-NAD(P)+ reductase; photosynthesis; electron transfer complex; X-ray crystallography; thioredoxin reductase *Corresponding authors E-mail addresses: dseo@cacheibm.s.kanazawa-u.ac.jp; gkurisu@protein.osaka-u.ac.jp † N.M and D.S contributed equally to this work Present address: T Shiba, Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Abbreviations used: FNR, ferredoxin-NAD(P)+ reductase; Fd, ferredoxin; GR, glutathione reductase; AdR, adrenodoxin reductase; ONFR, oxygenase-coupled NADH-ferredoxin reductase; RC, photoreaction center; TrxR, thioredoxin reductase; FO, flavin-oxidizing; FR, flavin-reducing; WT, wild type Introduction Ferredoxin-NAD(P)+ reductase (FNR, EC 1.18.1.2) is a ubiquitous ferredoxin (Fd)-dependent enzyme containing flavin adenine dinucleotide (FAD) as a prosthetic group, which generally catalyzes the reversible redox reaction between Fd and NAD(P)+/ NAD(P)H On the basis of phylogenic and structural information, FNRs are classified into two distinct families; plant-type and glutathione reductase (GR)type.1 FNR, which is representative of the plant type, is a plastidic-type localized in chloroplast2–4 or non- 0022-2836/$ - see front matter © 2010 Elsevier Ltd All rights reserved 404 photosynthetic tissues5 that is well characterized physiologically and biochemically High-resolution structures of several plant-type FNRs have shown that the FAD binding and NADPH binding domains are arranged sequentially on the primary structure In contrast to plant-type FNR, GR-type FNRs have much wider physiological functions, providing electrons for a broad range of metabolic processes as diverse as steroid hydroxylation in mitochondria, as well as methane oxidation and reductive activation of biosynthetic enzymes However, the structural reports of GR-type FNRs are limited to GR,6 AdR,7,8 Mycobacterium tuberculosis FprA,9 Pseudomonas sp BphA410 and putidaredoxin reductase.11 All of these proteins display essentially the same fold as that of GR Consideration of the phylogenic and structural aspects, GR-type FNRs have been assigned to two subdivisions; mitochondrial adrenodoxin reductase (AdR)-like and oxygenasecoupled NADH-ferredoxin reductases (ONFRs)-like FNRs.1 The green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum) is a moderate thermophilic anaerobe The bacterium carries out nonoxygenic photosynthesis with sulfur compounds as an electron donor Photosynthesis apparatus of the bacterium includes a type I photoreaction center (RC) homologous to photosystem I of the chloroplast, which converts light energy to redox chemistry.12 The RC in C tepidum photoreduces a Fd directly,13 and subsequently FNR catalyzes the reduction of NADP+ to NADPH with the reduced Fds Despite the physiological similarity, the amino acid sequence of C tepidum FNR is phylogenetically unrelated to those of their chloroplast counterpart C tepidum FNR exists in a homodimeric form that displays significant homology with NADPH-dependent thioredoxin reductase (TrxR) rather than conventional FNRs, and is reactive to NADPH and NADH.14 Thus, FNR from C tepidum is distinct from plant-type FNR On the basis of amino acid sequence comparisons, C tepidum FNR might be classified as a GR-type FNR However, it cannot easily be assigned to any of the subdivisions AdR-like and ONFRs-like FNRs possess extra helices or domains Recently, three homologues from Bacillus subtilis (YumC),15 Rhodopseudomonas palustris16 and Thermus thermophilus HB2717 have been purified in both native and recombinant forms All of these proteins conserve the homodimeric form and a high level of reactivity with Fd These FNRs, including C tepidum FNR, should be classified into the third subdivision of TrxR-like FNRs, as proposed by Mandai et al.17 TrxR is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin, having two cysteine residues as the redox active site, which plays several key roles in maintaining the redox environment of the cell There are two distinct types of TrxRs; Low-Mr TrxRs (Mr ∼ 35,000) typically found in prokaryotes, archaea, plants and lower eukaryotes, and High-Mr T r x R s ( M r ∼ 5, 000 ) o bserv e d i n higher Structure of Ferredoxin-NAD(P)+ Oxidoreductase eukaryotes.18 TrxR-like FNR shows high amino acid sequence homology to low-Mr TrxRs Escherichia coli TrxR, a representative of the Low-Mr TrxR, consists of two functional domains, one for FAD binding and one for NADPH binding The NADPH domain possesses the redox-active residues, Cys135 and Cys138, required for thioredoxin reduction E coli TrxR was crystallized in two conformations; one is the flavin-oxidizing (FO) form,19,20 and the other is the flavin-reducing (FR) form complexed with thioredoxin.21 In the FO form, the redox active disulfide adjoins the re-face of the flavin in an orientation that allows reduction of the disulfide with concomitant oxidation of the flavin However, NADPH binds far from the flavin ring and the active-site cysteines are buried where they cannot react with thioredoxin In the FR form structure complexed with thioredoxin, the NADPH domain rotates by 67° and moves toward the FAD domain by 1.4 Å A large rotation of the NADPH domain would move the active cysteine residues to the protein surface where it reacts with thioredoxin through the disulfide bond connection This rotational movement from the FO and FR conformation positions the pyridine ring of NADP+ analogue parallel with the flavin ring This kind of large conformational change has not been found in planttype or GR-type FNRs In this study, in order to understand the structural basis of the enzymatic reaction of newly recognized TrxR-like FNRs, we have examined the crystal structure of C tepidum FNR by X-ray crystallography with a view to determining its 3D structure.22 Our data reveal that C tepidum FNR retains its structural topology with TrxR but possesses several unique structural features Furthermore, mutational studies of the FAD-stacking Phe residue and flexible hinge region between the two functional domains suggest the structural basis for substrate binding Results The crystal structure of C tepidum FNR was determined by the single wavelength anomalous dispersion method using Se-Met-substituted recombinant protein and the wild type (WT) structure was refined to a resolution of 2.4 Å (Table 1) The C tepidum FNR is a dimer of two identical polypeptides, labeled A and B in Fig 1, that exist in the crystallographic asymmetric unit with disordered regions at the N- and C-termini of each chain (i.e residues – 12 of chain A and B, 349 – 360 of chain A and 331 – 360 of chain B) Each polypeptide chain is composed of two distinct functional domains; an FAD binding and an NAD(P)H binding domain An FAD molecule is non-covalently bound to each FAD domain at the carboxyl ends of the central parallel βsheet The NAD(P)H domain has no bound NAD(P)+ molecule in the crystal structure, but has essentially the same fold as that of E coli TrxR with a bound NADP+ molecule (PDB accession codes 1TDF and 1F6M).20,21 Therefore, despite the absence of a bound Structure of Ferredoxin-NAD(P)+ Oxidoreductase 405 Table Crystallographic data and refinement statistics A Crystallographic data X-ray source Space group Unit-cell parameters a (Å) b (Å) c (Å) Wavelength (Å) Resolution range (Å) Total reflections Unique reflections Completeness (%) Rmerge(I)a (%) I/δ No sites Phasing power FOM acentric FOM centric B Refinement statistics Resolution (Å) Rworkb (%) Rfreec (%) No atoms Protein Ligand/ion Water molecules RMSD from ideal Bond lengths (Å) Bond angless (°) Ramachandran plot Most favored (%) Additionally allowed (%) Generously allowed (%) Native Sepeak AR-NW12 C2221 AR-NW12 C2221 100.5 128.0 128.4 0.98306 39.57–2.40 (2.49–2.40) 242,503 32,978 100 (100) 8.0 (48.5) 12.0 (3.92) – - 101.12 128.49 128.41 0.97923 33.79–2.80 (2.90–2.80) 151,863 20,924 99.9 (99.9) 8.4 (53.9) 12.9 (2.6) 11 0.808 0.28529 0.08308 39.56–2.40 24.0 28.9 5013 106 -97 0.008 1.170 90.1 9.9 Values in parentheses are for the highest resolution shell a Rmerge(I) = Σǀ I (k) – bINǀ / Σ I (k), where I (k) is the value of the kth measurement of the intensity of a reflection, bIN is the mean value of the intensity of that reflection and the summation is the overall measurement b R-factor = ΣǀǀFobs(hkl)ǀ – ǀFcalc(hkl) ǀǀ / ΣǀFobs(hkl)ǀ c Rfree is the R-factor computed for a test set of reflections that were omitted from the refinement process NAD(P)+ molecule, we have denoted the corresponding region of the molecule as the NAD(P)H domain in the C tepidum FNR structure The chain topologies of the two functional domains resemble each other, having an α+β structure with a topological core of a Rossmann fold The FAD domain is composed of eight βstrands and five α-helices, and the NAD(P)H domain is composed of nine β-strands and two αhelices (Fig 2) The FAD domain consists of two discontinuous segments of the polypeptide (residues 13 – 131 and 262 – 329) The NAD(P)H domain is inserted between two segments of the FAD domain through a hinge region, which forms a short anti-parallel β-sheet The continuous loop region is long enough to prevent direct interaction between the two functional domains When the FAD domain of one protomer is superimposed on that of the other, one NAD(P)H domain is rotated by 39.8° and translated by 1.1 Å with respect to the other (Fig 3) Domain motion analysis23 suggests that this rotational and translational shift is a result of a distortion of the backbone at the hinge region, comprising residues 129 – 133 and 259 – 268 One significant structural feature of C tepidum FNR is the presence of a C-terminal sub-domain (residues 330 – 347) with a connecting long loop (Figs and 2) The sub-domain was seen only in chain A at the end of the FAD domain Although there is enough space for the sub-domain of chain B in the crystal lattice, no corresponding electron density was found probably due to a mobile character An amino acid sequence alignment indicated that this unique structure is conserved only in TrxR-like FNRs (Fig 2) Homodimeric C tepidum FNR is held together primarily by interactions between the two FAD domains Extensive hydrophobic interactions and 13 direct hydrogen bonds exist between the two chains Due to the structural asymmetry caused by the Cterminal sub-domain, interacting residues at the dimer interface are different in the two protomers, as illustrated in Fig Most of these residues are conserved among TrxR-like FNRs, but not in E coli TrxR A total of 10 water molecules (Wat A415, A426, A427, A439, A457, B403, B441, B446, B458 and B486) mediate the non-direct interactions between Fig Stereo view of the crystal structure of C tepidum FNR The chains are depicted by different colors All figures were generated using PyMOL (http://pymol.sourceforge.net/) 406 Structure of Ferredoxin-NAD(P)+ Oxidoreductase Fig Sequence comparison between C tepidum FNR, R paustris FNR and E coli TrxR The secondary structures of C tepidum FNR and E coli TrxR are shown above the sequence with rods (α-helices) and arrows (β-strands) The color code for each chain is the same as that in Fig 1a Residues in the FAD domain are colored gray and residues in the NAD(P)H domain are colored brown Residues at the interface between the two FAD domains are highlighted with colored background Residues highlighted in red are participating in dimerization from both chains, residues in green are from chain A and residues in blue are from chain B The active site cysteine residues in E coli TrxR are shown as red letters the two FAD domains (data not shown), suggesting that the main chain fold has the leading role in fixing the relative orientation of two FAD domains almost identical with those of TrxR One more remarkable interaction occurs between the C-terminal sub-domain in chain A and the FAD molecule in chain B The isoalloxazine ring of FAD is held by strong π-π stacking with Phe337 and Fig Comparison of the hinge-bending motion of C tepidum FNR and E coli TrxR Each gray allow shows the rotation axis from the left-hand panel to the right-hand panel obtained from domain motion analysis.23 Structure of Ferredoxin-NAD(P)+ Oxidoreductase hydrogen bonds from residues Ser338 and Ser339 (Fig 4) The two stacking ring systems are positioned almost in parallel with a separation of 3.5 Å Interestingly, among TrxR-like FNRs, an aromatic residue is conserved at this site followed by two aliphatic hydroxyl-containing residues (Ser or Thr) This could be a common structural feature of TrxRFNRs The total contact area of the dimeric C tepidum FNR is 8811.3 Å2 (cf 7274.8 Å2 for E coli TrxR) The increase in total contact area of C tepidum FNR is obviously due to the presence of the Cterminal sub-domain Consequently, the contributions of the two chains of C tepidum FNR in forming the total contact area (52 % for chain A and 48 % for chain B) are slightly different, whereas the contribution of the two identical TrxR protomers is almost equal The overall FAD molecule is bound in an extended conformation to the cleft formed by the two functional domains (Figs and 4) The re-face of the isoalloxazine ring of chain A is relatively exposed to solvent, and that of chain B is covered with the FAD-stacking sub-domain By contrast, the re-face of the ring in E coli TrxR is always covered by the NAD(P)H domain and never exposed to solvent.20,21 At the si-face of the isoalloxazine ring, N3 and O2 form hydrogen bonds with residues Asp64 and Ile309, which is found also in the E coli TrxR structure A strong π-π stacking between the phenol ring of Tyr57 and the isoalloxazine ring of FAD is present in C tepidum FNR (Fig 4) with the two ring systems in an almost parallel orientation In E coli TrxR, the corresponding Thr47 residue does not interact with the FAD molecules The Tyr57 of 407 FNR also provides an additional hydrogen bond to the ribitol O2 In common with Asp286 of TrxR, Asp298 of C tepidum FNR interacts with the ribitol moiety The pyrophosphate and adenine ribose moieties interact with residues Ile20, Gly21, Thr25, Ser45, Gly51, Gln52, Thr96, Val97, Ala126, Ala127, Gly130, Phe132 and Asp298 (Fig 4) In chain A, Phe132 provides an additional hydrogen bond to the AO2 oxygen atom of the ribose moiety (data not shown) In E coli TrxR, Glu38 provides a hydrogen bond to the same oxygen atom of the ribose moiety Gly130 and Phe132 in C tepidum FNR both interact with the adenine ribose moiety, but the corresponding residues in E coli TrxR not interact with the FAD molecules Another residue interaction different from that of E coli TrxR is Thr25, which corresponds to Ala16 in TrxR As described above, each C tepidum FNR protomer consists of two functional domains whose architecture is essentially identical with those of E coli TrxR except for the C-terminal sub-domain (Figs and 2) In contrast to the similarity in domain structures, the relative orientation of the two functional domains is quite different (Fig 3) Comparison of the two protomers in the crystallographic asymmetric unit implied that the NAD(P)H domain is able to rotate The hinge region of C tepidum FNR comprises anti-parallel β-strands Differences of dihedral angles of the hinge regions between the two protomers are given in Table First, we identified six residues (Ala131, Phe132, Glu133, Gly260, Asn264 and Gly266) showing a significant deviation in ϕ and φ angles, and a large displacement of Cα between each orientation Fig Close-up view of the FAD environments of C tepidum FNR The color codes are all the same as that in Fig Structure of Ferredoxin-NAD(P)+ Oxidoreductase 408 Table Differences of dihedral angles of the hinge regions C tepidum FNR a Gly128 Leu129 Gly130a Ala131 Phe132a Glu133 Pro134a Arg135 Lys136 Leu137 Ile259 Gly260a Phe261 Lys262 Ser263 Asn264 Leu265a Gly266a Pro267a Leu268 E coli TrxR Δϕ (°) Δφ (°) 5.56 –15.00 –40.58 –40.63 134.49 51.29 –4.96 –7.65 –3.90 –4.69 –7.41 –14.90 –33.91 32.59 –41.45 –25.99 12.39 59.77 –11.52 3.23 0.26 40.59 12.26 –89.69 –73.69 –6.28 1.24 7.05 16.96 –15.43 4.88 43.10 11.99 13.38 –38.03 64.97 20.46 –50.36 18.52 15.86 Δϕ (°) Δφ(°) Gly113 Ala114 Ser115 Ala116 23.27 4.70 41.09 –18.47 –42.75 –72.46 –16.27 4.07 Arg117 Tyr118 Leu119 Ile243 Gly244 His245 Ser246 Pro247 Asn248 Thr249 –3.96 7.14 –4.70 15.94 39.02 23.61 15.71 –6.73 0.15 –14.43 1.31 13.25 19.91 –40.38 –29.77 –19.58 –34.54 –9.08 –5.46 3.88 Ala250 Ile251 –0.36 0.75 12.76 –17.01 Differences between chains A and B in C tepidum FNR are shown on the left and differences between FO and FR conformations in E coli TrxR are shown on the right a Conserved residues in C tepidum FNR, T thermophilus FNR, B subtilis FNR and R palustris FNR Because there might be a common structural basis for Fd-dependence, we eliminated the non-conservative residues for potential mutational sites In order to investigate the importance of the flexible hinge enabling the NAD(P)H domains to rotate, we mutated the three conservative residues, Phe132, Gly260 and Gly266, to Pro and tested their effect on the catalytic activity The mutational effect on the reactivity with NADPH was assayed using ferricyanide as an artificial electron acceptor (Table 3) We reasoned that this hinge-bending motion might be involved also in Fd binding However, we were unable to measure reactivity with Fd because of the high cytochrome c reductase activity of C tepidum FNR Therefore, the effect was evaluated by measuring diaphorase activity The Gly260Pro variant showed drastically diminished reactivity with NADPH, while the Gly266Pro variant showed a considerable increase of Km value and decrease of kcat value for NADPH compared to the WT enzyme However, the Phe132Pro mutant did not display any drastic change in kinetic parameters (Table 3) Both Gly260 and Gly266 are present in the downstream β-strand of the anti-parallel β-sheet that comprises the hinge region connecting the two functional domains The upstream β-strand is three residues longer and the downstream is one residue longer than those of E coli TrxR (Fig 2, Table 3) A difference of hinge length between two homologous enzymes could explain this result Ala154 and Ser157 in the NAD(P)H-domain of C tepidum FNR, corresponding to the two active cysteine residues of TrxR, are far away from the isoalloxazine ring of FAD Instead of two cysteine residues in the FO structure of TrxR, the FADstacking Phe337 occupies the corresponding position in C tepidum FNR (Fig 4) This stacking geometry is rather similar to that of the C-terminal Tyr residue of plant-type FNR.4,24 In order to elucidate the functional role of this FAD-stacking aromatic residue in C tepidum FNR, we have introduced point mutations into Phe337, which has been done for pea25 and Anabaena FNRs.26 Comparisons of the UV-visible spectrum of the WT enzyme with those of the Phe337Ser, Phe337Tyr and Phe337His variants revealed that replacement of Phe337 in C tepidum FNR results in small changes in the absorption properties of the flavin prosthetic group (Fig 5a) The absorbance maxima of transition bands were shifted slightly by – nm toward the shorter wavelengths in all mutants These shifts presumably arise from alterations in the isoalloxazine ring environment upon replacement of Phe337 Compared to the plant-type FNRs, an equivalent substitution affected the enzymatic activity differently (Table 3) For Anabaena FNR, the replacement of the C-terminal Tyr by Ser led to a considerable decrease in kcat for the diaphorase activity,26 but the equivalent replacement of C tepidum FNR did not affect the kinetic parameters significantly Interactions of the different FNR variants with NADP+ were analyzed by difference spectroscopy (Fig 5b) Small but significant band shifts were found for the difference spectra of all Phe337 variants upon Table Steady-state kinetic parameters for diaphorase activity of wild type and mutants of C tepidum FNRs Diaphorase activity (NADPH) FNR form Vmax (s-1) Km (μM) Wild type Phe132Pro Gly260Pro Gly266Pro Phe337Ser Phe337His Phe337Tyr 303 ± 253 ± 1.2 4.5 ± 1.8a 122 ± 279 ± 277 ± 273 ± 51 ± 46.8 ± 0.9 a Observed rate at mM NADPH 660 ± 90 39 ± 1.4 25 ± 1.8 33 ± 1.8 NADPH-cyt c red (s-1) NADPH oxidase (s-1) ɛ (mM-1 cm-1) at λmax (nm) 6.1 ± 0.2 3.6 ± 0.3 0.09 ± 0.03 0.59 ± 0.04 7.5 ± 0.4 7.4 ± 0.3 7.3 ± 0.7 1.4 ± 0.16 3.5 ± 0.8 0.38 ± 0.02 1.5 ± 0.2 1.6 ± 0.2 1.9 ± 0.2 1.8 ± 0.2 10.0 / 467 10.2 / 458 9.8 / 469 9.5 / 467 10.6 / 459 10.9 / 460 10.3 / 459 Structure of Ferredoxin-NAD(P)+ Oxidoreductase Fig Spectroscopic properties of C tepidum FNR and its variants (a) Absorption spectra of WT (black), Phe337His (blue), Phe337Tyr (green) and Phe337Ser (red) variants of C tepidum FNR in the visible region (b) Difference spectra elicited by the binding of WT (black), Phe337His (blue), Phe337Tyr (green) and Phe337Ser (red) variants of C tepidum FNR (∼ 15 μM) with NADP+ (1 mM) addition of NADP+ (maxima at 498 nm and 501 nm, minima at 515 nm) Although the peak in the 500– 510 nm region in the difference spectra has been reported as an indication of stacking between the NADP+ nicotinamide ring and the isoalloxazine ring of FAD in the plant-type FNRs, we did not obtain any evidence of such stacking in the case of TrxRlike FNRs.26,27 Currently, we can conclude only that NADP+ binding modulates the local environment around the FAD molecule Discussion Our attempts to obtain crystals of C tepidum FNR complexed with NAD(P)+ by using a co-crystallization or soaking method were unsuccessful We therefore determined the NADP+-free structure of C tepidum FNR In the current crystal packing, the 409 size of water channels in the crystal lattice was too small for soaking NAD(P)+ molecules, and we found that the side chain of an aromatic residue, Phe337, occupied a plausible docking site for the nicotinamide ring of NAD(P)+ These results might explain why our attempts to obtain crystals of the productive complex with NAD(P)+ failed The FAD-stacking side chain of Phe337 is located in the re-face of the flavin where the conserved C-terminal tyrosine of plant-type FNR stacks In E coli TrxR, the re-face of the flavin is always occupied by the redox-active functional groups; the loop containing the redoxactive disulfide (Cys135–Cys138) in the FO state of TrxR or the pyridine ring of bound NADP+ analog in the FR state of TrxR In this study, site-directed mutagenesis of C tepidum FNR indicated that the FAD-stacking Phe337 interaction influenced the microenvironment of the isoalloxazine ring, but the enzymatic activity of Phe337 variants of C tepidum FNR did not exhibit drastic changes, as observed for the plant-type FNRs This is probably due to hydrogen bonds between the adjacent Ser338 and Ser339 in C tepidum FNR and the isoalloxazine ring (Fig 4), which help to maintain the interaction between the C-terminal sub-domain and FAD It is not clear why Phe337 neatly stacks the FAD, but the re-face of the flavin is functionally very important Therefore, we suggest the possible structural role of Phe337 in preventing unnecessary exposure of reduced FAD to the bulk solvent during the catalytic cycle It has been reported that the stacking aromatic residue has a different functional role in the case of GR28 and detailed studies of this homoplastic-like feature are required to understand its functional role in each NAD(P)H-dependent enzyme In the complex structure of E coli TrxR and NADP+ , the 2′-P-AMP half was bound to the enzyme in a well defined manner, while the electron density for the other NMN half was very poor,20 similar to that of plant-type FNRs.4,24 On the basis of the structural similarity in the NAD(P)H-domain, C tepidum FNR was assumed to share a common mode of NADP+ binding with that of E coli TrxR The amino acid residues required for NADP+ binding in E coli TrxR are essentially conserved in C tepidum FNR In this study, it was confirmed that the relative positions of these residues on the molecular surface are also conserved (Leu119, Ile151, Arg176, Arg181 and Ile243 for the 2′-P-AMP moiety; Thr156, Gly244 and Arg293 for the NMN moiety) Therefore, NADP+ presumably binds to C tepidum FNR or E coli TrxR in the same manner Based on superposition of the NADP+-free structure of FNR and the NADP+-bound structure of TrxR,20 we have modeled a plausible complex of FNR and NADP+ Because there are two different orientations of the NAD(P)H domain relative to the FAD domains within the dimeric structure, the two NADP + molecules are asymmetric In both model structures, the distances between the nicotinamide C4 and flavin N5 are 15.0 Å and 15.7 Å, respectively Therefore, C tepidum FNR must undergo a conformational change during the catalytic process The 410 simplest possibility is that the NAD(P)H domain rotates to the fully closed form as seen in the FR conformation of E coli TrxR (Fig 3).21 This would involve the displacement of the FAD-stacking side chain of Phe337 Such large-scale rotation between the open and closed conformations is structurally possible because there is no direct contact between the NAD(P)H and FAD domains, thereby giving the NAD(P)H domain a greater degree of freedom Structure of Ferredoxin-NAD(P)+ Oxidoreductase The trajectories of rotation of the NAD(P)H domain, presumed above, are different from those of E coli TrxR in terms of amplitude and angles (Fig 3).21 Mutational analysis of the flexible hinge region using Gly260Pro and Gly266Pro variants confirmed that the flexibility of the hinge is functionally important The hinge region of both E coli TrxR and C tepidum FNR is composed of a single antiparallel β-sheet and a comparison at the amino acid Fig The electrostatic surface potentials of C tepidum FNR Positive and negative surfaces are shown in blue and red, respectively (a) Top view with the same orientation as Fig (b) Bottom view rotated by 180 ° about the horizontal axis relative to panel a Structure of Ferredoxin-NAD(P)+ Oxidoreductase sequence level reveals that the length of the hinge of C tepidum FNR is different from that of E coli TrxR (Fig 2, Table 2) The NAD(P)H domain of C tepidum FNR did not contribute to dimerization, whereas that of E coli TrxR has some inter-subunit interactions These distinctive structural features of the domain interface could be one reason why the trajectories of the domain rotation are different between two structurally related enzymes During the catalytic cycle of E coli TrxR, two cysteine residues located in the NAD(P)H domain mediate the redox reaction between FAD and a thioredoxin molecule.20,21,29 C tepidum FNR lacks the corresponding cysteine residues, thereby allowing direct electron transfer between FAD and the FeS cluster of Fd The open form, found only in C tepidum FNR, could be required for productive docking of Fd that must be close enough to the flavin of the FAD molecule This large-scale domain motion of C tepidum FNR, which is distinct from that of TrxR, might confer Fd dependence on the ancestral structure in common with TrxR, even though the sizes of the thioredoxin and Fd molecules are similar Electron transfer between FNR and Fd requires the transient protein–protein complex formation in which their prosthetic groups (FeS cluster and FAD) are positioned at an appropriate distance and orientation with respect to each other In general, a hydrophobic core surrounded by electrostatic residues is involved in the formation of the electron transfer complex The electrostatic surface of C tepidum FNR is shown in Fig The top of the surface has basic patches, but the other side is highly acidic The vicinity around both FAD isoalloxazine rings is hydrophobic with the surrounding surface area displaying basic patches formed by residues Lys60, Arg135, Lys136, Lys262 and Arg321 Moreover, on the surface of chain B, the C-terminal subdomain of chain A provides an additional basic patch formed by Lys332, Arg334 and Lys344 In the well characterized plant-type30,31 and GR-type7,32 FNR systems, FNR provides the basic patch, and Fd provides the acidic patch to facilitate the correct geometry of interaction The positive patches of C tepidum FNR surrounding the flavin might complement ferredoxin because biochemical analysis shows that C tepidum Fds are highly acidic.13 Although preliminary docking calculation supports this docking geometry (data not shown), more experiments are needed to confirm the Fd-binding sites Recently, some C tepidum FNR homologs have been found and biochemically characterized.15–17 On the basis of amino acid sequence alignments, all homologous enzymes possess the C-terminal subdomain and have the same length of hinge region connecting the two functional domains In this structural analysis, we determined the crystal structure of C tepidum FNR as a representative of TrxR-like FNR subfamily In addition to the structural comparison with analogous plant-type FNRs and homologous E coli TrxR, mutational analysis on the FAD stacking aromatic residue and potentially flexible hinge showed a unique structural basis for 411 the substrate binding of TrxR-like FNR In the dimeric C tepidum FNR structure, one of the two C-terminal sub-domains is mobile and could not be seen in the X-ray structure Mobility of the Cterminal sub-domain might be important to make way for the nicotinamide ring stacking and sometimes to shield the reduced FAD from the bulk solvent It is possible that rotation of the NAD(P)H domain is reciprocally correlated to the displacement of the mobile C-terminal sub-domain This will be an interesting point to elucidate in the next step Experimental Procedures Purification, crystallization and data collection of C tepidum FNR C tepidum FNR was over-expressed, purified and crystallized as described.22 The orthorhombic and tetragonal forms of crystals were obtained under the same crystallization conditions The resolution limit of the orthorhombic crystals of C tepidum FNR was higher than that of the tetragonal crystals Thus, the orthorhombic crystals were used for structure determination The methionine auxotroph strain of E coli B834(DE3) was used to prepare Se-Met-substituted C tepidum FNR Crystallization conditions for C tepidum FNR and Se-Met C tepidum FNR were screened using the hanging-drop, vapor diffusion method at 293 K After refinement of the crystallization conditions, the best crystals of both FNRs were obtained using 0.1 M Mes buffer pH 6.0 containing 20 % (w/v) PEG 4000 and 0.1 M ammonium sulfate as precipitant For data collection under cryogenic conditions, crystals were soaked briefly in a reservoir solution containing 15 % (v/v) glycerol Crystals were mounted in a nylon loop and flash-cooled in a stream of gaseous nitrogen at 100 K Diffraction data from crystals of the native and Se-Met C tepidum FNR were collected by the oscillation method (Δϕ 1.0°) using synchrotron radiation at beamline NW-12 of the Photon Factory (Tsukuba, Japan) Diffraction images were collected at 100 K using an ADSC Quantum210 CCD detector and a Rigaku GN2 cryosystem The data were processed and scaled using the HKL2000 program package and the results and statistics are summarized in Table Structure determination of C tepidum FNR The phases of the Se-Met-substituted C tepidum FNR crystal were determined using the single wavelength anomalous dispersion method, and six selenium sites were identified with the programs SHELXC/D.33 Initial phases were calculated with the program SHARP.34 Phases were improved using density modification with the programs DM35 and SOLOMON,36 assuming a solvent content of 57% (v/v) The resultant electron density map was clear enough to build the initial model of Se-Met-substituted C tepidum FNR Automatic model building was done with the program RESOLVE.37 The initial model covered approximately 58 % of the amino acid residues in the protein Then, the initial model was transformed to the lattice of the native crystal, and iterative manual model building was done with the program COOT.38 The model was refined against the 2.4 Å data set of the native crystal with programs CNS39 and 412 Refmac540 in the CCP4 suite Finally, TLS refinements were done with Refmac5 after adding solvent molecules The C-terminal sub-domain of the B-chain was not modeled, even in the final electron density map The Nterminal 12 residues and the C-terminal 30 residues from positions 331 to 360 in chain A and the N-terminal 12 residues and C-terminal 12 residues from 349 to 360 in chain B could not be included in the final model, probably because of their significant flexibility In the final electron density map, 98 water molecules were assigned in the asymmetric unit The R-factor of the final model is 0.240 (Rfree 0.288) Statistical analysis with the program PROCHECK showed that the quality of the model was above the normal standard.41 Preparations of C tepidum FNR and its mutants The C tepidum FNR mutants Phe132Pro, Gly260Pro, Gly266Pro, Phe337His, Phe337Ser and Phe337Tyr were engineered in pETBlue-CT1512 plasmid by QuikChange site-directed mutagenesis using the following oligonucleotide primers (complementary primers are not shown): F132P, GGT CTC GGT GCG CCA GAG CCG CGC AAG; G260P, CTG CTG ATT CTG ATC CCC TTC AAA TCG AAT CTC; G266P, TTC AAA TCG AAT CTC CCA CCG CTG GCC CGG; F337H, GAG AAA ATT CGT AAT GTC CAC AGC AGC GTC AAG ATG; F337S, GAG AAA ATT CGT AAT GTC TCT AGC AGC GTC AAG ATG; F337Y, GAG AAA ATT CGT AAT GTC TAT AGC AGC GTC AAG ATG The DNA encoding each mutated protein was sequenced in full using a Hitachi SQ5500 sequencer (Hitachi, Tokyo, Japan) to verify that the anticipated mutations had been introduced Expression and purification procedures for WT C tepidum FNR and its mutants were as described.22 Steady state enzyme assays and spectroscopic characterization The NADPH diaphorase assay with potassium ferricyanide as an electron acceptor was done as described.26 The reaction mixture for the diaphorase assay (1 ml) was 100 mM potassium phosphate (pH 7.0), U of glucose-6phosphate dehydrogenase (G6PDH, Leuconostoc mesenteroides; Biozyme Laboratories, Blaenavon, UK), 10 mM glucose 6-phosphate (G6P, Calzyme Laboratories, San Luis, CA), mM potassium ferricyanide and 2–45 nM FNRs together with NADPH (Oriental Yeast Co., Ltd., Tokyo, Japan) The assay was done under aerobic conditions at 25 °C by monitoring the decrease in absorbance at 420 nm The NADPH oxidase assay was done under aerobic conditions at 25 °C by monitoring the decrease in absorbance at 340 nm The reaction mixture for the NADPH oxidase assay was 100 mM potassium phosphate (pH 7.0), 0.15 mM NADPH and 11–25 nM FNRs NADPH-dependent cytochrome c reduction activity was assayed under aerobic conditions by monitoring the increase in the absorbance at 550 nm with horse heart cytochrome c The reaction mixture (1 ml) was 0.1 mM cytochrome c from horse heart (Sigma-Aldrich, St Louis, MO), 10 mM G6P, U G6PDH, 10 μM NADPH and 11 – 15 μM FNRs in 100 mM potassium phosphate buffer (pH 7.0) The concentrations of NADPH (ɛ340 6.2 mM-1cm-1), potassium-ferricyanide (ɛ420 1.02 mM-1cm-1) and horse heart cytochrome c (Δɛ550 21 mM-1cm-1) stock solutions were determined by photometry In the assays, blanks Structure of Ferredoxin-NAD(P)+ Oxidoreductase consisted of all assay reagents except FNR Turnover rates are expressed as the number of NADPH molecules consumed by one molecule of native-form FNRs per second Each datum point is the average of three or four independent measurements Kinetic constants were obtained by a fit to the Michaelis–Menten equation by non-linear regression data analysis using Igor 5.0.2 software (WaveMetrics, Lake Oswego, OR) Absorption coefficients for WT and mutated C tepidum FNRs were determined by the heat denaturation method.42 Protein solutions containing approximately 10 μM FNR in 10 mM Hepes-NaOH buffer (pH 7.0) were incubated in a boiling waterbath for 10 in the dark Denatured proteins were removed by centrifugation at 15,000g for 15 The concentration of FAD in the supernatant was determined from: e450 = 11:3 mM−1 cm−1 The UV-visible absorption spectra were measured at 25 °C with a double beam spectrophotometer V-560 (JASCO, Tokyo, Japan) Difference spectra were obtained by subtracting the control spectrum, recorded prior to addition of substrates, from the corrected spectrum Protein 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degree of freedom Structure of Ferredoxin-NAD(P)+ Oxidoreductase The trajectories of rotation of the NAD(P)H domain, presumed above, are different from those of E coli TrxR in terms of amplitude... superposition of the NADP+-free structure of FNR and the NADP+-bound structure of TrxR,20 we have modeled a plausible complex of FNR and NADP+ Because there are two different orientations of the NAD(P)H... Structure of Ferredoxin-NAD(P)+ Oxidoreductase sequence level reveals that the length of the hinge of C tepidum FNR is different from that of E coli TrxR (Fig 2, Table 2) The NAD(P)H domain of