Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Stockman, Brian J.; Westler, William M.; Mooberry, Eddie S.; Markley, John L.

doi:10.1021/bi00401a021

Cited by 47 publications

(34 citation statements)

References 32 publications

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“…Earlier NMR studies had shown that the 15N chemical shift of N5 in A . 7120 flavodoxin is similar to that of free FMN and different from that of other flavodoxins (Stockman et al, 1988), suggesting that N5 might be exposed to solvent. However, the partial NMR structure showed N5 to be solvent inaccessible as found in the X-ray structure.…”

Section: Crystal Packingmentioning

confidence: 81%

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Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution

et al. 1992

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The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A . 7120 flavodoxin belong to the monoclinic space group P2, with a = 48.0, b = 32.0, c = 51.6A, and 0 = 92", and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2a and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the 04' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A . 7120, whereas in A . nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A , respectively, whereas in the third flavodoxin the distance is 3.1 A , close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent. In all three long-chain flavodoxins, a water molecule bridges the ends of the inserted loop in the os strand and minimally perturbs its hydrogen bonding with p4. Many of the water molecules in these proteins interact with the flavin binding loops. The conserved &core of the three long-chain and two short-chain flavodoxins superpose with root mean square deviations ranging from 0.48 A to 0.97 A .

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Section: Crystal Packingmentioning

confidence: 81%

“…The cells were isolated by centrifugation, and the flavodoxin was purified by the method of D.W. Krogmann and C. Harper (Harper, 1988) as described by Stockman et al (1988).…”

Section: Proteinmentioning

confidence: 99%

Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution

et al. 1992

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“…The potential for the semiquinone/hydroquinone couple in E. coli flavodoxin, -440 to -455 mV at pH 7 (Vetter & Knappe, 1971; Hoover, 1997), is typical for flavodoxins. In many flavodoxins, including the flavodoxin from Anabaena 7120 that resembles E. coli flavodoxin, "N chemical shifts of the flavin N(1) atom indicate that N( 1) is not protonated in the hydroquinone state (Franken et al, 1984;Stockman et al, 1988). Thus, the reduced isoalloxazine bound to flavodoxins is anionic, and the low sq/hq potential can be attributed to unfavorable electrostatic interactions between the negatively charged flavin and its protein environment (Ludwig et al, 1990;Swenson & Krey, 1994;Zhou & Swenson, 1995.…”

Section: Oxidation-reduction Potentialsmentioning

confidence: 99%

A flavodoxin that is required for enzyme activation: The structure of oxidized flavodoxin from Escherichia coli at 1.8 Å resolution

Hoover

Ludwig

1997

Protein Science

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In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B 12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2,2,2,, a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwc>rk of 0.196 and an Rrree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 8,) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 8, ( R = 0.191; = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus injluenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.Keywords: cryocrystallography; electron transfer; Escherichia coli; flavodoxin; reductive activation; X-ray structure Flavodoxins are small flavin mononucleotide-containing proteins found in many microorganisms, where they serve to transfer electrons at low oxidation-reduction potentials. The FMN is bound tightly but noncovalently to a single polypeptide chain, and its one-electron redox potentials are shifted substantially in the presence of the apoprotein so that reduction occurs in two discrete one-electron steps. Among known flavodoxins, the oxidized/ semiquinone potentials vary from -50 to -260 mV, whereas potentials for the hydroquinone/semiquinone couple are in the range

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“…Distance restraints for the calculations were obtained from proton NMR data (van Mierlo et al, 1991b,c). The flavin binding site and the protein flavin interactions in the flavodoxin from Anabaena 7120 were investigated with 'H, I3C and 15NNMR spectroscopy (Stockman et al, 1988(Stockman et al, , 1990. 'H and I5N resonances of oxidized flavodoxin from Anacystis niduluns were assigned using "N edited three-dimensional (3D) NMR experiments (Clubb et al, 1991).…”

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confidence: 99%

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements

et al. 1993

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Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequencespecific assignment of all backbone and most of the side chain 'H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOES, together with information about slowly exchanging amide hydrogens and HN-W spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel B-sheet and four a-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the P-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.Desulfovibrio vulgaris flavodoxin is a member of a group of small microbial electron-transfer proteins which contain a molecule of non-covalently bound FMN as their redox-active group. It consists of 148 amino acid residues and has a molecular mass of 16.3 kDa (Dubourdieu and Fox, 1977). The protein-bound flavin can occur in three distinct redox states : an oxidized (quinone), a one-electron-reduced (semiquinone) and a two-electron-reduced (hydroquinone) form (Ghisla and Massey, 1989). In most physiological reactions the flavodoxins shuttle between the one-electron-reduced and the twoelectron-reduced state (Simondson and Tollin, 1980). The redox potentials for this transition vary from -320 mV to -518 mV among flavodoxins from different organisms (Vervoort, 1991 ;Deistung and Thorneley, 1986), whereas the value for the free FMN is -124 mV (Anderson, 1983). This indicates that the binding of the flavin to the apoflavodoxin causes significant changes in its redox properties. Ring strain, the burial of charge in a hydrophobic environment,

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Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Cited by 47 publications

References 32 publications

Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution

Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution

A flavodoxin that is required for enzyme activation: The structure of oxidized flavodoxin from Escherichia coli at 1.8 Å resolution

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements

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