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CLOSTRIDIUM BEIJERINCKII FLAVODOXIN MUTANT: G57T OXIDIZED
OverviewOverview
X-ray analyses of wild-type and mutant flavodoxins from Clostridium, beijerinckii show that the conformation of the peptide Gly57-Asp58, in a, bend near the isoalloxazine ring of FMN, is correlated with the oxidation, state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of, three conformations: trans O-up, in which the carbonyl oxygen of Gly57, (O57) points toward the flavin ring; trans O-down, in which O57 points, away from the flavin; and cis O-down. Interconversions among these, conformers that are linked to oxidation-reduction of the flavin can, modulate the redox potentials of bound FMN. In the semiquinone and reduced, forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up, conformation and accepts a hydrogen bond from the flavin N5H [Smith, W., W., Burnett, R. M., Darling, G. D., & Ludwig, M. L. (1977) J. Mol. Biol., 117, 195-225; Ludwig, M. L., & Luschinsky, C. L. (1992) in Chemistry and, Biochemistry of Flavoenzymes III (Muller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper confirm that, in crystals, of wild-type oxidized C. beijerinckii flavodoxin, the Gly57-Asp58 peptide, adopts the O-down orientation and isomerizes to the cis conformation. This, cis form is preferentially stabilized in the crystals by intermolecular, hydrogen bonding to Asn137. Structures for the mutant Asn137Ala indicate, that a mixture of all three conformers, mostly O-down, exists in oxidized, C. beijerinckii flavodoxin in the absence of intermolecular hydrogen, bonds. Redox potentials have been manipulated by substitutions that alter, the conformational energies of the bend at 56M-G-D-E. The mutation, Asp58Pro was constructed to study a case where energies for cis-trans, conversion would be different from that of wild type. Intermolecular, interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is, cis, and O-down, when the flavin is oxidized. Reduction of the flavin, induces rearrangement to the trans O-up conformation. Redox potential, shifts reflect the altered energies associated with the peptide, rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol)., Further, the results of mutation of Gly57 agree with predictions that a, side chain at residue 57 should make addition of the first electron more, difficult, by raising the energy of the O-up conformer that forms when the, flavin is reduced to its semiquinone state. The ox/sq potentials in the, mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by, approximately 60 mV (1.3 kcal/mol). Introduction of the beta-branched, threonine side chain at position 57 has much larger effects on the, conformations and potentials. The Thr57-Asp58 peptide adopts a trans, O-down conformation when the flavin is oxidized; upon reduction to the, semiquinone, the 57-58 peptide rotates to a trans O-up conformation, resembling that found in the wild-type protein. Changes in FMN-protein, interactions and in conformational equilibria in G57T combine to decrease, the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol), and to increase the sq/hq potential by 80 mV (-1.7 kcal/mol). A, thermodynamic scheme is introduced as a framework for rationalizing the, properties of wild-type flavodoxin and the effects of the mutations.
About this StructureAbout this Structure
1FLD is a Single protein structure of sequence from Clostridium beijerinckii with FMN as ligand. Structure known Active Site: FMN. Full crystallographic information is available from OCA.
ReferenceReference
Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes., Ludwig ML, Pattridge KA, Metzger AL, Dixon MM, Eren M, Feng Y, Swenson RP, Biochemistry. 1997 Feb 11;36(6):1259-80. PMID:9063874
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