CONTROL OF OXIDATION-REDUCTION POTENTIALS IN FLAVODOXIN FROM CLOSTRIDIUM-BEIJERINCKII - THE ROLE OF CONFORMATION CHANGES

X-ray analyses of wild-type and mutant flavodoxins from Clostridium be ijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxida tion state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen o f Gly57 (O57) points toward the flavin ring; trans O-down, in which O5 7 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 tran s O-up conformation and accepts a hydrogen bond from the flavin N5H [S mith, 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 4 27-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper co nfirm that, in crystals of wild-type oxidized C. beijerinckii flavodox in, the Gly57-Asp58 peptide adopts the O-down orientation and isomeriz es to the cis conformation. This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137. Structu res for the mutant Asn137Ala indicate that a mixture of all three conf ormers, mostly O-down, exists in oxidized C. beijerinckii flavodoxin i n the absence of intermolecular hydrogen bonds. Redox potentials have been manipulated by substitutions that alter the conformational energi es of the bend at (56)M-G-D-E. The mutation Asp58Pro was constructed t o study a case where energies for cis-trans conversion would be differ ent from that of wild type. Intermolecular interactions with Asn137 ar e precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when t he flavin is oxidized. Reduction of the flavin induces rearrangement t o the trans O-up conformation. Redox potential shifts reflect the alte red energies associated with the peptide rearrangement; E(ox/sq) decre ases by similar to 60 mV (1.3 kcal/mol). Further, the results of mutat ion of Gly57 agree with predictions that a side chain at residue 57 sh ould make addition of the first electron more difficult, by raising th e energy of the O-up conformer that forms when the flavin is reduced t o its semiquinone state. The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by similar to 60 mV (1.3 kca l/mol). Introduction of the beta-branched threonine side chain at posi tion 57 has much larger effects on the conformations and potentials. T he Thr57-Asp58 peptide adopts a trans O-down conformation when the fla vin 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 conformation al 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 introduc ed as a framework for rationalizing the properties of wild-type flavod oxin and the effects of the mutations.