Conformational energetics of a reverse turn in the Clostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials.

A surface loop in the flavodoxin from Clostridium beijerinckii comprised of residues -Met(56)-Gly-Asp-Glu(59)- forms a four-residue reverse turn which undergoes a conversion from a mix of cis/trans peptide configurations that approximate a type II configuration in the oxidized state to a type II' turn upon reduction of the bound flavin mononucleotide (FMN) cofactor. This change results in the formation of a new hydrogen bond between the N(5)H of the reduced cofactor and the carbonyl group of Gly57 of the central peptide bond of the turn, an interaction that is thought to contribute to the modulation of the oxidation-reduction potentials of the cofactor [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. In this study, the direct linkage of the conformational energetics of this turn to the stabilization of the FMN semiquinone was established by systematically replacing the second and third residues of the turn (Gly57 and Asp58) with the -Gly-Gly-, -Gly-Ala-, -Ala-Gly-, and -Ala-Ala- dipeptidyl sequences. On the basis of published position specific preferences for residues with side chains (mimicked by Ala) and glycine, a strong correlation was observed between E(ox/sq) and the calculated free-energy differences between the type II and type II' conformations of each of these sequence combinations. The -Ala-Gly- sequence, which favors the type II turn configuration primarily adopted in the oxidized state, displays a E(ox/sq) value that is about 150 mV more negative than that for the wild-type-like -Gly-Ala- sequence, which prefers the type II' conformation observed in the reduced states. The -Gly-Gly- and -Ala-Ala- mutants exhibit intermediate E(ox/sq) values consistent with their ambivalent turn preferences. The potential changes are primarily the result of alterations in the stability of the semiquinone state. These results provide more conclusive evidence for the crucial role of this conformational change in the modulation of the redox potentials of this flavodoxin. Furthermore, this study establishes a direct association between the conformational energetics of the protein, induced in this case by the sequence specificity of a beta-turn, and the differential thermodynamic stabilization of specific redox states of the cofactor, demonstrating another means by which flavoproteins can modulate the redox potentials of the bound cofactor.