ELECTROSTATIC EFFECTS OF SURFACE ACIDIC AMINO-ACID-RESIDUES ON THE OXIDATION-REDUCTION POTENTIALS OF THE FLAVODOXIN FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovale ntly bound flavin mononucleotide (FMN) cofactor. These proteins functi on as low-potential one-electron transferases in bacteria. A distingui shing feature of these flavoproteins is the dramatic decrease in the m idpoint potential of the semiquinone/hydroquinone couple of the FMN up on binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physio logical function. The structural basis of this phenomenon is not yet t horoughly understood. In this study, the contribution of six acidic re sidues (Asp62, Asp63, Glu66, Asp95, Glu99, and Aspl06) to the perturba tion of the redox properties of the cofactor has been investigated. Th ese residues are clustered about the FMN binding site within 13 Angstr om of the N(1) atom of the cofactor. Using oligonucleotide-directed mu tagenesis, these residues were neutralized in various combinations thr ough the substitution of asparagine for aspartate and glutamine for gl utamate. Seventeen mutant flavodoxins were generated in which one to a ll six acidic residues were systematically neutralized, often in vario us spatial configurations. There was no obvious correlation between th e midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. Howev er, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. The se increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substit ution. Collectively, the unfavorable electrostatic environment provide d by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone coupl e that typifies the flavodoxin family, apparently through the destabil ization of the flavin hydroquinone anion.