Regulation of oxidation-reduction potentials through redox-linked ionization in the Y98H mutant of the Desulfovibrio vulgaris [Hildenborough] flavodoxin: Direct proton nuclear magnetic resonance spectroscopic evidence for the redox-dependent shift in the pK(a) of histidine-98

Flavodoxin from Desulfovibrio vulgaris is a low molecular weight (15 000 Da) acidic flavoprotein that contains a single flavin mononucleotide (FMN) cofactor. A distinguishing feature of the flavodoxin family is the exceptionally low midpoint potential of the semiquinone/hydroquinone couple. Tyrosine-98, which flanks the outer or si face of the FMN, plays an important role in establishing the oxidation-reduction properties of the bound cofactor as demonstrated by the substitution of a number of amino acids at this position [Swenson, R. P., & Krey, G. D. (1994) Biochemistry 33, 8505-8514]. The midpoint potential for the semiquinone/hydroquinone couple increases substantially when basic residues are introduced at this position. The pH dependency in the Y98H mutant is consistent with a redox-linked ionization model in which the favorable electrostatic coupling between the imidazolium cation and the flavin hydroquinone anion is responsible for the higher potential. Such a model predicts an increase in the pK(a) of 1.5 units for His98 upon complete reduction of the FMN. In this study, proton nuclear magnetic resonance spectroscopy was used to directly determine the intrinsic pK(a) of His98 as a function of the redox state of the cofactor in this flavodoxin. Values for the pK(a) of His98 in the oxidized and fully reduced flavodoxin are 7.02 +/- 0.08 and 8.43 +/- 0.11, respectively, an increase in the pK(a) by 1.41 units, which conforms with the previous prediction. These results provide direct experimental proof of the redox-linked ionization of this residue and provides further evidence of the crucial role of electrostatic interactions, in this case, in the stabilization of the flavin hydroquinone anion. This phenomenon may represent a general mechanism in the modulation of the reduction potential of the flavin cofactor within flavoenzymes in which ionizable groups such as histidine in the active center change ionization states during the catalytic cycle.