REGULATION OF OXIDATION-REDUCTION POTENTIALS THROUGH REDOX-LINKED IONIZATION IN THE Y98H MUTANT OF THE DESULFOVIBRIO-VULGARIS [HILDENBOROUGH] FLAVODOXIN - DIRECT PROTON NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPICEVIDENCE FOR THE REDOX-DEPENDENT SHIFT IN THE PK(A) OF HISTIDINE-98

Flavodoxin from Desulfovibrio vulgaris is a low molecular weight (15 0 00 Da) acidic flavoprotein that contains a single flavin mononucleotid e (FMN) cofactor. A distinguishing feature of the flavodoxin family is the exceptionally low midpoint potential of the semiquinone/hydroquin one couple. Tyrosine-98, which flanks the outer or si face of the FMN, plays an important role in establishing the oxidation-reduction prope rties of the bound cofactor as demonstrated by the substitution of a n umber of amino acids at this position [Swenson, R. P., & Krey, G. D. ( 1994) Biochemistry 33, 8505-8514]. The midpoint potential for the semi quinone/hydroquinone couple increases substantially when basic residue s are introduced at this position. The pH dependency in the Y98H mutan t is consistent with a redox-linked ionization model in which the favo rable electrostatic coupling between the imidazolium cation and the fl avin 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 re duced 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 re dox-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 re present a general mechanism in the modulation of the reduction potenti al of the flavin cofactor within flavoenzymes in which ionizable group s such as histidine in the active center change ionization states duri ng the catalytic cycle.