EVALUATION OF THE ELECTROSTATIC EFFECT OF THE 5'-PHOSPHATE OF THE FLAVIN MONONUCLEOTIDE COFACTOR ON THE OXIDATION-REDUCTION POTENTIALS OF THE FLAVODOXIN FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH)

Two mutants of the Desulfovibrio vulgaris flavodoxin, T12H and N14H, w ere generated which, for the first time, place a basic residue within the normally neutral 5'-phosphate binding loop of the flavin mononucle otide cofactor binding site found in all flavodoxins. These,histidine residues were designed to form an ion pair with the dianionic 5'-phosp hate, either altering its ionization state or offsetting its negative charge to allow evaluation of the magnitude of its electrostatic effec t on the redox properties of the cofactor, The midpoint potential for the oxidized/semiquinone couple was not significantly altered in eithe r mutant. However, the midpoint potentials for the semiquinoe/hydroqui none couple (E(sq/hq)) were less negative than that of the wild type, increasing by 28 and 15 mV relative to that of the wild type for the T 12H and N14H mutants, respectively, at pH 6. P-31 NMR spectroscopy sug gests that, just as for wild type, the phosphate group in each mutant does not change its ionization state between pH 6, and 8, Therefore, t he small increases in midpoint potential must be linked to the protona tion of the histidine residues, either through favorable interactions with the anionic hydroquinone or by the partial compensation of the ch arge on the 5'-phosphate. Values for the pK(a) of His12 and His14 in t he oxidized flavodoxin were determined by H-1 NMR spectroscopy to be 6 .71 and 6.93, respectively, which are only modestly elevated relative to the average value for histidines in proteins. This suggests that th e histidines do not form strong ion-pairing interactions with the phos phate and/or that the effective charge on the 5'-phosphate may be subs tantially less than the reported formal dianionic charge. Either way, the data provide evidence for the rather weak electrostatic interactio n between a charged group at this site and the anionic flavin hydroqui none. In contrast, E(sq/hq) reported for the apoflavodoxin-riboflavin complex, which lacks the 5'-phosphate group, is 180 mV less negative t han that of the native flavodoxin, The re-evaluation of the redox and cofactor binding properties of the riboflavin complex generated values for the dissociation constants for the riboflavin complex in the oxid ized, semiquinone, and hydroquinone oxidation states that are 2100-, 6 3000-, and 54-fold higher, respectively, than that for the naturally o ccurring flavin mononucleotide complex, The large redox potential shif ts observed for both redox couples in the riboflavin complex are prima rily the consequence of a decreased stabilization of the semiquinone r ather than the result of the absence of the negative charge of the 5'- phosphate. It is concluded from this study that the negative charge on the phosphate group of the cofactor does not play a disproportionate role in decreasing E(sq/hq), at most contributing equivalently with th e acidic amino acid residues clustered around the flavin to an unfavor able electrostatic environment for the formation of the flavin hydroqu inone anion.