Crystallographic investigation of the role of XXXX 95 in the modulation of the redox potentials of @entity31539 flavodoxin.

@entity31539 @entity70585 @entity2227 @entity1785 @entity535 @entity8156 @entity70584 @entity2734 @entity13478 @entity3971 @entity70583 @entity492 @entity3338 @entity493
The side chain of @entity492 95 in flavodoxin from @entity31539 provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute @entity493 , @entity2734 , or @entity2227 for this @entity535 to assess the effect of this charge on the @entity13478 / @entity8156 redox potential (E(1)) of the FMN cofactor. The @entity70583 mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/ @entity13478 midpoint redox potential (E(2)). The crystal structures of the oxidized and @entity13478 forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the @entity70583 mutant in the @entity8156 form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the @entity70583 mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with @entity2227 causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the @entity70584 mutant reveals a large movement of the 60-loop (residues 60-64) away from the @entity1785 in the oxidized structure. Reduction of this mutant to the @entity8156 causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the @entity70584 mutant imply that electrostatic repulsion between a @entity3338 on the side chain at position 95 and the @entity3971 ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the @entity70583 mutant. Replacement of D95 with @entity2734 has no effect on E(2) but causes E(1) to change by 45 mV. The @entity70585 mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.
