| The molecular basis for the mechanism of the binding of riboflavin-based cofactors to flavoproteins is not well understood. In this thesis, a model is proposed for the binding of flavin cofactors to the Desulfovibrio vulgaris flavodoxin. In the presence of inorganic phosphate, flavin mononucleotide (FMN) binds to the flavodoxin in two ways; phosphate-first, where the 5'-phosphate group makes the initial contact with the flavodoxin, and ring-first, where contact is initiated by the isoalloxazine ring. Riboflavin, which lacks the 5'-phosphate group of FMN, can only bind via the ring-first mechanism. In the absence of inorganic phosphate, FMN can only bind via the phosphate-first mechanism and riboflavin binds very weakly. Thus a phosphate molecule, either the 5'-phosphate group of FMN or inorganic phosphate, is required for ring-first binding to occur. Based on nuclear magnetic resonance (NMR) studies, the environment of several residues changes upon phosphate binding. In particular, the side chain of Trp60 in the ring-binding site, is experiencing different environments in the presence of phosphate. More time is spent in the alternate environments as the phosphate concentration increases. This suggested a role for Trp60 in the communication between the phosphate- and ring-binding subsites, and that Trp60 was involved in an "aromatic gate" similar to one observed in the Anabaena apoflavodoxin (Genzor et al., 1996). Examination of the D. vulgaris holoflavodoxin structure suggested a role for Ser58 in this gate; this was confirmed by mutating this residue to alanine, effectively removing the hydrogen bond "latch" that was available to hold the Trp60 "gate" open.;It was noted in the D. vulgaris experiments that riboflavin binding ability was altered when either of the two aromatics on the faces of the isoalloxazine ring was removed. However, the two aromatics are not the sole determinant of riboflavin binding because riboflavin binding was not induced in mutants of Clostridium beijerinckii flavodoxin that added a second aromatic residue, even in the presence of inorganic phosphate. Also, the S58A mutant of D. vulgaris flavodoxin binds riboflavin weakly, even though both aromatics are present. Therefore, there must be other factors that determine the riboflavin binding ability of apoflavodoxins. |
