A mechanism has been proposed for the binding of flavin mononucleotide (FMN) and riboflavin to the apoflavodoxin from Desulfovibrio vulgaris [Murray, T. A., and Swenson, R. P. (2003) Biochemistry 42, 2307-2316]. In this model, the binding of the flavin isoalloxazine ring is dependent on the presence of a phosphate moiety in the phosphate-binding subsite, suggesting a cooperative interaction between that region and the ring-binding subsite. In the absence of inorganic phosphate, FMN can bind through the initial association of its 5'-phosphate group in the phosphate-binding subsite followed by insertion of the flavin ring. Because riboflavin lacks the 5'-phosphate group, it is unable to bind to this apoprotein in the absence of inorganic phosphate in solution. However, inorganic phosphate can enhance the rate of ring binding by occupying the phosphate-binding subsite. In this paper, NMR, near-UV circular dichroism (CD), and fluorescence spectroscopy provide evidence for a phosphate-induced conformational change within the isoalloxazine ring-binding subsite. Phosphate-dependent changes in the chemical shifts of 22 amide groups were observed in (1)H-(15)N HSQC NMR spectra. The majority of these groups are proximal to the phosphate-binding subsite or the loops that constitute the isoalloxazine ring-binding site. Also, a phosphate-dependent change in the environment or position of the Trp60 side chain was apparent in the NMR data and was confirmed by associated changes in the near-UV CD and tryptophan fluorescence spectra when compared to the spectra of the W60A mutant. These data suggest that phosphate, either the 5'-phosphate of the FMN or inorganic phosphate from solution, facilitates the movement of the side chain of Trp60 out of the isoalloxazine ring-binding site and other associated conformational changes, creating a population of apoflavodoxin that is capable of binding the isoalloxazine ring. This conformational switch may explain why some apoflavodoxins cannot bind riboflavin and also supports the "aromatic gate" model proposed from the crystal structure of the Anabaena apoflavodoxin [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332].