Using Raman spectroscopy to monitor the solvent-exposed and "buried" formsof flavin in p-hydroxybenzoate hydroxylase

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine r ing system of FAD is capable of adopting in two positions on the protein. I n one, the "in" form, the ring is surrounded by protein groups and has litt le contact with solvent; in the second, "out" form, the ring is largely sol vent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment o f isoalloxazine ring modes whose normal mode origin can be assigned by usin g density functional theory and ab initio calculations. Further insight int o the sensitivity of these modes to changes in environment is provided by t he Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous s olution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided ex amples of the "in" conformation. These data are compared to those for flavi n bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show th at the flavin is "out" There are several spectral regions where characteris tic differences exist for flavin in the "in" or "out" conformation, these o ccur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-1). These spectral fea tures can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in these pop ulations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallog raphic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly " out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenz oate there is clear evidence from the Raman marker bands that a mixed popul ation of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Mass ey. V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).