Using Raman Spectroscopy To Monitor the Solvent-Exposed and "Buried" Forms of Flavin in p-Hydroxybenzoate Hydroxylase

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• 作者：Yuangang Zheng ; Jian Dong ; Bruce A. Palfey ; and Paul R. Carey
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：December 21, 1999
• 年：1999
• 卷：38
• 期：51
• 页码：16727 - 16732
• 全文大小：77K
• 年卷期：v.38,no.51(December 21, 1999)
• ISSN：1520-4995

文摘

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoatehydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting intwo positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has littlecontact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman differencespectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformationalstates for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ringmodes whose normal mode origin can be assigned by using density functional theory and ab initiocalculations. Further insight into the sensitivity of these modes to changes in environment is provided bythe Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the proteincomplexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate,p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those forflavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out".There are several spectral regions where characteristic differences exist for flavin in the "in" or "out"conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm^-1. These spectral featurescan be used as empirical marker bands to determine the populations of "in" and "out" for any complexof PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changingtemperature or pH. Thus, it will now be possible to determine the conformational state of the flavin inPHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data arealso 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-hydroxybenzoate there is clear evidencefrom the Raman marker bands that a mixed population of "in" and "out" exists with the majority beingin the "out" state. This is consistent with the conclusions drawn from crystallographic studies on thiscomplex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M.L. (1994) Science, 266, 110-114).