Zheng Y, Dong J, Palfey B A, Carey P R
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106-4935, USA.
Biochemistry. 1999 Dec 21;38(51):16727-32. doi: 10.1021/bi9918893.
X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent 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 of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, 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 for flavin 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 features 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 those populations 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 crystallographic 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-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population 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., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).
对几种涉及与对羟基苯甲酸羟化酶（PHBH）结合的黄素腺嘌呤二核苷酸（FAD）的复合物进行的X射线晶体学研究表明，FAD的异咯嗪环系统能够在蛋白质上占据两个位置。在一种位置，即“内”形式中，环被蛋白质基团包围，与溶剂接触很少；在第二种“外”形式中，环在很大程度上暴露于溶剂中。利用拉曼差光谱法，可以获得溶液中不同复合物处于两种构象状态下黄素环的拉曼光谱。这些光谱由丰富多样的异咯嗪环模式组成，其正常模式起源可通过使用密度泛函理论和从头计算来确定。固态、二甲基亚砜（DMSO）和水溶液中发光黄素的拉曼光谱进一步深入了解了这些模式对环境变化的敏感性。对于蛋白质复合物，与野生型PHBH结合的黄素以及野生型PHBH加底物对羟基苯甲酸的拉曼差光谱提供了“内”构象的实例。这些数据与与野生型PHBH加2,4 - 二羟基苯甲酸结合的黄素的数据进行了比较，其中X射线分析表明黄素是“外”的。在几个光谱区域中，黄素处于“内”或“外”构象时存在特征差异，这些差异出现在1700、1500、1410、1350、1235和1145 cm⁻¹附近。这些光谱特征可作为经验性标记带，用于确定PHBH任何复合物中“内”和“外”构象的比例，并监测这些比例随系统扰动（例如通过改变温度或pH）的变化。因此，对于那些无法进行X射线晶体学分析的复合物，现在有可能确定PHBH中黄素的构象状态。还给出了Tyr222Phe突变体的拉曼差数据。拉曼数据表明，Tyr222Phe突变体的异咯嗪环主要是“外”构象。然而，在存在底物对羟基苯甲酸的情况下，从拉曼标记带可以明确看出存在“内”和“外”构象的混合群体，其中大多数处于“外”状态。这与对该复合物的晶体学研究得出的结论一致（加蒂，D. L.，帕尔菲，B. A.，拉赫，M. S.，恩奇，B.，梅西，V.，巴卢，D. P.，和路德维希，M. L.（1994年）《科学》，266，110 - 114）。
