Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein.
Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein.
复制标题
甲基营养细菌 W3A1 电子转移黄素蛋白中黄素辅因子及其相互作用的非共振拉曼研究。
作者：
Kunyun Yang;R. Swenson
用非共振拉曼光谱研究了来自甲基营养细菌W3A1(WETF)的电子转移黄素蛋白(WETF)在溶液中氧化态和阴离子态与黄素的相互作用。拉曼光谱数据揭示了氧化态WETF的几个独特特征。黄素的C(4)=O的拉曼光谱的异常高频率表明，在WETF中与C(4)O的氢键作用很弱或不存在。相反，C(2)=O的氢键是迄今为止研究过的黄素蛋白中最强的。根据晶体结构，αSer254的侧链羟基是wETF中氧化黄素辅因子中N(5)原子的氢键供体。用半胱氨酸取代αSer254的定点突变导致了蛋白质氧化和阴离子半醌状态中与N(5)相关的拉曼光谱的移动。这些结果证实了N(5)处存在氢键相互作用，这在氧化蛋白质的晶体结构中是明显的，并且它持续处于单电子还原状态。这些数据表明，这些谱带可以作为黄素蛋白两种氧化态中N(5)相互作用的有用的拉曼标记。WETF显示黄素环I(邻二甲苯环)相关条带的频率异常低，这表明环I微环境不同于大多数其他黄素蛋白。拉曼数据表明，αS254C突变改变了I环的环境，可能是由于wETF的FAD结构域的迁移率改变的结果。这些不寻常的黄素-蛋白质相互作用可能与wETF独特的氧化还原特性有关。
Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.