Watmough N J, Kiss J, Frerman F E
B. F. Stolinsky Laboratories, Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Eur J Biochem. 1992 May 1;205(3):1089-97. doi: 10.1111/j.1432-1033.1992.tb16877.x.
Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the alpha-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in procine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar.
从反硝化副球菌中纯化出电子传递黄素蛋白（ETF），并研究了其与猪和人ETF的结构及氧化还原关系。这三种蛋白质的亚基质量基本相同，细菌和猪ETF的α-螺旋含量非常相似，表明整体结构相似。一种与人类大小亚基发生交叉反应的抗（猪ETF）多克隆抗体也与副球菌ETF的大亚基发生强烈交叉反应。然而，与小亚基的交叉反应非常弱。尽管如此，细菌小亚基的一个氨基末端肽和四个内部肽与人类小亚基显示出广泛的序列同一性。猪和副球菌ETF的固有色氨酸荧光发射光谱也表明了环境中的局部相似性。虽然猪和副球菌ETF的可见光谱几乎相同，但细菌蛋白中的黄素荧光仅为哺乳动物蛋白的15%。此外，细菌蛋白中黄素的圆二色光谱明显更强，表明异咯嗪环在两种蛋白质中的微环境不同。副球菌ETF的酶促或光化学还原迅速产生阴离子半醌；细菌ETF中完全还原黄素的形成非常缓慢。根据细菌ETF黄素半醌的歧化平衡判断，细菌ETF中黄素偶联的氧化还原电位间距与猪ETF基本相同。综合酶促还原和歧化平衡表明，两种ETF的黄素电位一定非常接近。数据表明，细菌和哺乳动物蛋白质的结构特性以及蛋白质中黄素辅基的热力学特性非常相似。
