Calcaterra N B, Picó G A, Orellano E G, Ottado J, Carrillo N, Ceccarelli E A
Department of Biological Sciences, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Biochemistry. 1995 Oct 3;34(39):12842-8. doi: 10.1021/bi00039a045.
The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners.
利用定点突变研究了酪氨酸308对豌豆铁氧还蛋白-NADP⁺还原酶稳定性的贡献。不变的羧基末端酪氨酸的酚侧链与FAD辅因子的异咯嗪部分共面堆积。荧光测量表明，这种相互作用在还原酶脱辅基蛋白对FAD的荧光猝灭中起重要作用。用色氨酸或苯丙氨酸取代酪氨酸只会使结合的FAD的量子产率略有增加，而用丝氨酸和甘氨酸进行非芳香族取代则导致荧光大幅增强。NADP⁺滴定实验的结果支持了最近的一个假说[卡尔普斯等人（1991年）《科学》251，60 - 66]，表明在没有核苷酸的情况下，Tyr 308的酚环可能填充烟酰胺结合口袋。通过热变性和尿素诱导变性研究判断，定点突变体的稳定性相对于野生型酶有所降低。带有非芳香族取代的FNR变体表现出更广泛的不稳定。热力学稳定性的降低与催化活性的损害相关[奥雷拉诺等人（1993年）《生物化学杂志》268，19267 - 19273]。结果表明，异咯嗪环附近富含电子的芳香侧链的存在对于FNR全酶的最大稳定性至关重要，从而导致一种黄素构象，该构象优化了辅基与其氧化还原伙伴之间的电子流动。
