Flavocytochrome P450BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set
Flavocytochrome P450BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set
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DOI：
10.1074/jbc.m401716200
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发表时间：
2004-05-28
影响因子：
4.8
通讯作者：
Munro, AW
中科院分区：
文献类型：
--
作者：
Girvan, HM;Marshall, KR;Munro, AW
保守的谷氨酸将血红素共价连接到真核细胞CYP 4 P450酶的蛋白质骨架上。在相关的巨大芽孢杆菌P450 BM 3中，相应的残基是Ala（264）。产生A264 E突变体，并通过动力学和光谱方法进行表征。与野生型酶相比，A264 E具有改变的吸收光谱（Soret最大值在类似于420.5 nm处）。脂肪酸底物产生了一个类似走廊的光谱变化，Soret带移动到426 nm。用长链脂肪酸进行光学滴定，表明A264 E比野生型酶具有更高的亲和力。血红素铁中点还原电位在无底物A264 E是更积极的比野生型P450 BM 3，并没有改变后，底物结合。EPR，共振拉曼，和磁性CD光谱表明，A264 E保持在低自旋状态基板结合后，不像野生型P450 BM 3。EPR光谱显示在无衬底的A264 E中有两个主要物种。第一个有正常的Cys水铁结扎。第二个类似于甲酸盐结扎的P450 cam。饱和脂肪酸增加人口的后一种物种，这表明，底物的力量谷氨酸促进Cys-Glu配体集，目前在较低的量在无底物的酶。在近红外磁性CD光谱中的一种新的电荷转移跃迁提供了新的A264 E血红素铁连接状态的光谱特征。A264 E保留加氧酶活性，尽管谷氨酸配位的铁，表明结构重排发生后血红素铁还原，使双氧结合。血红素铁的谷氨酸配位通过A264 E突变体的结构研究得到证实（Joyce，M.G.，Girvan，H.M.，蒙罗，AW，和Leys，D.（2004）J.Biol.Chem.279，23287-23293）。
A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala(264). The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at similar to420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M.G., Girvan, H.M., Munro, A.W., and Leys, D. ( 2004)J. Biol. Chem. 279, 23287-23293).