Evaluation of the functional role of the heme-6-propionate side chain in cytochrome P450cam
Evaluation of the functional role of the heme-6-propionate side chain in cytochrome P450cam
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DOI：
10.1021/ja077902l
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发表时间：
2008-01-16
影响因子：
15
通讯作者：
Hayashi, Takashi
中科院分区：
文献类型：
--
作者：
Harada, Katsuyoshi;Sakurai, Keisuke;Hayashi, Takashi
细胞色素 P450cam (P450cam) 通过非共价相互作用将原血红素 IX 作为辅基结合。 6-丙酸血红素，两个丙酸血红素侧链之一。与 Arg112 和其他亲水性氨基酸残基形成氢键相互作用。在这里，我们使用具有 6-去丙酸 6-甲基化原血红素 IX（单腿下摆）的重构蛋白展示了 P450cam 中 6-丙酸侧链的结构和功能率。光谱数据和酶活性表明，去除 6-丙酸盐对酶性质没有明显影响。从天然 radox 伴侣 Putidaredoxin (Pdx) 到 P450cam 的电子转移速率没有显着变化。然而，6-丙酸的去除使 Pdx 的亲和力降低了 3.5 倍，支持了 Arg112 作为 Pdx 结合位点的基本组成部分的作用。共振罗马实验表明，去除 6-丙酸酯会削弱 Fe-S 键强度。重构蛋白的 X 射线结构在 1.55A 分辨率下与野生型蛋白的 X 射线结构高度重叠，而重构蛋白中 Cys357 hems 配体的硫醇盐由于缺乏 6-丙酸盐，从蛋白表面可见。 Fe-S 键的延长和水的可及性可以促进硫醇盐阴离子质子化为硫醇，解释了在温和条件下观察到的非活性 P420 物种的形成。因此，d-樟脑羟基化反应需要 6-丙酸酯-蛋白质基质相互作用来维持活性 P450 物质。
Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovelent interactions. Heme-6-propionate, one of the two hems-propionate side chains. forms hydrogen-banding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional rates of the 6-propionate side chain in P450cam using a reconstituted protein with 6- depropionate 6-methylated protoheme IX (one-legged hems). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate has no clear influence on the enzyme property. The rate of electron transfer from putidaredoxin (Pdx), a natural radox partner, to P450cam was not significantly changed. whereas, the removd of the 6-propionate decreased the affinity of Pdx by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. Resonance Roman experiments indicate that removal of the 6-propionate weakens the Fe-S bond strength. The Xray structure of the reconstituted protein at 1.55A resolution is highly superimposable with that of the wild-type protein, whereas the thiolate of the Cys357 hems ligand in the reconstituted protein is visible from the protein surface owing to the lack of the 6-propionate. Lengthening of the Fe-S bond and the water accessibility could facilitate protonationof thiolate anion to thiol, explaining the observed formation of the inactive P420 species under the mild conditions. Therefore, the d-camphor hydroxylation reaction requires a 6-propionate-protein matrix interaction to maintain an active P450 species.