From the Institutes of Inorganic and.
Organic Chemistry, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland.
J Biol Chem. 2017 Dec 29;292(52):21481-21489. doi: 10.1074/jbc.M117.813683. Epub 2017 Nov 6.
Cytochrome P450cam (CYP101Fe) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO] Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH] (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe, with a of 9.6 × 10 s All processes are complete within 40 μs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeOpor] (compound 1) or CYP101[FeO] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe, yielding a Δ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 μs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.
细胞色素 P450cam（CYP101Fe）对樟脑进行区域选择性羟化。在非常低的温度和分流反应条件下进行的实验基础上，提出了这种经过充分研究的酶的催化循环中的可能羟化中间体，但在正常催化循环中，在 0°C 以上的温度下，它们的存在尚未得到验证。在这里，我们通过使用脉冲辐射来快速向樟脑结合的 CYP101[FeO]提供催化循环的第二个电子，从而证明有可能模拟 CYP101Fe 的自然催化循环。根据在 440nm 处出现的吸收最大值，我们得出结论，CYP101[FeOOH]（化合物 0）在 5μs 内积累，并迅速分解为 CYP101Fe，半衰期为 9.6×10s。所有过程在 4°C 下 40μs 内完成。重要的是，不能将瞬态吸收带分配给 CYP101[FeOpor]（化合物 1）或 CYP101[FeO]（化合物 2）。然而，从酪氨酸自由基形成和衰减的动力学中获得了涉及化合物 1 的间接证据。定量形成了 5-羟基樟脑，并且在脉冲辐射实验期间暴露于辐射不会损害酶的催化活性。化合物 0 的快速衰减使我们能够计算化合物 0→化合物 1→化合物 2→CYP101Fe 转化的吉布斯活化能的极限，分别产生 45、39 和 39kJ/mol 的值。在 37°C 下，从化合物 0 到铁（III）态的步骤只需 4μs。我们在 4°C 下的动力学研究通过增加时间维度来补充规范机制。
