Sharonov Iu A
Mol Biol (Mosk). 1992 Nov-Dec;26(6):1251-62.
Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.
记录并分析了来自恶臭假单胞菌的无樟脑和结合樟脑的还原型细菌细胞色素P450cam在索雷特区域（360 - 480 nm）的磁圆二色性（MCD）光谱，温度范围为2 K至290 K。底物与酶的结合定性地改变了MCD强度的温度依赖性。在没有樟脑的情况下，在T < 4.2 K时MCD强度随1/T呈线性增加，这证明了基态电子态的简并或接近简并。在有底物的情况下，简并被消除，温度曲线在T < 4.2 K时显示出饱和行为，并且其高温部分具有波长依赖性。索雷特带长波长区域的温度曲线在大约15 K处有一个最大值，而在短波长区域MCD强度单调增加直至饱和。温度曲线的波长依赖性证明了两种不同形式的结合底物的还原型P450cam共存。从温度曲线的理论分析中得出了以下结论。在没有底物的情况下，血红素铁处即使有菱形畸变也非常小，如果有的话，并且轴向零场分裂参数D为负（P450cam和P450LM2的D分别为 - 8.3 cm-1和 - 6.2 cm-1）。在有底物的情况下，两种形式的还原型P450cam具有正的参数D但值不同（D1 = 12 cm-1和D2 = 28 cm-1），并且血红素铁处有大的菱形畸变。D值之间超过两倍的差异使得能够从总的MCD光谱中分离出两种酶形式的温度依赖性贡献，并模拟在有底物存在时还原型P450cam的MCD光谱随温度的变化。考虑到底物结合对还原型P450cam基态电子态的剧烈影响，可以认为底物结合诱导了酶从无活性状态转变为活性状态。
