Cytochrome P450BM-3 in complex with its substrate: Temperature-dependent spin state equilibria in the oxidized and reduced states
Cytochrome P450BM-3 in complex with its substrate: Temperature-dependent spin state equilibria in the oxidized and reduced states
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DOI：
10.1007/bf03166593
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发表时间：
2007-01-01
影响因子：
1
通讯作者：
McDermott, A. E.
中科院分区：
文献类型：
--
作者：
Jovanovic, T.;Harris, M.;McDermott, A. E.
在细胞色素P450酶的结构研究中，底物以各种方式结合；重要的是确定哪些底物与选择性氧化时采用的构型最相似。在此，我们试图确定与N-棕榈酰甘氨酸饱和的细胞色素P450 BM-3的催化结合模式高度密集的条件。当底物直接结合在为氧化做好准备的血红素上时，水配体的置换是必要的，因此，铁的血红素通常从低自旋转变为高自旋。使用光学光谱和固态核磁共振，研究全长酶和分离的血红素结构域，我们表明，在室温和更高温度下，可以看到高自旋形式的大量存在，但在低温下则不能。相反，在整个温度范围内，还原态表现出高自旋。与酶的氧化和还原形式结合的底物中的各向同性化学位移与温度有关，这与附近顺磁中心的存在是一致的，但对于抗磁性的CO结合形式和化合物的自由形式，则与温度无关。还原的(亚铁血红素)物种在-54℃到+35℃的温度范围内对H-2底物的化学位移表现出居里定律依赖性，而氧化的(铁血红素)物种在底物甲基的H-2和C-13位移中都表现出明显的非居里依赖关系，低温下顺磁性血红素的影响大大减弱。这些数据与快速平衡中至少两种结合模式的混合物是一致的，其中血红素在室温下是高自旋的，但在低温下是低自旋的。
In structural studies of cytochrome P450 enzymes, substrates have been seen to bind in a variety of modes; it is important to identify those with the closest resemblance to the configurations adopted during selective oxidation. We attempt here to identify conditions in which the catalytic binding mode of cytochrome P450 BM-3 saturated with N-palmitoylglycine is highly populated. When the substrate binds directly atop the heme, primed for oxidation, displacement of the water ligand is necessary, and thereby the ferric heme is generally converted from low-spin to high-spin. Using both optical spectroscopy and solid-state nuclear magnetic resonance, studying both the full-length enzyme and the isolated heme domain, we show that a high population of the high-spin form is seen at room temperature and above, but not at reduced temperatures. In contrast, the reduced state exhibits high spin throughout the temperature range. The isotropic chemical shift of deuterons in the substrate bound to the oxidized and reduced forms of the enzyme was temperature-dependent, consistent with the presence of a nearby paramagnetic center, but temperature-independent for the diamagnetic CO-bound form, and for the free form of the compound. The reduced (ferrous heme) species shows Curie law dependence of the H-2 substrate chemical shift with respect to temperature from -54 to +35 degrees C, but the oxidized (ferric heme) species showed a pronounced non-Curie dependence in both the H-2 and the C-13 shift of the substrate's methyl group, with the effect of the paramagnetic heme at low temperatures being much reduced. These data are consistent with a mixture of at least two binding modes in rapid equilibrium wherein the heme is high-spin at room temperature but low-spin at cryogenic temperatures.