Comparative Fourier transform infrared studies of the secondary structure and the CO heme ligand environment in cytochrome P-450cam and cytochrome P-420cam
Comparative Fourier transform infrared studies of the secondary structure and the CO heme ligand environment in cytochrome P-450cam and cytochrome P-420cam
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DOI：
10.1021/bi9700173
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发表时间：
1997-07-01
期刊：
影响因子：
2.9
通讯作者：
Simonneaux, G
中科院分区：
文献类型：
--
作者：
Mouro, C;Jung, C;Simonneaux, G
首次将傅里叶变换红外光谱技术应用于细胞色素P-450蛋白质二级结构的分析。从傅立叶自反卷积和拟合酰胺I‘区(1600-1700 cm(-1))的红外光谱，我们估计无底物细胞色素P-450 cam的α-螺旋含量为44%，β-折叠含量为31%，转数为18%。在樟脑存在下，得到54%的α-螺旋和3(10)-螺旋，21%的β-折叠和21%的转角，这与53%的α-螺旋，19%的β-折叠和16%的转角[Poulos，T.L.，Finzel，B.C.，&Howard，A.J.(1987)J.Mol]的结晶学数据一致。比奥尔。195、687-700]。细胞色素P-420cam由无底物细胞色素P-450cam以两种方式产生：(I)通过温度升高到60℃和(Ii)通过暴露在KSCN中直到1.5M。二级结构组成被确定为每种温度和KSCN浓度，并与在1900到2000 cm(-1)之间观察到的铁配体CO伸缩振动带的变化进行比较。热诱导细胞色素P-420的α-螺旋含量为19%，β-折叠含量为53%，转数为14%，5%的反平行β-折叠来自蛋白质聚集体中的分子间氢键。细胞色素P-420的形成与KSCN浓度的函数关系表明细胞色素P-420有两种类型。当KSCN浓度为1M时，细胞色素P-420的二级结构变化不大，而当KSCN浓度为1.5M时，细胞色素P-420的二级结构发生了较大的变化，85%的细胞色素P-420没有蛋白质沉淀，含有30%的α-螺旋、48%的β-折叠和17%的转角。铁配体CO伸展区的红外光谱显示了细胞色素P-420的几个亚构象。在细胞色素P-420形成过程中，与无底物的细胞色素P-450cam-CO相比，CO伸展模式向更高的频率移动了3-11 cm(-1)，主要特征是约1964 cm(-1)。
For the first time, Fourier transform infrared spectroscopy has been applied to cytochrome P-450 to analyze the protein secondary structure. From Fourier self-deconvolution and fitting the infrared spectra in the amide I' region (1600-1700 cm(-1)), we estimate 44% alpha-helix, 31% beta-sheet, and 18% turns for substrate-free cytochrome P-450cam. In the presence of camphor, 54% alpha-helix and 3(10)-helix, 21% beta-sheet, and 21% turns are obtained which agree with the crystallographic data of 53% alpha-helix, 19% beta-sheet, and 16% turns [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. Cytochrome P-420cam is produced from substrate-free cytochrome P-450cam in two ways: (i) by temperature elevation up to 60 degrees C and (ii) by exposure to KSCN up to 1.5 M. The secondary structure composition is determined for each temperature and KSCN concentration and compared with the changes observed in the iron Ligand CO stretch vibration bands appearing between 1900 and 2000 cm(-1). Thermally induced cytochrome P-420 has an alpha-helix content of 19%, a beta-sheet content of 53%, 14% turns, and 5% antiparallel beta-sheets from intermolecular hydrogen bonds within protein aggregates. The formation of cytochrome P-420 as a function of the KSCN concentration indicates two types of cytochrome P-420. Up to 1 M KSCN, the induced cytochrome P-420 displays only Little modification of the secondary structure, whereas at 1.5 M KSCN, larger changes are observed, resulting in 85% cytochrome P-420 without protein precipitation and containing 30% alpha-helix, 48% beta-sheet, and 17% turns. Infrared spectra in the iron ligand CO stretch region show several subconformers for cytochrome P-420. During the cytochrome P-420 formation, the CO stretch modes are shifted to higher frequencies by 3-11 cm(-1), with a main feature at about 1964 cm(-1), compared to those of substrate-free cytochrome P-450cam-CO.