细胞色素P-450cam和细胞色素P-420cam二级结构及一氧化碳血红素配体环境的傅里叶变换红外光谱比较研究。

Comparative Fourier transform infrared studies of the secondary structure and the CO heme ligand environment in cytochrome P-450cam and cytochrome P-420cam.

作者信息

Mouro C, Jung C, Bondon A, Simonneaux G

机构信息

Max Delbrück Center for Molecular Medicine Berlin-Buch, Robert-Rössle-Strasse 10, D-13125, Berlin, Germany.

出版信息

Biochemistry. 1997 Jul 1;36(26):8125-34. doi: 10.1021/bi9700173.

DOI:10.1021/bi9700173

PMID:9201961

Abstract

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 310-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.

摘要

傅里叶变换红外光谱首次被应用于细胞色素P - 450，以分析蛋白质二级结构。通过傅里叶自去卷积并拟合酰胺I'区域（1600 - 1700 cm⁻¹）的红外光谱，我们估计无底物细胞色素P - 450cam的α - 螺旋含量为44%，β - 折叠含量为31%，转角含量为18%。在樟脑存在的情况下，得到54%的α - 螺旋和310 - 螺旋、21%的β - 折叠以及21%的转角，这与53%的α - 螺旋、19%的β - 折叠和16%的转角的晶体学数据相符[普洛斯，T. L.，芬泽尔，B. C.，& 霍华德，A. J.（1987年）《分子生物学杂志》195卷，687 - 700页]。细胞色素P - 420cam可通过两种方式由无底物细胞色素P - 450cam产生：（i）升温至60摄氏度；（ii）暴露于高达1.5 M的KSCN中。确定了每种温度和KSCN浓度下的二级结构组成，并与在1900至2000 cm⁻¹之间出现的铁配体CO伸缩振动带中观察到的变化进行比较。热诱导产生的细胞色素P - 420的α - 螺旋含量为19%，β - 折叠含量为53%，转角含量为14%，并且由于蛋白质聚集体内的分子间氢键形成了5%的反平行β - 折叠。细胞色素P - 420的形成与KSCN浓度的关系表明存在两种类型的细胞色素P - 420。在KSCN浓度达到1 M时，诱导产生的细胞色素P - 420的二级结构仅有微小变化，而在KSCN浓度为1.5 M时，观察到较大变化，产生了85%的细胞色素P - 420，且无蛋白质沉淀，其α - 螺旋含量为30%，β - 折叠含量为48%，转角含量为17%。铁配体CO伸缩区域的红外光谱显示细胞色素P - 420有几种亚构象。在细胞色素P - 420的形成过程中，与无底物细胞色素P - 450cam - CO相比，CO伸缩模式向高频方向移动了3 - 11 cm⁻¹，主要特征在约1964 cm⁻¹处。