Nozawa T, Shimizu T, Hatano M, Shimada H, Iizuka T, Ishimura Y
Biochim Biophys Acta. 1978 Jun 21;534(2):285-94. doi: 10.1016/0005-2795(78)90011-9.
Magnetic circular dichroism spectra of oxidized, reduced and carbonmonoxy reduced forms of cytochrome P-450 from D-camphor grown Pseudomonas putida (P-450cam) were studied in the near infrared region (650 to 1200 nm) at various temperatures in the presence of D-camphor. Oxidized P-450cam with camphor exhibited positive (+) and negative (-) magnetic CD bands at 825 and 970 nm, respectively, and both of them were assigned to Faraday B terms. The magnetic CD spectrum of reduced P-450cam in the presence of D-camphor exhibited at least five components in the region between 650 to 1175 nm and one of them at 760 nm showed considerably smaller magnitude than that of the corresponding band of deoxymyoglobin. These results were interpreted to mean that the heme-iron in both oxidized and reduced P-450cam has a ligand field symmetry lower than C4v, i.e. a strong rhombic character of the heme in cytochrome P-450. Carbonmonoxide complex of reduced P-450cam exhibited no detectable magnitude of magnetic CD in the near infrared region but showed CD bands at 710 (-) and 850 (+) nm. The results were compared and discussed with those of carbonmonoxy hemoglobin and myoglobin. In addition, temperature dependent changes in the spin state of oxidized P-450cam from high to low by decrease of temperature were observed by measuring both magnetic CD and absorption spectra in the near ultraviolet and visible regions (300 to 650 nm), provided that the temperature of the sample was varied slowly (approximately 3 degrees C/min) between room and liquid nitrogen temperature in a 0.03 M phosphate buffer (pH 7.2) containing a saturated amount of D-camphor and 70% (v/v) glycerol. The significance of this phenomenon is also discussed.
研究了从以D-樟脑为生长底物的恶臭假单胞菌中提取的细胞色素P-450（P-450cam）的氧化态、还原态和一氧化碳还原态在近红外区域（650至1200nm）、不同温度以及存在D-樟脑情况下的磁圆二色光谱。氧化态的P-450cam与樟脑结合时，在825nm和970nm处分别呈现正（+）和负（-）磁圆二色带，二者均归属于法拉第B项。还原态的P-450cam在存在D-樟脑时的磁圆二色光谱在650至1175nm区域至少有五个组分，其中在760nm处的一个组分的强度明显小于脱氧肌红蛋白相应谱带的强度。这些结果被解释为意味着氧化态和还原态的P-450cam中的血红素铁具有低于C4v的配体场对称性，即细胞色素P-450中血红素具有很强的菱形特征。还原态的P-450cam的一氧化碳复合物在近红外区域未检测到磁圆二色性，但在710（-）和850（+）nm处呈现圆二色带。将这些结果与一氧化碳血红蛋白和肌红蛋白的结果进行了比较和讨论。此外，通过在含有饱和量D-樟脑和70%（v/v）甘油的0.03M磷酸盐缓冲液（pH7.2）中，在室温和液氮温度之间缓慢改变样品温度（约3℃/分钟），同时测量近紫外和可见光区域（300至650nm）的磁圆二色光谱和吸收光谱，观察到氧化态的P-450cam的自旋态随温度降低从高到低的变化。还讨论了这种现象的意义。
