Magnetic circular dichroism of Pseudomonas putida cytochrome P-450 in near infrared region

Abstract

Magnetic circular dichroism spectra of oxidized, reduced and carbonmonoxy reduced forms of cytochrome P-450 from d-camphor grown Pseudomonas putida ( P-450 cam) were studied in the near infrared region (650 to 1200 nm) at various temperatures in the presence of d-camphor. Oxidized P-450 cam 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-450 cam 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-450 cam has a ligand field symmetry lower than C 4v, i.e. a strong rhombic character of the heme in cytochrome P-450. Carbonmonoxide complex of reduced P-450 cam 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-450 cam 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 (≈3° 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.