Electron spin resonance studies of wild-type and mutant cytochromes P-450d: effects of mutations at proximal, aromatic and distal sites on g values.

Low-temperature (6-40 K) electron spin resonance (ESR) spectra of cytochrome P-450d (P-450d) and its 17 mutants have been measured. The spectra of the wild-type and all mutant P-450ds showed signals at around g = 8, 3.7 and 1.7, while they didn't show any signal at around g = 2 up to 40 K. It was thus suggested that all of these P-450ds essentially take the ferric high-spin form. The g values of the proximal mutants were closer to those of the wild-type than those of the distal and aromatic mutants, suggesting that mutations at the distal and aromatic sites influence the electronic state of the heme more profoundly than those of the proximal site. The distal multiple mutants whose distal sequences are the same as those of the low-spin type P-450s such as rat P-450c, mouse P1-450 and P3-450 showed only high-spin ESR signals. Thus the spin state of P-450ds (the wild-type and all mutants) may not be solely due to specific characteristics of the distal site, but to the unique nature of the whole heme environment of P-450d. It is also suggested that the amino acids at the distal region of P-450d may be located close to the heme, so that the water molecule cannot bind to the heme, thus taking the high-spin state. Both the aromatic mutants showed rather large deviations of the g values from those of wild-type P-450d, suggesting that the aromatic region somehow interacts with the heme.

Effects of 50 ppm NO2 gas exposure on physiological functions of rats.

When rats were exposed to 50 ppm NO2 gas for 36 h, remarkable changes in some biochemical levels compared with those of control rats were observed. Namely, levels of total cholesterol, ester cholesterol, total lipids, triglycerides, nitrogen of urea, uric acid, glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and cytochrome P-450 of the exposed rats were decidedly different from those of the control rats. Thus, it was suggested that functions of liver are acutely injured upon exposure to 50 ppm NO2 gas, although extensive pulmonary injury resulting from such an exposure may also be responsible for some of the abnormal serum values.