NADH- and oxygen-dependent multiple turnovers of cytochrome P-450-CAM without putidaredoxin and putidaredoxin reductase

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Abstract
Phenazine methosulfate (PMS) was used to mediate electron transfer from NADH to camphor-hydroxylating cytochrome P-450 (P-450-CAM) [from Pseudomonas putida] in the absence of putidaredoxin and putidaredoxin reductase under aerobic conditions. Identification and quantitation of exo-5-hydroxycamphor, the only product, was accomplished by gas chromatography. When P-450-CAM was absent or when other heme proteins (Hb, myoglobin, horseradish peroxidase) were substituted for P-450-CAM, no exo-5-hydroxycamphor was detected. Product formation was not inhibited by the addition of catalase, superoxide dismutase or hydroxyl radical scavengers. Significant inhibition was observed with CO and metyrapone, known inhibitors of the fully reconstituted P-450 system. Addition of 2,3-dimercaptopropanol to the NADH/PMS/P-450 system led to a 4-fold increase in product formation; when putidaredoxin was added (without dimercaptopropanol), a 20-fold increase in product formation was observed. Constant bubbling with O2 resulted in a further increase in the amount of product (150-fold increase overall). Evidently, PMS can substitute for the electron-transfer proteins putidaredoxin and putidaredoxin reductase in the transfer of electrons from NADH to P-450-CAM, resulting in multiple turnovers. O2-dependent multiple turnovers of cytochrome P-450 have not been previously observed within the fully reconstituted, 3-protein system.