NADH- and oxygen-dependent multiple turnovers of cytochrome P-450-CAM without putidaredoxin and putidaredoxin reductase.
Phenazine methosulfate (PMS) has been successfully used to mediate electron transfer from NADH to cytochrome P-450-CAM in the absence of putidaredoxin and putidaredoxin reductase under aerobic conditions. Identification and quantitation of exo-5- hydroxycamphor , the only product, has been accomplished by gas chromatography. In the absence of cytochrome P-450-CAM, or when other heme proteins (hemoglobin, myoglobin, horseradish peroxidase) are substituted for P-450-CAM, no exo-5- hydroxycamphor is detected. Product formation is not inhibited by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers; however, significant inhibition is observed with carbon monoxide and metyrapone, known inhibitors of the fully reconstituted P-450 system. Addition of 2,3-dimercaptopropanol to the NADH/PMS/P-450 system leads to a 4-fold increase in product formation; when putidaredoxin is added (without dimercaptopropanol), a 20-fold increase in product formation is observed. Constant bubbling with oxygen results in a further increase in the amount of product (150-fold increase overall). Our results show that 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. Molecular oxygen dependent multiple turnovers of cytochrome P-450 have not been previously observed without the fully reconstituted, three-protein system.
D002164:Camphor / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins; D006900:Hydroxylation; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010100:Oxygen; D011549:Pseudomonas / Q000201:enzymology
D005288:Ferredoxins; D009243:NAD; D008773:Methylphenazonium Methosulfate; C100137:putidaredoxin; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase; D010100:Oxygen