NADPH oxidase/cytochrome 554 complex

All oxidases need to have a cofactor to which electrons can be transferred. O2 is a direct electron receiver for some reactions while NADPH and flavoprotein complexes known as the cytochrome Ps can be involved in a complex transfer of reducing and oxidizing power. Cytochrome P450 is a well-studied oxidizing system and is illustrated in Figure 9. (The number which is added as a subscript to the P family refers to the wavelength of maximum absorbance for the species.)... 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

The system depends on an electron transport pathway that transfers electrons from NADPH through a flavoprotein (NADPH cytochrome P-450 reductase) to cytochrome P-450 that is the terminal oxidase of the chain (10). The xenobiotic first forms a complex with the oxidized form o cytochrome P-450 which is reduced by an electron passing down the chain from NADPH. The reduced cytochrome P-450/substrate complex then reacts with and activates molecular oxygen to an electrophilic oxene species (an electron deficient species similar to singlet oxygen) that is transferred to the substrate with the concommitant formation of water. Cytochrome P-450 thus acts primarily as an oxene transferase (2). Substrate binding is a relatively nonspecific, passive process that serves to bring the xenobiotic into close association with the active center and provide the opportunity for the oxene transfer to occur. 

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