CPR

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Abstract

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) utilizes oxygen and NADPH to mediate the production of various indigenous and xenobiotic molecules in various cells and organs. Curiously, when a 1:1 ratio is considered optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100:1 or higher. Furthermore, the NADPH equivalents consumed in these in vitro or in situ assemblies often far exceed the amount of metabolized substrate. It has been reported that CPR can activate molecular oxygen and generate diffusible reduced oxygen species (DROS). Furthermore, CPR consumes peroxide through diffusible radical mediated process, resulting in the formation of water (but without significant oxygen evolution).  

The researchers also quantitatively demonstrated that the oxygen activation rate and peroxide depletion rate of CPR are the main reactivities in the CYP+CPR mixture. They show that CPR can modulate the concentration of diffusible reducing oxygen species in the reaction milieu. These studies point out that since CPR inevitably generates and depletes DROS, there must be energetically ‘wasteful’ and potentially ‘hazardous’ in CPR-mediated reactions. So, this also explains why CPR is distributed at low density in cells. Some activity primarily attributable to the CYP heme-center is now identified to be a facet of the flavins of CPR. 

Cytochrome P450 and cytochrome P450 reductase

Cytochrome P450s (CYPs), as a diverse family of heme-thiolate proteins, are responsible for the metabolism of several indigenous molecules and xenobiotics in vivo. CYPs are considered to have great green chemistry potential due to their ability to selectively oxidize relatively non-reactive moieties and generate chiral synthons. CYP works in concert with the highly conserved cytochrome P450 reductase (CPR, a diflavoenzyme). The terminal redox equivalents are obtained from reduced nicotinamide adenine dinucleotides (NADPH and NADH), and molecular triplet oxygen is used as the main oxidant. The entire CYP+CPR reaction was found to be "decoupled", resulting in the production of diffusible reduced oxygen species (DROS) of superoxide and peroxide in the reaction system. Substrate binding to the active site of CYP can increase the redox potential of heme iron, and this protein-protein interaction can promote the reduction of CYP by CPR.

Figure 1. Erstwhile mechanism for DROS and water formation at heme center (Manoj, K.M.; et al. 2010)

Since "uncoupling" results in a loss of NADPH redox equivalents, this is considered an energy-wasting process. Theoretically, one molecule of NADPH can give rise to one molecule of peroxide or one molecule of hydroxylated product through the overall two-electron process. However, there are experimental data showing that the amount of NADPH consumed far exceeds the total amount of hydroxylated product and DROS formed in the reaction medium. To explain this stoichiometric imbalance, a mechanism based on the heme center is proposed, in which a two-electron deficient oxygenated CYP catalytic intermediate called compound I consumes another NADPH molecule (with the aid of CPR), thereby producing water.

Figure 2. The reactions involved in CYP+CPR mixtures (Manoj, K.M.; et al. 2010)

Involvement of radical reactions in CPR mediated depletion of peroxide

Figure 3 shows the effect of the inclusion of ascorbic acid palmitate (AAP), a potent radical scavenger, on CPR-mediated peroxide degradation reactions. A positive control reaction with CPR showed that more than 97% of the initial peroxide was degraded within 40 minutes. The control with AAP alone gave about 35% background peroxide degradation, while the test group including both CPR and AAP gave 47% degradation over the same period. Considering the autocatalytic degradation of peroxides, the AAP-containing CPR reaction mixture only gave about 19% of the CPR-containing reactivity. This result positively suggests that the CPR mediated depletion of peroxide involves diffusible radicals.

Figure 3. The effect of a radical scavenger in CPR mediated peroxide depletion (Manoj, K.M.; et al. 2010)