The study demonstrates that CPR and P450 3A4 can be prepared to highly pure state by the use of detergent CHAPS. Optimisation of published methods led to pure flavoprotein, ~90% full-length with a small amount of truncated CPR. The reconstitution of CPR and P450 3A4 into liposomes using CHAPS and Superose 6 column purification has achieved a homogenous highly functional proteoliposomes with good catalytic activity and almost completely reducible P450 3A4 in a simple controllable system. The spectroscopic data has shown that reduction of P450 3A4 in proteoliposomes was at least 10 fold higher than in the simple reconstituted system suggesting that isolation of proteoliposomes from unbound protein aggregates had marked effect on the catalytic activity of P450. Negative staining electron microscopy has revealed proteoliposomes of having mean diameter of 200 ±15 nm in size; the lipid:protein ratio indicated that they incorporated 350 proteins per liposome. Type 1 difference spectral changes were observed upon binding of testosterone and erythromycin. Measurements of the first electron transfer have shown that the reduction of P450 3A4 is highly dependent on the presence of substrate. P450 3A4 reduction in proteoliposomes in the presence of testosterone was rapid and biphasic, with 90% of the P450 reduced in the fast phase, whereas reduction in the presence of erythromycin was monophasic, but substantially slower. Changes in the CPR:P450 molar ratio did not alter the rate of reduction and thus the data strongly indicates that first electron transfer occurs through preformed CPR:P450 complexes in the proteoliposomes that have a lifetime of about order of hundreds of milliseconds. The origin of the slow phase of reduction of P450 is not conclusive from our experiments, but it may be due to P450 heterogeneity. NADPH oxidation and 6β-hydroxytestosterone formation results have revealed that P450 3A4 is highly uncoupled enzyme with rates limited by CPR to P450 3A4 ratio. Hyperbolic plots of rates of NADPH consumption and 6β hydroxytestosterone formation vs CPR concentration indicate apparent Ks of 0.4 μM. This suggests that CPR:P450 complex can dissociate and reform between first and second electron transfer, which in turn indicates that second electron transfer occurs by diffusion mechanism
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