Reduction of Cytochrome P‐450 LM₂ by NADPH in Reconstituted Phospholipid Vesicles is Dependent on Membrane Charge

Abstract

The kinetics of the reduction of cytochrome P-450 LM, mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochronie P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction ; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of 0-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH -cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.The resolution of the liver microsomal hydroxylase system into phospholipid, NADPH -cytochrome P-450 reductase and cytochrome P-450 [I] and their purification to homogeneity provided the experimental basis to study component interactions at a molecular level. Studies of this kind required the reconstitution of the isolated components which was achieved e.g. by mixing the two protein components in the presence of LauzGroPCho. Applying a membranous system based on egg yolk PtdCho or OlezGroPCho to the reconstitution of hydroxylation reactions catalyzed by cytochrome P-450 LM, or P-450 LM4 was not successful in comparison to the system with Lau,GroPCho; much lower enzyme activites were obtained [2,3]. However, by introducing negatively charged phospholipids into the membranous system, catalytic activities comparable to those reached in the system with Lau2GroPCho were observed [3]. The catalytic activation mediated by negatively charged phospholipids was indeed found to be linearily dependent upon the net amount of negative charges in the reconstituted membrane. Evaluatio...

Mechanism of rate control of the NADPH-dependent reduction of cytochrome P-450 by lipids in reconstituted phospholipid vesicles

“…The dissociation constants as given in Table 1 refer to the specified experimental conditions without changes in incubation volume and column procedure. P and L were kept constant in concentration whereas changes in the RIP ratio were achieved by variations of R. A constant LIP ratio was maintained because of a dependence of the P-450 reduction and catalytic activity, as well, on LIP [2]. Specifications of components concentrations refer to vesicle suspension, a transformation to lipid surface concentration e.g.…”

“…At constant reductase/P-450 stoichiometry (RIP ratio = 0.2) the physiologically most relevant rapid reaction phase (ql) increased in amount with the net negative charge of the reconstituting lipid. The apparent rate constants, on the other hand, proved to be rather less variable [2].…”

“…At constant reductase/P-450 stoichiometry (RIP ratio = 0.2) the physiologically most relevant rapid reaction phase (ql) increased in amount with the net negative charge of the reconstituting lipid. The apparent rate constants, on the other hand, proved to be rather less variable [2].The present contribution deals with comprehensive studies on the reduction reaction with different protein stoichiometries (RIP ratio) in order to analyze the reaction mechanism in detail. For reconstitution three different lipid systems were selected : according to preceding investigations [2] the most active negatively charged Ole,GroPEtn/PtdSer (3 :I, w/w) system was compared with the significantly less active neutral Ole,GroPCho system.…”

“…The availability of isolated proteins and Iipids, respectively, enabled the investigation of reconstituted systems of selected composition and stoichiometry and this way studies of the catalytic mechanism on a molecular level. In preceding papers it could be shown that the lipid control of the P-450 reduction and of substrate conversion depends on membrane charges which correspond to the electrophoretic mobility of respective vesicles [ 1,2].…”

“…For reconstitution three different lipid systems were selected : according to preceding investigations [2] the most active negatively charged Ole,GroPEtn/PtdSer (3 :I, w/w) system was compared with the significantly less active neutral Ole,GroPCho system. Additionally a microsomal lipid mixture was included.…”