The NADPH-supported reduction of cytochrome P-450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two-exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P-450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1 :I complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 pM for a microsomal lipid extract, 0.051 pM for a 3:l (wiw) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 pM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase/P-450 ratio the amount of the rapidly reduced P-450 exceeds the equilibrium concentration of a 1 :I complex. Preformed 1 :I associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P-450 association does correspond to the data.The hepatic, endoplasmic cytochrome P-450 monooxygenase system is rate-determined by specific interactions between the three main components, the terminal oxidase cytochrome P-450, the NADPH-dependent cytochrome P-450 reductase as electron-donating flavoprotein, and phospholipids which constitute the membrane tnatrix. 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].The microsomal P-450 reduction exhibits at least two reaction phases, but up to even four partial reactions have been discriminated [3]. As yet the structural origin of this functional behaviour is rather unknown. However, two main approaches are generally accepted. A rather homogeneous model suggests randomly distributed proteins in the membrane, the interaction of which is regulated by diffusion control; the slow partial reaction is taken as to indicate some structurally unfavoured P-450 molecules [4 -91. The cluster hypothesis, on the other hand, relates the fast phase to clustered P-450 molecules which further associate with reductase, whereas the slow phase is assigned to unclustered P-450 molecules which Abbreviations. P-450 LM, liver microsomal cytochrome P-450; P-450 LM2 and P-450 LM4, homogeneous isozymes of rabbit liver microsomal cytochrome P-450 designated according to their electrophoretic mobilities ; Ole,GroPCho, dioleoylglycerophosphocholine; Ole,GroPEtn, dioleoylglycerophosphoethanolamine; Lau,GroPCho, dilauroylglycerophosphocholine; PtdSer, phosphatidylserine.Enzymes. NADPH-cytochrome P-450 reductase (EC 1.6.2.4); flavoprotein-linkedmonoxygenease or cytochrome P-450 (EC 1.14...
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...
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