The FMN domain of CPR has clusters of negatively charged residues around the FMN binding site and a striking patch of positively charged residues at the opposite end of the domain. The mutation V233P/E234P, involving two residues at the very beginning of the flexible “hinge,” remote from the linker and FAD domains, leads to a substantial increase in the R g and D max values, indicating a larger population of the extended conformation, a 3-fold decrease in the K M for cytochrome c and a slight increase in k cat. The positions of the residues involved are shown in Figure 3, and the shapes of these mutants and their kinetic constants for cytochrome c reduction are given in Table 2. To further test the importance of the extended conformation for the activity of CPR, we have constructed mutants involving residues at interdomain interfaces or in the flexible hinge. As the ionic strength is varied, we observe a striking correlation between k cat and D max ( Figure 4C) this can readily be understood if the extended form of the enzyme, favored at high ionic strength, is required for intermolecular electron transfer. These results are consistent with the idea that oxidized CPR exists in equilibrium between compact and extended conformations, the position of which is sensitive to the ionic strength of the solution. The effect of high salt is completely reversible on decreasing the salt concentration by dialysis. Increasing the ionic strength up to 0.24 M has no significant effect, but further increases lead to a marked elongation of the molecular envelope until, by I = 0.54 M, R g has increased from 26.4 Å to 32.5 Å and D max has increased from 74 Å to 108 Å ( Figure 4A Table S1). SAXS experiments show that CPR becomes more elongated at high ionic strength. ) show that the domain-domain interface around the FAD and FMN cofactors includes a number of ion-pair interactions, raising the possibility that increasing ionic strength would affect the position of the conformational equilibrium. Comparing our model of the extended conformation to the crystal structure, it is apparent that a major difference between the two arises from a rotation of the FMN domain over the surface of the linker domain, leading to the FMN becoming solvent accessible ( Figure 3). Thus, the loss of the interdomain interactions of the loops around the FMN is compensated by the much more extensive interactions between helices B and F in the FMN domain and helix I in the linker domain. The residues of these helices, particularly those involved in these interdomain interactions, are highly conserved ( Figure S5). In the model of the extended conformation, these three helices interact much more extensively, involving almost the whole length of helix I. In the crystal structure, these primarily involve loops around the FMN, but, in addition, two residues near the N termini of helices B and F (E96, E216) interact with residues (R285 and T386) at the N terminus of helix I in the linker domain ( Figure 3, shown in magenta). Regions of the FMN domain involved in interdomain interactions are shown in dark blue. The model of the extended conformation is compared to the crystal structure in Figure 3. ![]() This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |