Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

Abstract

ABSTRACT:Conformational transitions between closed and open states in the NADPH-cytochrome P450 oxidoreductase (POR) play a critical role in its electron-transport function. In this study, we determined rotational diffusion coefficients of the EDANS fluorophore attached to the cytosolic POR construct lacking the N-terminal transmembrane region. We identified two dynamic modes, slow and fast, which are interpreted as the rotational diffusion of POR as a whole and the local domain motion, respectively. Timescale of the local rotational diffusion component suggests that it may correspond to the transient opening of the fully oxidized POR structure.NADPH-cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to multiple cytochromes P450 and other proteins in the endoplasmic reticulum of liver cells and other tissues 1 . Conformational changes in POR molecule are required for productive interaction with cytochromes P450, and the "open" and "closed" structures of POR were identified by X-ray crystallography 2-4 . Because the complex of POR with cytochrome P450 has never been structurally resolved, how these conformational states of POR relate to the steps in the functional cycle-remains debated. For example, oxidized POR has been demonstrated to assume closed conformation (blue ribbon in Figure 1) in solution by NMR spectroscopy and small-angle X-ray scattering 5-7 , while other reports indicated it is in an open form in the oxidized state 8,9 . In this study, we probed conformational states of the oxidized cytosolic fragment of POR, residues 57-678, (in the following text-POR) from viewpoint of the local conformational freedom measured by rotational diffusion constants. We used a single cysteine mutant of POR with otherwise cysteine-less background to introduce a longlived EDANS fluorophore in the FMN domain of POR. Two distinct time constants observed in fluorescence anisotropy decays allowed us to propose that FMN domain might have some degree of local mobility within the oxidized POR structure.