Abstract
NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.
Highlights
Monooxygenase enzymes occur in all kingdoms of life and cytochromes cytochrome P450 (P450) (P450s) represent the largest superfamily of them (Lamb and Waterman, 2013; Munro et al, 2013)
Microsomal P450s catalyze the oxidation of a wide variety of essential endogenous and xenobiotic compounds (Coon, 2005; Rendic and Guengerich, 2015) by a two-electron activation of molecular oxygen, whereby one atom of oxygen is inserted into the organic substrate and the other is reduced to water (Guengerich, 2007)
Electrons flow through cytochrome P450 reductase (CPR) from NADPH to oxidized flavin adenine dinucleotide (FAD) as a hydride ion transfer to the FAD N5 atom, one by one, from the FAD to the flavin mononucleotide (FMN), from the FMN hydroquinone to external acceptors, with the FMN cycling mainly between the hydroquinone and the blue semiquinone states (Murataliev et al, 1999)
Summary
Monooxygenase enzymes occur in all kingdoms of life and cytochromes P450 (P450s) represent the largest superfamily of them (Lamb and Waterman, 2013; Munro et al, 2013). The FMN domain is in a different position, more and more distant from the rest of the protein, whereas their connecting and FAD-binding domains are strictly superimposable, demonstrating a marked reorientation of the FMN domain relative to the FAD domain (Hamdane et al, 2009) Another evidence for domain mobility has come from the structural study of a chimeric enzyme consisting of the FMN-binding domain of yeast CPR and the remainder of the molecule, including the hinge segment, from human CPR (Aigrain et al, 2009). This chimeric enzyme, which is active in NADPH reduction of cytochromes c and P450, displays a single, well-defined, molecule in the asymmetric unit. By analyzing the salt-dependent changes of the cytochrome c reductase activity in the context of both soluble and membrane-bound forms of CPR, we propose several hypotheses on the role played by these residues on the transition between the locked to the unlocked state, and in the electron transfer mechanism of CPR
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