**1. Introduction**

Microsomal cytochrome P450 (CYP) metabolism requires a coupled supply of electrons, which are donated by the auxiliary protein NADPH cytochrome P450 oxidoreductase (CPR). CPR, encoded by the *POR* gene, is a ~78-kDa electron-transferring diflavin enzyme anchored to the membrane of the endoplasmic reticulum [1]. CPR mediates a two-electron transfer (ET) per reaction cycle, originated from NADPH enabling CYP-mediated metabolism of many compounds. These include endobiotics, e.g., steroids, bile acids, vitamins and arachidonic acid metabolites, as well as many xenobiotics, including therapeutic drugs and environmental toxins [2,3]. Moreover, CPR is the unique electron supplier of heme oxygenase, squalene monooxygenase and fatty acid elongase [4], sustaining exclusively the activity of these enzymes. Cytochrome *b*<sup>5</sup> (CYB5) can donate the second electron to CYP, competing with CPR for the binding site on the proximal side of CYP [5]. CYB5's interaction may have either a stimulating, inhibiting or having no effect over CYP catalytic activity, which seems to be CYP isoform and even substrate dependent [6].

CPR comprises a number of structurally distinct domains namely an N-terminal hydrophobic membrane anchoring domain; two flavin binding domains for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN); a linker domain joining the FMN and FAD domains, as the provider of structural flexibility; and an NADPH binding domain [7]. The hinge segment, a highly flexible stretch with no defined secondary structure links the FMN and the connecting/FAD domain [8–10]. Electrons are transferred from NADPH through FAD (reductase) and FMN (transporter) coenzymes of CPR to redox partners, such as to the heme group in the reactive center of CYP [11].

Initial structural studies of CPR identified compact conformations that allowed internal (inter-flavin) ET, but were unable to reduce external acceptors [8,12,13]. Subsequently, three separate studies identified different open structures of CPR that allowed ET to redox partners, indicative of domain motion of CPR [10,14,15]. It is now fairly established that CPR exists in a conformational equilibrium between open and closed states in its ET function, which is highly dependent on ionic strength conditions [10,16,17]. The transition between these states appears to occur through a rapid swinging and rotational movement [17,18]. Certain residues in the hinge region have been suggested to be of importance for these large conformational changes [19], and seem to form a conformational axis, involved in a partial rotational movement of the FMN domain relative to the remainder of the protein [10,18].

Analysis of CPR domain dynamics is pertinent to understand its role in the interactions with its natural redox partners and its gated ET function. The affinities between CPR and CYP have been indicated among the factors modulating the protein dynamics of CPR. Different CYP isoforms may be differently served by CPR gating its ET differentially [20,21]. Although advances obtained during the last decade, CPR's structural features controlling ET are not yet properly identified. CPR mutations may perturb specific structural requisites, necessary for the optimal transition between open and closed conformations, as well as disturb the interaction of CPR with its redox partners [10,20–22].

Previously, we have studied the effect of mutations in CPR on its redox partners [20,22,23] and the effect of alterations in the hinge segment in CPR-dependent cytochrome *c* reduction [24]. These hinge mutations showed differential effects on the conformational equilibrium of CPR and ET efficiency to cytochrome *c*, a non-physiological redox partner of CPR. Through modulation of the ionic strength conditions we demonstrated that electrostatic and flexibility properties of the hinge are critical for ET function, in which CPR's membrane anchoring was shown to play an important role [24]. Although frequently used as a surrogate, the soluble cytochrome *c* has been indicated to interact differently with CPR, when compared with interactions of natural membrane-bound partners, such as CYP [25]. The use of cytochrome *c* as redox partner may have obscured additional important clues on structural features of the hinge segment involved in CPR's open/closed dynamics and its gated ET function. To address this issue, three hinge mutants were selected from the initial eight mutants of our former study, based on their specific phenotypes in cytochrome *c* reduction. Human membrane bound CPR mutants S243P, I245P and R246A (numbering according to the human CPR consensus amino acid sequence NP\_000932) were each combined with three different human CYPs, namely CYP1A2, 2A6 or 3A4, representatives of three major CYP families involved in drug metabolism [2,3]. The effect of the structural deviations of the three mutants was probed to obtain further insights on the role of the hinge segment of CPR in the interaction and ET with these physiological redox partners, using different experimental approaches.

#### **2. Results**

#### *2.1. Bacterial Coexpression of CPR Mutants and CYP*

Wild-type CPR and CPR hinge mutants S243P, I245P and R246A were separately introduced in the *E. coli* BTC strain and co-expressed with CYP1A2, 2A6 or 3A4, using methods described previously [26,27]. CYP expression levels were determined in bacterial whole-cells (Table 1). When co-expressed with CPR variants, CYP expression levels varied between 109–241 nM, 96–130 nM and 105–143 nM for CYP1A2, 2A6 and 3A4, respectively. Expression levels for these CYPs were comparable with those found previously with BTC strains [22,28,29]. More importantly, no large deviations were found in the CPR:CYP ratio between the four CPR variants, when expressed with each of the three CYPs (see Table 1). This enabled us to ascribe differences in activities of the CPR variants to the mutations, and not to variations in the stoichiometry between the two enzymes. These ratios were actually similar to those observed in our previous studies [22,28,29] and are in the range of those observed in human liver microsomes [30,31].


**Table 1.** Microsomal cytochrome P450 (CYP) and NADPH cytochrome P450 oxidoreductase (CPR) contents of BTC cultures and membrane fractions.

<sup>1</sup> CYP and CPR contents are mean ± sd.
