Biochimica et Biophysica Acta 1814 (2011) 132–138

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Review

Conformational changes of the NADPH-dependent cytochrome P450 reductase in thecourse of electron transfer to cytochromes P450

Tomas Laursen, Kenneth Jensen, Birger Lindberg Møller ⁎Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, DenmarkVKR Research Centre “Pro-Active Plants”, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, DenmarkUNIK Synthetic Biology, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

⁎ Corresponding author. Plant Biochemistry LaboratorandBiotechnology, University of Copenhagen, 40ThorvaldC, Copenhagen, Denmark.

E-mail address: blm@life.ku.dk (B.L. Møller).

1570-9639/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbapap.2010.07.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 March 2010Received in revised form 9 June 2010Accepted 1 July 2010Available online 17 July 2010

Keywords:NADPH-cytochrome P450 oxidoreductaseFMN-binding domainSwinging modelRotating modelNitric oxide synthase

The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromesP450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of theprosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR undergoes largeconformational changes. This mini-review discusses the new evidence provided for such conformationalchanges involving a combination of a “swinging” and “rotating” model and highlights the molecularmechanisms by which formation of these conformations are controlled and thereby enables CPR to serve asan effective electron transferring “nano-machine”.

y, Department of Plant Biologysensvej,DK-1871 Frederiksberg

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

NADPH-dependent cytochrome P450 oxidoreductase (CPR) is a~78 kDa membrane anchored flavoprotein localized with its catalyticsite at the cytoplasmic surface of the endoplasmic reticulum [1,2]. CPRhas evolved as the result of an ancestral fusion of two differentflavoproteins, enabling it to bind one molecule each of FAD and FMN[3–5]. The tertiary structure of CPR is well conserved throughout thedifferent kingdoms demonstrating its catalytic effectiveness andimportance [6]. As inferred from its name, the main in vivo redoxpartners of CPR are cytochromes P450 (CYPs). CYPs constitute amultienzyme family of monooxygenases that catalyze regio- andstereospecific hydroxylations of a wide variety of substrates [7]. Theclassic catalytic cycle of P450 enzymes is based on heterolyticcleavage of dioxygen resulting in hydroxylation of the substrate andsimultaneous formation of water and includes two one-electrondonations by CPR [8]. Electrons are transferred from NADPH throughthe FAD and FMN coenzymes of CPR to the iron atom in the prostheticheme group of the CYPs [9]. CPR is able to transfer electrons to otherredox proteins than CYPs such as cytochrome c [2], cytochrome b5[10], heme oxygenase [11] as well as to the fatty acid elongationsystem [12]. CPR has also been identified in the outer membranes ofthe nuclear envelope [13]. The observed lower specific activity of CPR

in the nuclear membrane preparations compared to CPR from theendoplasmic reticulum could indicate that the endoplasmic reticulumis the physiological relevant environment.

The crystal structure of the solubilized rat CPR lacking the 6 kDaN-terminalmembraneanchor clearly shows thepresence of structurallydistinct domains (Fig. 1). The N-terminal part is positioned just abovethe membrane and harbours the FMN-binding domain. A ~15 aminoacid long hinge connects the FMN-binding domain to a linker domain.The linker domain is positioned adjacent to the entwined FAD andNADPH-binding domains. The architecture of CPR enables the FAD andFMN coenzymes to be positioned in close proximity for optimal inter-flavin electron transfer with a minimum distance between theisoalloxazine rings of the FAD and FMN coenzymes of 4 Å. The linkerdomain is suggested to somehow control the relative orientation of theFAD and FMN coenzymes. Interactions at the interface between thelinker and FMNdomains are likely to contribute to the tertiary structureof CPR (Fig. 1). The amino acid residues at the interface are mainlyhydrophilic and the interaction between the domains is primarilyelectrostatic [14].Whereas the “compact” structure of CPR is optimal forFAD–FMN electron transfer, it is incompatible with a proper interactionbetween the FMN-binding domain and the CYPs. This reflects that theCYP interacting residues of CPR are localized in the near surroundings ofthe FMN-coenzyme [15]. In the closed conformation of CPR, theseresidues are buried within the protein. Electron transfer from NADPHthroughCPR to theCYPheme iron thuswouldappear to require thatCPRundergoes a major conformational change. Direct evidence for themechanisms controlling and mediating the conformational change isstill elusive. The membrane anchor also affects the function of CPR as

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illustrated by the observation that soluble CPR is able to reducecytochrome c but incapable of donating electrons to membrane boundCYPs [16]. This adds to the mechanistic complexity of CPR function.

2. Inter-flavin electron transfer in CPR

In the presence of substrate and dioxygen, the monooxygenationreaction of the CPR–CYP enzyme system is dependent on the bindingof NADPH to CPR. Electrons, in the form of a hydride anion aretransferred from NADPH to FAD. Reduced FAD then transfers singleelectrons to FMN, which in turn reduces the prosthetic heme iron ofthe CYP [16,17]. NADPH coenzyme binding is regulated by aC-terminal located tryptophan or tyrosine residue. The ring systemof this aromatic residue was found to stack against the isoalloxazinering of FAD and thereby block electron transfer from NADPH to theFAD coenzyme. In the equivalent NADPH binding domain offerredoxin-NADP+ oxidoreductase, an aromatic residue fulfils thesame function [18]. Deletion or substitution of the C-terminaltryptophan residue in rat CPR (W677) resulted in a much strongerbinding of NADP+ in comparison to wild type CPR, indicating thatrepositioning of W677 can regulate the binding of NADPH and releaseof NADP+ [19]. NADP+ adopts multiple conformations in the crystalstructure of CPR. A low electron density of the ribose-nicotinamidemoiety of NADP+ indicates that this part is flexible and changesconformation during inter-flavin electron transfer [14]. The reducedFAD coenzyme transfers single electrons to the FMN coenzyme onceappropriate orientation and proximity between the coenzymes areobtained. NADP+ coenzyme binding has been shown to enhanceinter-flavin electron transfer [20]. NADP+ binding also inducesconformational changes which are not observed in the presence ofNAD+. The 2′-phosphate group of NADP+ is therefore consideredcritical for optimal electron transfer. Themaximum rate of inter-flavinelectron transfer in a two electron NADPH reduced semiquinone CPRis ~55 s−1 [21], which is considerably slower than would be predictedfrom the orientation and proximity of the FAD and FMN coenzymes

Fig. 1. Crystal structure of rat CPR following removal of the N-terminal membrane anchor byterminal the FMN binding domain (blue), linker domain (red) and FAD and NADPH binding(yellow), FAD coenzyme (orange) and NADPH (blue). The FMN domain is connected to the rinterface between the FMN (blue) and linker (red) domains comprises mainly hydrophilicbetween the isoalloxazine rings of the FMN and FAD coenzymes is 4 Å, which is optimal fo

seen in the crystal structure. The maximum transfer rate has beencalculated to be 1010 s−1 for redox centers in this particular arrange-ment [22]. Another interesting observation is the negative effect ofglycerol on electron transfer rate, suggesting that the rate is limited byconformational changes, in this case due to the more viscous nature ofglycerol [20,21,23].

3. Coenzyme binding and redox state regulates domain movement

Nuclear Magnetic Resonance and Small-Angle X-ray Scattering(SAXS) studies have recently provided direct experimental evidencethat CPR exists in equilibrium between two conformations termed“open” and “compact” [24]. In the open conformation, the solventexposed FMN coenzyme favours efficient transfer of electrons fromFMN to the heme iron. The compact conformation serves to facilitateFAD to FMN electron transfer. The equilibrium between the open andcompact form of CPR is dependent on the redox state and NADP+

coenzyme binding. Controlled by these regulators, CPR is able toshuttle between the compact conformation with a radius of gyrationof ~27 Å and the open conformation with a radius of gyration of~33 Å. The radius of gyration is a measure of the overall spread of theprotein based on the rootmean square of the distances to the center ofthe protein. The CPR conformationwas examined in the oxidized formCPRox, and in the 2-electron reduced form CPRsq or in the 4-electronreduced form CPRhq. The reduced forms of CPR were generated byaddition of NADPH and consequently have NADP+ coenzyme bound.Analyses of CPRox show an equal distribution between the open andcompact conformations whereas the fully reduced coenzyme-boundCPRhq is found predominantly in the compact conformation in a ratioof 85:15. An increase in the proportion of the compact form is seen byreduction of CPRox to the CPRsq form, but not to the same extent asseen for CPRhq. CPR reduced to the hydroquinone level by NADH doesnot alter the equilibrium, indicating that coenzyme binding isrequired before a conformation shift may take place. The weaklybinding NAD+ did not stay bound to CPR at the concentrations used.

trypsin treatment. CPR comprises a number of structurally distinct domains, from the N-domain (green). The coenzymes are shown as ball and sticks, with the FMN coenzymeest of the protein via a ~15 amino acid loop structure termed the “hinge” (yellow). Theresidues, which show electrostatic interactions. In this structure, the minimal distancer inter-flavin electron transfer. The structure is based on the PDB-file 1AMO.

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Binding of 2′,5′-ADP to CPRox resulted in a shift in conformationsimilar to the one observed for CPRsq. This shows that coenzymebinding alone is sufficient for a conformation shift but does not aloneaccount for the ratio shift from 50:50 to 85:15 between the compactand open conformations seen for CPRox and CPRhq, respectively. Onlythe fully reduced CPRhq with NADP+ bound leads to this shift inequilibrium. SAXS data were used to model a structure of the openconformation. Only a mixture of CPR in compact and open conforma-tions was able to describe the dimensions obtained from the SAXSdata (Fig. 2). In the open and compact conformations of CPR, theFMN–binding domain is envisioned to retain a position just above themembrane surface. In contrast, the FAD/NADPH-binding domain isthought to rise further above the membrane during the transitionfrom a compact to open conformation (Fig. 3).

The hinge connecting the FMN and linker domains is critical indetermining the mobility of the FMN-binding domain with respect tothe remaining part of the protein. The length of the hinge isevolutionarily fairly conserved to be ~15 amino acids. In rat CPR,deletion of four amino acids ΔTGEE in the hinge results in severestructural changes of the mutated protein. The mutant proteintransfers electrons from FMN to heme with the same rate as wildtype CPR, but is incapable of inter-flavin electron transfer. Althoughthe two Glu residues deleted in the mutant protein are known to formsalt-bridges with the FMN domain, it appeared to be the reducedlength of the hinge and not the lack of interaction that caused thedramatic structural changes. The hinge was proposed to act as a leashproviding flexibility between the FMN and FAD domains as requiredduring the conformational changes in the course of the NADPH toheme electron transfer [25,26].

A soluble yeast–human chimeric CPR (YH–CPR) has been shown tobe functionally competent with respect to FMN-heme electron transfer.The YH–CPR is composed of a yeast derived FMN-binding domain andhuman CPR derived hinge–linker–FAD–NADPH domains. YH–CPR

Fig. 2.Oxidized CPR (CPRox) exists in equilibrium between a compact and open conformationenvelope of CPR in a compact conformation with the crystal structure superimposed. Bsuperimposed. C. The extended molecular envelope with a new extended structure superthought to be involved in a new interface between the FMN and linker domain.This figure is from [24].

adopts an extraordinary open conformation in which the FMN andFAD coenzymes are separated by 86 Å compared to 4 Å in the rat CPRcompact structure [27]. The hinge was proposed to be responsible forthe reorganization of YH–CPR. However, the linker domain might alsobe envisioned to be involved in control of domainmovements. The openconformation of YH–CPR could reflect improper interaction of the yeastFMNdomain and the human linker domain [20]. The establishment of anew inter-domain interface during the conformational change fromcompact to an open form involving the FMN-binding domain, the hingeand the linker domain was proposed [24]. The putative inter-domaininteractionsbetween the FMNand linker domain support the regulatoryeffect of the linker domain in domainmovement. The open structure ofYH–CPR is not consistent with the open structure described by Ellis et al2009 and is probably not naturally occurring but the divergentconformation of the chimeric protein reflects the potential of CPR toundergo drastic conformational changes to accommodate the interac-tion with its redox partner.

The studies on the conformational changes of CPR during thecourse of NADPH to heme electron transfer have so far been biasedtowards a “swinging” model where CPR occupies an open andcompact conformation (Fig. 3). Based on the disordered FMN domainin the crystal structure of rat CPR [19], a new model for domainmovement was suggested. In this “rotating”model, the disorder foundin the FMN domain is postulated to be a consequence of a horizontalrotational domainmovement of CPR. This rotation of the FMN domainaround the flexible hinge, of CPR in the compact form, would renderthe FMN domain solvent exposed and accessible for the CYP redoxpartner and electron donation to the heme iron. In the previouslydescribed ΔTGEE mutant, three distinct open conformations of CPRwere found [26]. In the most compact form, the FMN domain wasspeculated to rotate by pivoting at the C-terminal end of the hinge. Forvisualization see http://www.molmovdb.org/cgi-bin/morph.cgi?ID=234385-8941 and Refs. [28,29]. If this horizontal rotating

. SAXS data was used to construct a model of the extended structure of CPR. A. Molecular. The extended molecular envelope with the crystal structure of the compact CPRimposed. The FMN domain is shown in light blue and residues highlighted in red are

Fig. 3. A model of CPR in open and closed conformations in a lipid bilayer. In this model, the FMN-binding domain is positioned at the membrane surface, and the open and closedconformations are illustrated as predicted to be oriented in vivo. The open structure (left) is compatible with FMN-heme electron transfer. The closed structure (right) is optimal forFAD-FMN electron transfer. The FMN domain is shown in blue, linker domain in purple, FAD and NADPH binding domain in green, FMN coenzyme as yellow spheres, FAD coenzymeas orange sticks and NADPH as blue sticks.This figure is from [24].

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movement takes place in vivo, it opens the possibility that compact aswell as open conformations of CPR may be active in electron donationfrom reduced FMN to the heme iron.

4. Hinge phosphorylation may direct CPR domain rearrangement

Nitric oxide synthase (NOS) is a diflavin protein present inmammalian cells and is structurally very similar to CPR [6,30]. InNOS, binding of calmodulin and phosphorylations regulate inter-flavin electron transfer and domain movements. Calmodulin activateselectron transfer between the FAD and FMN coenzymes andphosphorylation of a C-terminal tail “unlocks” the protein, therebyallowing the FMN domain to move and interact with the heme groupof the redox partner [31,32]. NOS have also been found to be in aconformational equilibrium between open and compact forms. Thesestudies led to the discovery that the equilibrium ratios are similar tothose reported for CPR [24]. However, whereas binding of NADP+ wasthe main regulator for CPR, calmodulin binding and phosphorylationregulates the equilibrium between the open and compact forms ofNOS [33]. Kinase mediated phosphorylation of typically Tyr, Ser or Thrresidues is a general mechanism to alter the activity or structure ofproteins [34] as well as of carbohydrate polymers [35,36]. A localconformational change resulting from a phosphorylation event andthe introduction of a highly negatively charged phosphate group canlead to dramatic changes in the overall tertiary structure of a protein.

Alternatively, phosphorylation may serve to modulate the overallsurface charge of the protein and thereby alter interaction with otherprotein domains [37]. NOS and CPR are structurally closely relatedand knowledge obtained from one protein can often be projected tothe other but CPR does not contain a regulatory binding site like thecalmodulin binding site of NOS and regulation by phosphorylationhas so far not been described. However, a search for phosphorylationsites using the NetPhos 2.0 Server [38] of the identical hingesequences from rat and human CPR revealed several putativephosphorylation sites. In rat CPR, the hinge sequence consists ofresidues 231–245 [14]. Three residues within this sequence werefound to be likely targets for phosphorylation and are highlighted inthe sequence 231FGVEATGEESSIRQY245. The equivalent hinge se-quence in the plant CPR from Sorghum bicolor CPR2b consists ofresidues 254–268 and includes two putative phosphorylation sites atthe residues highlighted in the sequence 254LLRDENDASTGTTYT268[39]. The X-ray crystallographic analysis showed that the hinge is adisordered solvent exposed structure [14]. In analogy to thestructural importance of phosphorylation of the hinge sequence inNOS, it seems reasonable to assume that phosphorylation of thehinge would have a major influence on the overall conformationalchanges of CPR. In numerous other types of proteins, missing regionsof electron density have been shown to carry out important functions[40]. Disruption of phosphorylation sites may be part of theexplanation for the open structures in the ΔTGEE mutant [25,26].

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5. Interaction of CPR and CYPs

Solvent exposure of the FMN coenzyme is a main criterion forsuccessful interaction with CYPs. The interaction between CPR andCYPs is essentially based on electrostatic forces. An uneven surfacecharge distribution makes the FMN-binding domain a strong dipole[41], with a negative face comprised of several Glu and Asp residues,known to be critical for proper interaction with CYPs [42]. It isexpected that the negatively charged carboxyl groups, organized inclusters, interact electrostatically with positively charged residuesnear the heme group. The heme group is situated in a domain on theproximal face of the CYP protein, which include six alpha-helices anda random coil. This domain comprises a considerable number ofpositively charged residues, mainly Arg and Lys residues. Mutations inthe negatively charged face cleft of CPR revealed that the binding sitesfor cytochrome c, b5 or P450 are not identical but do overlap to someextent [15,43–46]. Negatively charged residues at the surfacesurrounding the FMN coenzyme form a cleft allowing a less than9 Å distance, during CPR–CYP interactions, between the FMNcoenzyme and the heme [14]. Since electrons can travel up to 14 Åbetween protein redox centers, this docking model of CPR and CYPseems reasonable for efficient electron transfer [22]. The docking siteis found to be solvent exposed in the open conformation of CPR, arequirement for CYP interaction [24]. The course of electron transfer ishere speculated to involve either of two conformational changes, oralternatively a combination of the two. Both models share a commoncompact structure of CPR, which facilitate interflavin electron transferleading to the two electron reduced FMN coenzyme. In the course ofelectron transfer from FMN to heme CPR undergoes a conformationalchange ultimately leaving the FMN coenzyme solvent exposed andaccessible for CYP interaction. The “swinging” model involves majorconformational changes resulting in an opening of the enzyme. In thismodel, the NADPH, FAD and linker domain rises above the membraneand leaves the FMN coenzyme solvent exposed (Fig. 3). Alternatively,the “rotating”model involves a horizontal rotation of the FMN domainallowing the FMN coenzyme to shuttle between being embedded inthe CPR enzyme and being solvent exposed. The overall conforma-tional change of CPR is considerable less dramatic in this modelcompared to the “swinging” model. Finally, we speculate that thedomain movements may involve a combination of the two models.Thus, during electron transfer from FMN to heme, CPR may partly riseabove the membrane while the FMN domain rotate horizontallyultimately leaving the FMN coenzyme solvent exposed.

5.1. Metabolon formation, pros and cons

Eukaryotic CYP proteins belong to one of the largest enzyme familiesand carry out essential functions inmammalian aswell as plant cells [7].Mammalian CYPs are involved in key endogenous reactions like steroidmetabolismaswell as in themetabolismof xenobiotics [47]. In addition,plant CYPs are involved in the synthesis of an incredible variety of bio-active natural products, currently numbering ~200.000 different knownstructures [48,49]. Some of the pathways involved in biosynthesis ofbio-active natural products in plants are thought to be organized asmetabolons, in which substrates are channelled within the enzymecomplexes [50]. This serves to increase catalytic efficiency and to avoidleakage of potential toxic or labile intermediates [51]. Metabolonformation may be facilitated by small scaffolding proteins devoid ofseparate catalytic activities [52–54].The biosynthesis of dhurrin, acyanogenic glucoside derived from tyrosine, involves two CYP proteins(CYP79A1 and CYP71E1) [55–58], CPR [59] and a soluble glycosyltransferase (UGT85B1) [60]. Dhurrin formation is highly channelled andtheproteins involved in its synthesis are envisioned to formametabolon[61–63]. Association of CYPs is a fundamental feature that significantlyincreases their individual catalytic rates [64]. In a recent study of theorganization of CYPc17 and CYParom catalyzing steroid synthesis in

humans, both CYPs were observed to form homodimers whereasheterodimer formation was not observed. Complex formation betweeneach of the CYPs and CPR was observed [65]. These conclusions wereobtained using living cells or biomimeticmembranes and a combinationof fluorescence resonance energy transfer, quartz crystal microbalanceand atomic force microscopy studies. Nano disc technology providesanother excellent experimental system to study the assembly of CYPand CPR complexes by atomic force microscopy [66,67] and SAXS[68,69]. CPR is the donor of reducing equivalents to CYPs and wouldtherefore need to efficiently interact with CYPs integrated intometabolons whether these are organized as monomeric, homodimericor oligomeric structures [8]. The up to three paralogs of CPR found invascular plants opens another possibility for metabolon formation [6].No reason has so far been forthcoming forwhyplants encodemore thanone CPR, but maybe the different paralogs are functionally specializedwith respect to their ability to engage in metabolon formation? Theputative ability of CPR paralogs to control the formation of differentmetabolons would help to organize the numerous biosynthetic path-ways involved in producing the large number of bio-active naturalproducts found in plants. Unfortunately, no results have so far beenpublished to support this theory.

5.2. CPR interactions with CYPs, stable or transient?

Metabolons may be transient or stable structures and may also befunctioning-dependent structures, the assembly of which is dependenton the presence of substrate [51,70]. Transient metabolon formationoffers a way to optimize biological processes in response to intra- andextracellular compounds. Formation of transient metabolons may bededuced from the experiments involving differential acetylation ofLys residues of CYP17α in the presence and absence of CPR [71].MALDI-TOF mass spectrometry revealed that the presence of CPRleads to a steric hindrance of acetylation of specific Lys residues for upto 15 min when the experiment is carried out at 4 °C. The stableinteraction at 4 °C could be an artefact derived from the reducedfluidity of themembrane at the low temperature.When incubated at amore physiological temperature (20 °C for 15 min), the presence ofCPR had no effect on the acetylation, meaning that the interaction istransient not stable under these conditions [71], which supports thetheory that CPR interacts transiently with CYP metabolons.

The ratio between CYP and CPR proteins of approximately 15:1observed in mammalian cells also speak for a transient interactionbetween CPR and CYPs [72]. This stoichiometric imbalance wouldimply that CPR is constantly shuttling between different CYPs andnot bound integral partners of CYP metabolons. However, the twomodels of conformational changes during electron transfer fromNADPH to heme, the “swinging” model and the “rotating” modeldescribed above, speaks for either a transient or stable interaction,respectively. The architecture of CPR in the “swinging”model arguesfor a transient interaction whereas the “rotating” model, in theory,would allow a stable CPR–CYP interaction while the FMN domainshuttles the FMN coenzyme between the FAD coenzyme and theheme of the CYP.

5.3. Microdomains, a way to fine tune metabolon formation

Co-location of CPR and CYPs within the same membrane systemserves to counteract the overall stoichiometric imbalance. Thusoverexpression of CYPs in plants does generally not result inphenotypic changes e.g. dwarfed phenotypes as a result of reducedactivity of CYPs involved in gibberellin synthesis [61,73,74]. Thisimplies that the shuttling of CPR between different CYPs is not a ratelimiting factor for the flux of carbon through CYP catalyzed pathways.

Microdomains present within the endoplasmic reticulum couldserve to further optimize CPR interaction with specific CYPs. The lipidbilayer has been suggested to play a role in the interactions between

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CPR and CYPs [54]. In addition to the membrane spanning anchor ofCPR, several basic amino acid residues at the membrane facing part(lower surface) of the FMN domain form a patch, which maycontribute to the membrane association of CPR. The contribution ofthe patch towards membrane binding is probably minor, given thattrypsin treatment of many CPRs results in cleavage of the membraneanchor and solubilisation of CPR. Instead, a weak interaction betweenthe positively charged residues of the patch and negatively chargedlipids [14,41] could affect the redox potential of CPR, makinginteractions with CYPs more favourable [75]. Studies of the humanCYP3A4 showed that negatively charged membranes facilitate theinteraction with CPR [76]. The membrane interacting patch is notdirectly involved in CYP interaction rather than the membrane chargefavours electron transfer [77] but may also serve the function to directCPR into microdomains with specific lipid compositions and therebyact as a guidance to arrange CPR with selected CYP partners. Lipidmembrane microdomains, lipid rafts, which are rich in cholesterol,saturated fatty acids and sphingolipids form stable platforms in theliquid disordered membrane that in response to environmentalchanges can rapidly dissociate and associate [78]. In the formationof metabolons it has been demonstrated that substrates may mediateassociation of the enzymes involved [79–82]. The dhurrin metabolonincludes two CYPs, however, it is not known whether one or two CPRproteins are complexed in this metabolon or if the role of CPR isstrictly transient [63]. Based on the “swinging” model the CPR–CYPinteraction is destined to be somewhat transient because of the largeconformational changes now envisioned to take place during electrontransfer as previously discussed. Arrangement of CPR and CYPs inspecific membrane microdomains would significantly increase thecoincidence of interaction and thereby optimize turnover rate. Withinthese microdomains, membrane charge, substrate accessibility andphosphorylation might all be ways to activate CYPs and CPR bymodulating the conformation and redox potential.

6. Conclusion

It has been known for decades that CPRmediates electron transfer toeukaryotic CYPswith a highly diverse range of substrates. As outlined inthis review, the knowledge gained within the last few years has greatlyimproved our understanding on howCPR and CYP interact. The rotatingmovement of the FMN-binding domain as mediated by the flexiblehinge could be a key mechanism to enable tight binding of CPR to CYPsin aNADPH-hemeelectron transfer compatible conformation.A rotationwould allow CPR to remain bound to the CYP and shuttle electrontransfer between the FADcoenzyme to the FMNcoenzyme and from thereduced FMN to the heme. So far there are no indications that otherdomains are involved in interactions with CYPs and the patchesproposed to be responsible for CPR–CYP interaction are located closeto the catalytic centers. However, future studies may reveal that CPR islocalized within specific microdomains in the endoplasmic reticulum,which could facilitate interaction with CYPs. Furthermore, additionalbut yet undiscovered dynamic features in the CPR structure could serveto catalyze the required electron transfer fromNADPH to the heme ironrequired for CYPs to carry outmonohydroxylations. It will be exciting tounderstand more of the secrets behind this versatile “nano-machine”.

Acknowledgements

The authors gratefully acknowledge financial support from theDanish Council on Technology and Production Sciences, the VillumKann Rasmussen Foundation to the research center “Pro-ActivePlants” and the research initiative “UNIK Synthetic Biology” fundedby the Danish Ministry of Science, Technology and Innovation. TheFaculty of Life Sciences, University of Copenhagen is acknowledged forgranting a PhD stipend to Kenneth Jensen.

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