protein  protein 
 interactions are essential to the function 
 of the ubiquitous xenobiotic - metabolizing cytochrome p450 monooxygenases , 
 or cyps .
classic studies have demonstrated 
 the importance of cyp interactions with the accessory proteins cytochrome 
 p450 reductase ( cpr ) and cytochrome b5 ( b5 ) for both electron transfer 
 and allosteric modulation .
however , it has recently become 
 clear that cyp enzymes may participate in additional interactions 
 with other protein partners , such as progesterone receptor membrane 
 component 1 ( pgrmc1 ) , human serum albumin ( hsa ) , and even other cyp 
 isoforms .
these interactions have been shown to alter cyp function 
 and , in some cases , lead to the production of toxic metabolites . in 
 this review
, we attempt to summarize the available evidence from the 
 primary literature and offer some future perspectives on the study 
 of noncanonical protein  protein interactions with cytochrome 
 p450 enzymes .
cytochrome 
 p450 reductase ( cpr ) was identified as the primary cyp electron transfer 
 partner by the late 1960s .
the term cytochrome 
 p450 reductase is a bit of a misnomer , as this enzyme participates 
 as a reducing partner in a number of different reactions , including 
 those catalyzed by heme oxygenases i and ii , squalene monooxygenase , 
 and certain fatty acid desaturases . initially characterized as a cytochrome c reductase , cpr was eventually found to be localized in 
 the endoplasmic reticulum , where it functions as a redox partner with 
 cyps that are also located there .
cpr is a diflavin ( fmn , fad ) containing 
 reductase that receives its electron - reducing equivalents from nadph .
the cpr electron transfer protein shuttles two electrons to the 
 cyp during the reaction cycle , with the second electron transfer thought 
 to be the rate - limiting step .
cpr is known to be post - translationally modified and has an apparent 
 molecular weight between 76 and 80 kda , depending on post - translational 
 modifications , the species - specific isoform , and detergent solubilization 
 procedures .
in addition to the fmn , fad , 
 and nadph binding domains , cpr also contains a 60 amino acid hydrophobic 
 n - terminal membrane anchoring region , which is essential for full 
 activity with native redox partners , such as cytochrome p450 enzymes .
a number of important crystal structures 
 of cpr and several of its mutants have been produced , including those 
 demonstrating important conformational changes that take place upon 
 reduction and cyp binding .
the first crystal structure of cpr was obtained using an n - terminally 
 truncated sequence of the rat isoform .
this structure 
 demonstrated that the nadph  fad binding domain also contained 
 an important linker sequence that could help position the fmn and 
 fad domains in close proximity for efficient electron transfer .
the 
 linker sequence distinguishes cpr from the closely related , single - domain 
 ferrodoxin reductases .
it is worthwhile to note that this particular 
 n - terminally truncated construct was not able to support cyp reduction , 
 even though it successfully reduced cytochrome c. more recent x - ray 
 crystal structures have provided some insight into cyp 
the crystal structure of a cpr 
 mutant with a four amino acid deletion in the hinge region between 
 the fmn binding domain and the rest of the protein demonstrated remarkable 
 flexibility in this region by crystallizing in three different conformations .
although this particular mutant was not able 
 to catalyze intramolecular electron transfer between the fad and fmn 
 prosthetic groups , it was able to successfully reduce cyps , given 
 enough reducing equivalents .
the three different conformations observed 
 suggest that cpr inherently possesses a high degree of conformational 
 plasticity , especially in the c terminus of the hinge region , allowing 
 it to efficiently interact with a variety of cyp isoforms and other 
 enzymes .
other structures obtained from protein x - ray crystallography 
 and nmr have confirmed this conformational flexibility and the ability 
 of cpr to transition between closed and open forms , which had also been predicted by computational 
 molecular dynamics studies .
another intriguing 
 possibility is that subtle structural differences between cyp isoforms 
 may regulate interaction with cpr and cytochrome b5 .
seminal work examining structural and functional variation 
 in the 
 cpr gene has been conducted by both the masters and kranendonk groups , underscoring the importance 
 of cpr in drug disposition and disease .
several mutations in the gene 
 encoding cpr are known to be associated with specific disease states , 
 such as a rare form of congenital adrenal hyperplasia known as antley 
amino acid 
 changes that are implicated in these conditions include y181d , a457h , 
 y459h , v492e , and r616x .
surprisingly , despite the large number 
 of cyps present in the human 
 genome ( 57 , excluding possible pseudogenes ; http://drnelson.uthsc.edu/hum.html ) , there is only one gene encoding cpr .
additionally , the total cyp 
 content of the human liver is estimated to be between 5- and 25-fold 
 higher than that of cpr , suggesting differential expression .
the fact that cyps and cpr interact in a 1:1 stoichiometry raises 
 an unusual conundrum : exactly how do cyps and cpr interact to promote 
 efficient oxidation of substrates ? from a structural perspective , 
 it stands to reason that all of the different cyp isoforms must share 
 a common functional cpr binding surface .
indeed , several studies have 
 proposed that the binding interaction between cyp and cpr takes place 
 between the positively charged proximal surface near the cyp heme 
 and the negatively charged fmn and fad / nadph domains of cpr , suggesting 
 that their interaction is primarily driven by electrostatics .
however , there is some controversy in this regard , as other studies 
 have suggested that hydrophobic interactions are more predominant .
these discrepancies may be the result of the different experimental 
 approaches employed ( e.g. , x - ray crystallography vs site - directed 
 mutagenesis and stopped - flow fluorescence spectroscopy ) .
cpr 
 complex is remarkably low , between 5 and 110 nm , depending on the 
 particular isoform used and whether substrate is present , implying 
 a very high - affinity interaction . upon initial 
 examination ,
a tight - binding interaction may seem counterintuitive ; 
 however , a rapid kon or koff rate would allow the system to work more efficiently 
 due to swift equilibration .
clearly , 
 the interaction can be modulated by the presence of substrates and/or 
 inhibitors , as well as other cyp isoforms , as noted below .
another 
 compounding factor is the variety of methods , often indirect , that 
 have been used to determine cyp 
separate from its 
 function as an electron transfer partner , cpr 
 may also act as an allosteric effector to induce conformational changes 
 in cyps that modulate the rate of electron transfer .
a number of useful structural studies conducted with the bacterial 
 model cyp isoform , cyp101 ( p450cam ) , have been useful to inform our 
 understanding of how cpr might interact with mammalian cyps in order 
 to modulate electron transfer .
trans isomerization redox switch in cyp101 ( p450cam ) 
 that is triggered by binding to the reduced form of cyp101 s 
 cognate electron transfer partner , putidaredoxin ( pdx ) .
these conformational changes account for most 
 of the significant structural perturbations observed by nmr upon complex 
 formation .
conformational plasticity 
 may be one mechanism by which the binding affinity of a cyp and its 
 redox partner are modulated : if the subset of conformational states 
 occupied by the reduced electron transfer protein is the same as that 
 selected upon binding to a cognate cyp , then the entropic cost ( i.e. , 
 free energy ) of binding would be lowered , resulting in a higher affinity . in a unique approach to examine cyp 
electron 
 transfer partner 
 interactions , hiruma and colleagues utilized paramagnetic nmr in conjunction 
 with a site - specific lanthanide label to solve the solution structure 
 of the cyp101pdx complex .
this 
 cyp101pdx complex structure was , in turn , validated by simultaneous 
 determination of the x - ray crystal structure of the same complex . 

in contrast to reports of the interaction between mammalian cyps and 
 cpr , here hydrophobic networks dominated the binding interface between 
 the two proteins .
the data suggested a minor conformational substate 
 ( or substates ) that could represent an encounter complex between the 
 two proteins , as postulated by an earlier study .
additionally , the retrieved structural parameters were 
 consistent with an electron transfer rate theoretically faster than 
 that observed experimentally , suggesting the involvement of a gating 
 process in electron transfer .
similar allosterically 
 induced conformational changes have also been observed with mammalian 
 cyps and cytochrome b5 .
taken together , 
 these studies underscore the dynamic nature between cyps and their 
 electron transfer partners , both from a functional standpoint as well 
 as a structural one .
seemingly small structural changes , such 
 as a single amino acid 
 substitution , could have dramatic functional consequences on the ability 
 of cpr to efficiently deliver its electrons to a particular cyp isoform . 

recent studies have examined how genetic variation in the human cpr 
 gene can manifest itself in the form of alterations 
 in the isoform dependent cyp - mediated metabolism of certain substrates 
 ( table 1 ) .
interestingly , the effects of specific 
 cpr mutations seem to be cyp isoform dependent , suggesting a unique 
 interaction surface between individual cyp isoforms and cpr . in a 
 study examining the effect of the 35 most common cpr mutations on 
 the activity of cyp1a2 and cyp2c19 , miller and colleagues discovered 
 that while most mutations either decreased the activity of both cyp1a2 
 and cyp2c19 , or eliminated activity all together ( most notably the 
 a287p and r457h mutations ) , certain mutations enhanced cyp activity . in most cases , the enhancements 
 were specific to the cyp isoform examined and the particular cpr mutation . 
 for cyp1a2 , activity enhancement was observed with cpr mutants g213e , 
 r406h , and a462 t . in the case of cyp2c19 , increased activity was reported 
 for cpr mutants m263v and g504r .
most surprisingly , the q153r cpr 
 mutation , a rare disease - causing mutation that inhibits steroidogenic 
 activity , increased the activity of both cyp1a2 and cyp2c19 to 144 
 and 284% , respectively .
the authors rationalized these results by 
 suggesting that many of the cpr mutants with reduced activity had 
 decreased fad binding or lay in a region known to be important for 
 cpr  cyp contacts . the cpr gain - of - function
mutants were more 
 difficult to explain , but miller and colleagues suggest that , at least 
 in the case of the q153r mutant , electron transfer rates to the cyp 
 may be affected by the close proximity of glutamine residue 153 to 
 the fmn moiety .
the change in electrostatic 
 charge in the q153r mutant may accelerate electron transfer from cpr 
 to the cyp . whatever the mechanism involved , it is clear that there 
 are important functional consequences for subtle alterations in the 
 interactions between cpr and cyp isoforms .
a follow - up study examined 
 the effects of cpr mutations on the ability of cyp2d6 to metabolize 
 different substrates .
interestingly , 
 for any particular cpr mutation , different effects were seen depending 
 on the substrate used . the q153r cpr mutation mentioned above
had 
 a slight stimulatory effect on the cyp2d6-mediated metabolism of 2h-1-benzopyran-3-carbonitrile,7-(ethoxy - methoxy)-2-oxo-(9cl ) 
 ( eomcc ) , yet it almost doubled the rate of metabolism of dextromethorphan . 

this observation may speak to the promiscuous nature of drug - metabolizing 
 cyps and the multiple substrate binding sites within the active site .
additional work with the cyp3a4 isoform also indicated that it was 
 susceptible to functional variation due to cpr mutations .
consistent with the previous 
 findings for cyp2d6 and cyp1a2 , cyp3a4 activity is decreased in combination 
 with most of the cpr mutants examined , but it is increased with the 
 q153r mutant .
this effect was again substrate - specific 
 for the substrates testosterone and quinidine , and nominal activity 
 was observed for midazolam and erythromycin oxidation .
a study utilizing the full - length 
 cyp and cpr mutant isoforms simultaneously 
 expressed in insect sf9 cells via a baculovirus vector recapitulated 
 the results obtained with the bacterially expressed isoforms ( referenced 
 above ) .
a result unique to this study 
 was the observation that some of the reduced - function cpr mutants 
 regained activity when coexpressed with cyp isoforms in the sf9 insect 
 cells .
this suggests that mutant cpr association with a cyp may help 
 to stabilize the cpr protein into an active conformation , thereby 
 rescuing activity .
this is an intriguing possibility , given the postulate 
 by pochapsky regarding the ability of a cyp to select the active conformation 
 of a particular electron transfer partner .
additional studies have pointed to the importance of the n - terminal 
 hydrophobic anchoring sequence in supporting cyp - mediated oxidation . 

hayashi and colleagues found that an n - terminally truncated form of 
 rat cpr failed to support cyp1a1-mediated 7-ethoxycoumarin o - deethylation , although it efficiently reduced cytochrome 
 c and rat heme oxygenase i. furthermore , 
 structural studies have demonstrated that crystallized , yet catalytically 
 active , yeast cpr has a substantially different structure than the conventional n - terminally truncated 
 catalytically inactive rat enzyme , reinforcing 
 the importance of this sequence in supporting efficient electron transfer 
 from cpr to cyp .
cpr genetic variants may also have effects 
 on different cyp isoform 
 alleles as well . in a study examining 

the cyp2c9 alleles cyp2c9.1 , cyp2c9.2 , and cyp2c9.3 , tracy and colleagues 
 found increased metabolic oxidation of the substrates diclofenac , 
 flurbiprofen , and tolbutamide with four different mutant cpr enzymes 
 tested ( q153r , a287p , r457h , and a503v ) .
this was in contrast to the predominate effect seen previously with 
 cyp1a2 , cyp2d6 , cyp2b6 , and cyp3a4 , where a decrease in activity was 
 reported with these cpr mutants .
the authors noted that since the 
 effects they observed led to an increase in the vmax , but not a change in the km , it was most likely that the increase in activity observed was due 
 to enhanced electron transfer and not an increase in cyp substrate 
 affinity .
variation in the cpr 
 gene can lead directly to toxicity for certain 
 drugs that are metabolized through reduction .
the herbicide 
 paraquat produces toxicity through the generation of superoxide anion 
 during redox cycling .
examined the effect of six common cpr mutations 
 in patients with impaired steroidogenesis on the redox cycling of 
 paraquat in a cho cell model system .
in 
 five out of the six cpr mutants examined , paraquat toxicity was diminished . 
 although not explicitly confirmed in this study , the authors inferred 
 that the mutants either affected the catalytic activity or expression 
 levels of cpr , thereby reducing its ability to activate paraquat . 
 in the c569y mutant ,
the observed paraquat toxicity was equivalent 
 to that with the wild - type enzyme , implying a rescue of function with 
 this particular mutation .
the authors suggest that variation in the 
 cpr gene may be a primary susceptibility determinant in paraquat toxicity . 

similarly , in a study investigating the toxicity of mitomycin c , wang 
 et al . found that cpr mutations may be useful as biomarkers to predict 
 the therapeutic response to this anticancer drug .
mitomycin c is a common chemotherapeutic agent that is 
 metabolically activated by either a one- or two - electron reduction 
 step .
cpr is the primary enzyme responsible 
 for the one electron reduction , which produces the semiquinone anion 
 radical that is thought cause damage to cellular dna . of six cpr mutants 
 that were examined ( y181d , a287p
, r457h , v492e , c569y , and v608f ) , 
 cellular toxicity of mitomycin c was decreased in all with the exception 
 of the c569y mutant , demonstrating that a high degree of cpr functional 
 activity is needed to activate mitomycin c. in particular , the v608f and y181d mutants lacked any ability to 
 activate mitomycin c , presumably due to altered association with the 
 flavin moiety .
in addition to mitomycin c , the impact of cpr 
 variants has also 
 been examined with tacrolimus , warfarin , and other drugs , illustrating the importance 
 of this particular enzyme in drug metabolism and elimination .
cytochrome b5 ( b5 ) 
 is a small ( 17 kda ) heme - containing integral membrane protein 
 located on the cytosolic side of the endoplasmic reticulum and was originally identified 
 in 1950 in silkworm larva . although the 
 majority of b5 is membrane - bound , a certain fraction is present as 
 a soluble , cytosolic form in erythrocytes , where it catalyzes the 
 reduction of methemoglobin .
although 
 mutations in the b5 gene itself are quite rare , a few have been traced 
 to the congenital abnormality of 17,20-lyase deficiency . despite the fact that it can act as an electron donor to cyp enzymes , 
 b5 is primarily involved in lipid biosynthesis , delivering electrons 
 to microsomal desaturases that synthesize steroids , fatty acids , and 
 plasmalogens . in the early 1970s ,
estabrook 
 and mannering first demonstrated that b5 stimulated the metabolism 
 of several cyp substrates .
however , at this time , 
 the functional role b5 played in cyp - catalyzed oxidation was still 
 somewhat unclear .
early reports suggested that it could be involved 
 in delivery of the second electron in the cyp catalytic cycle , thereby 
 accelerating the rate - limiting step in cyp - mediated catalysis .
remarkably , a more contemporary 
 report from the wolf laboratory in a hepatic cpr deletion mouse model 
 established that b5 and b5 reductase can act as efficient cyp electron 
 donors , without the need of cpr . despite 
 its role in electron transfer ,
the effects seen from b5 are highly 
 variable and dependent on the particular reaction conditions used , 
 as addition of b5 can either inhibit or have no effect on some reactions 
 rather than stimulate them . furthermore , several studies have demonstrated that addition of 
 apo ( heme free ) b5 can stimulate the activities of many cyps , including 
 cyp3a4 , cyp2c9 , and cyp17 .
it has been postulated that , in certain cases at least , b5 exerts 
 the majority of its effects in the absence of electron transfer .
it is still not completely clear if b5 functions solely as an electron 
 donor , allosteric modulator , electron sink , or an uncoupling inhibitor .
it is entirely plausible that 
 its function may be contextual , depending on the particular isoform / substrate 
 combination with which it is interacting .
a number of elegant 
 studies over the years have established that electrostatic interactions 
 are the basis for formation of the cyp 
waskell and colleagues mapped out the binding interface between 
 cyp2b4 and b5 through extensive site - directed mutagenesis of both 
 proteins and identified r122 , r126 , r133 , k139 , and k433 of cyp2b4 
 as residues critical to their interaction .
recently , a number of structural and dynamic 2d nmr studies 
 have 
 provided additional insight into the cyp 
embedded in lipid bicells , 
 ramamoorthy , waskell , and colleagues were able to take advantage of 
 rotating frame separated local field ( slf ) nmr .
the authors employed two - dimensional proton evolved local - field 
 ( 2d pelf ) pulse sequences to accurately measure a broad range of heteronuclear 
 dipolar couplings , allowing for a complete mapping of b5 protein dynamics 
 in a native lipid bilayer environment .
notably , the authors discovered that the hydrophobic n - terminal 
  helix of b5 precessed in a cone with an approximate 8 
 fluctuation in the tilt of the helix , which is estimated to be 13 , 
 suggesting that the lipid - embedded hydrophobic n - terminus is more 
 mobile than originally thought .
this 
 added flexibility may allow this region of the protein to interact 
 with a diverse variety of cyp isoforms .
there is a growing appreciation 
 of how inherent protein flexibility can allow proteins to adopt non - native 
 states , which may allow them to gain new functions or interact with 
 different protein partners .
, ramamoorthy and colleagues 
 utilized a modified inept technique , dipolar enhanced polarization 
 transfer ( drept ) , to detect an increased range of residual dipolar 
 couplings from isotopically labeled b5 that allowed them to characterize 
 the amino acid side chain motions of b5 while embedded in lipid bicells .
two - dimensional nmr methodologies have 
 also been useful in providing 
 explanations for the specific rate enhancements seen with certain 
 cyp  substrate pairs .
the bifunctional steroidogenic enzyme 
 cyp17a1 performs both steroid hydroxylation , which is unaffected by 
 b5 , and an androgen - forming lyase reaction that is enhanced 10-fold 
 in its presence .
work by the scott group examining 
 the interactions between an isotopically labeled b5 construct and 
 cyp17a1 led to the observation that the cyp17a1b5 interaction 
 is stronger when the hydroxylase substrate pregnenolone is present 
 in the cyp17a1 active site than when the lyase substrate 17-hydroxypregnenolone 
 is bound .
this suggests that , at least 
 in this case , the b5 reaction rate enhancement may be primarily driven 
 by substrate affinity . in a follow - up 

study , in which cyp17a1 was isotopically labeled and the b5 construct 
 was unlabeled , titration of b5 into cyp17a1pregnenolone complex 
 induced a set of conformational substates closely resembling those 
 of the cyp17a117-hydroxypregnenolone complex without 
 b5 .
this result implied that b5 may 
 also be able to allosterically induce catalytically productive conformations 
 in cyp17a1 , even in the absence of substrate .
these studies 
 as well as others have culminated in a complete structural and catalytic model for 
 the cyp 
ahuja 
 et al . determined the structure of full - length , membrane bound b5 
 in the presence of cyp2b4 .
this structure , 
 determined for the first time in the presence of a phospholipid bilayer , 
 confirmed the electrostatic nature of the cyp 
b5 interaction , 
 revealing a large number of charge pairings between surface residues 
 on cyp2b4 and b5 that facilitate the interaction between the two proteins . 
 however , it also hinted at the importance of hydrophobic interactions 
 for the complex that are mediated by the phospholipid bilayer .
interestingly , 
 the authors described an increase in the affinity between cyp2b4 and 
 b5 in the presence of a small molecule substrate or inhibitor , similar 
 to that observed by estrada et al .
for the cyp17a1b5 interaction . additionally , their results suggest a pathway 
 for electron transfer between cyp2b4 and b5 mediated through a salt 
 bridge with arg125 of cyp2b4 and the heme propionates of b5 .
this study illustrates the power of 2d nmr techniques 
 to obtain detailed structural data for the interactions between two 
 protein partners .
finn and colleagues created a liver - specific 
 b5 conditional knockout 
 mouse in order to better define the functional importance of the cyp 
b5 
 interaction.in vitro metabolism studies demonstrated the complete lack of nadh - mediated 
 metabolism and a 50 to 90% decrease in nadph - mediated metabolism for 
 most of the drug substrates examined . additionally , a dramatic increase 
 in the half - life for these particular drug substrates was noted , thus 
 establishing that b5 is a major contributor to the in vivo metabolism of many drugs and xenobiotics , albeit in a substrate- 
 and isoform - selective manner .
a follow - up 
 study demonstrated altered clearance of several anticancer agents 
 in the same knockout mouse model .
therefore , 
 mutant b5 alleles that are deficient in their ability to interact 
 with , and transfer electrons to , hepatic cyps could lead to altered 
 clearance rates for certain drugs , which may contribute to drug - induced 
 toxicities .
furthermore , varying levels of b5 function important 
 for drug oxidation 
 can also produce altered metabolite ratios , potentially increasing 
 the production of toxic products in certain instances . in a study 
 conducted by the hollenberg group ,
b5 was determined to be essential 
 for the formation of protein adducts to cyp3a5 from metabolic activation 
 of the contraceptive agent 17-ethynylestradiol . in the absence of b5 ,
the reactive 17-oxirene - related 
 species was not formed , leading to cyp enzyme protection .
an additional 
 concern from a toxicological point of view is the potential for the 
 generation of reactive oxygen species ( ros ) due to increased uncoupling . indeed , using a reconstituted system containing 
 both lipid and cpr , b5 has been shown to generate superoxide and hydroxyl 
 radicals in the presence of cr(vi ) , leading to double - stranded dna 
 breaks in vitro.(109,110 ) the fact 
 that cpr was needed to observe the effect suggests the importance 
 of the interaction between b5 and cpr to generate these ros .
additionally , 
 ros generated by b5 have been demonstrated to increase lipid peroxidation 
 in reconstituted biological membranes , which may in turn lead to cellular 
 damage and death .
therefore , there are 
 multiple mechanisms through which altered b5 levels and/or function 
 can exert a toxicological effect .
surprisingly , cyps also exhibit protein  protein 
 interactions 
 with other cyps . in the early 1990s ,
research by coon , davydov , and 
 others demonstrated that cyps can form homomeric complexes .
work in the ensuing 2 decades has established that multiple cyp 
 isoforms can interact with one another in both homomeric and heteromeric 
 complexes and that these complexes can often have significant effects 
 on cyp - mediated oxidation of substrates ( for recent reviews , please see references ( 117 ) and ( 118 ) ) . for simplicity
, we 
 have grouped the cyp  cyp interactions into those described 
 by homomeric ( two or more molecules of the same isoform ) and heteromeric 
 ( two or more molecules of different isoforms ) .
pioneering studies of homomeric 
 cyp  cyp interactions were 
 conducted by davydov in the early to mid-1990s , examining the response 
 of cyp2b4 to pressure induced conformational changes .
davydov and his colleagues discovered that approximately 35% of 
 the ferrous cyp2b4 co - bound complex was refractory to conversion into 
 the inactive p420 form upon application of pressures in excess of 
 2 kbar .
however , this refractory population 
 was reduced to less than 5% upon addition of 0.2% triton n-101 , a 
 detergent that created conditions known to produce monomers .
these 
 results implied that a significant portion of active cyp ( i.e. , cyp 
 able to be reduced and bind co ) was present in the oligomeric state
. 
 later work by this same group would demonstrate that sodium dithionite 
 or cpr induced reduction kinetics in cyp3a4 are multiphasic under 
 conditions favoring cyp3a4 oligomers but monophasic under conditions 
 favoring monomers .
these studies are 
 suggestive of active cyp arranged in homomeric oligomers in the phospholipid 
 bilayer . confirmation of a direct physical interaction between 
 individual cyps in a live cell membrane was achieved in seminal studies 
 by kemper and colleagues using fluorescently labeled cyp2e1 and cyp2c2 
 in cells transfected with murine cyp cdna .
they found that while fluorescence resonance energy transfer ( fret ) 
 occurred between individual cyp2c2 molecules in a membrane , it could 
 not be detected between cyp2e1 monomers , representing a homomeric 
 self - association with cyp2c2 but , interestingly , not cyp2e1 .
the cyp2c2 dimers were later confirmed to exist 
 in murine hepatocyte endoplasmic reticulum membranes , indicating the in vivo relevance of such associations . taken together , these studies laid the groundwork 
 for analysis 
 of the functional consequences of cyp  cyp homomeric interactions . 
 in an elegant kinetic study conducted by jamakhandi et al .
, the authors 
 found that metabolism data for cyp2e1 fit best to either a quaternary 
 ( cyp  cpr  cyp  cpr ) , trimeric ( cyp  cyp  cpr ) , 
 or dimeric ( cyp  cyp ) complex model where only the binary ( cyp  cpr ) 
 complex was active .
the authors found 
 that simply varying the individual concentrations of cyp or cpr in 
 a reconstitution system produced contradictory results , which did 
 not correspond to the simplest functional ( 1:1 ) complex .
simultaneously , 
 job titration analysis revealed an asymmetric plot , indicative of 
 higher - order molecular complexes . in an attempt to identify the catalytically 
 relevant species ,
both cyp and cpr concentrations were titrated simultaneously , 
 resulting in a two - dimensional data surface . when the data were globally 
 fit to 32 different candidate models ( including binary , ternary , and 
 quaternary cyp 
cpr complexes ) , it revealed that the most likely 
 model was one composed of the binary complex ( cyp  cpr ) , the 
 quaternary complex ( cyp  cpr  cyp  cpr ) , and the 
 homodimer ( cyp  cyp ) . a second , although less likely , possibility 
 was a model that involved a weakly bound ternary complex ( cyp - cyp 
interestingly , the results implied that the binary complex was the 
 only catalytically active state . therefore , in this case , even though 
 cyp2e1 was shown to be capable of forming cyp 
cyp complexes 
 that could have an effect on catalytic activity , the binary cyp 
more recently , a comprehensive study by the backes 
 group examining 
 the effect of homomeric cyp complex formation on the activity of cyp1a2 , 
 cyp2b4 , and cyp2e1 found that , at least in the case of cyp1a2 , cyp 
cyp 
 homomeric complexes contribute to altered catalytic activity seen 
 with this enzyme under conditions that would promote dimer formation . to understand how cyp homomeric complexes might 
 affect function ,
the catalytic activities of several individual cyps , 
 including cyp2b4 , cyp2e1 , and cyp1a2 , were assessed in reconstituted 
 systems as a function of cyp concentration .
interestingly , although 
 oxidation mediated by cyp2b4 as a function of cyp  cpr best 
 fit to a simple hyperbolic michaelis  menten equation , cyp2e1 
 and cyp1a2 demonstrated atypical ( non - michaelis  menten ) kinetics 
 that fit best to a sigmoidal curve , indicating a high degree of cooperativity 
 with these particular isoforms . this non - michaelis  menten kinetic 
 behavior could be converted back to a hyperbola simply by increasing 
 the ionic strength of the buffer , suggesting that the cyp 
cyp complex in the membrane 
 using a variety of biophysical techniques , including chemical cross - linking 
 and bioluminescence resonance energy transfer ( bret ) . in yet another study ,
cyp 
 interaction was found to affect 
 enzymatic function by a direct modulation of allosteric ( non - michaelis  menten ) 
 enzyme kinetics .
both homotropic and heterotropic cooperativity have 
 long been known to occur with hepatic drug - metabolizing cyp isoforms , 
 particularly cyp3a4 .
recently , davydov and colleagues reported that stimulation of cyp3a4-mediated 
 oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin ( 7-bfc ) by -naphthoflavone 
 in human liver microsomes ( hlm ) was highly dependent on the degree 
 of cyp3a4 oligomerization .
the amount 
 of enzyme activation seen was directly correlated with the surface 
 density of the enzyme .
while no activation was detectable at high 
 lipid / cyp ratios ( 750 ) , activation reached greater than 225% 
 at a lipid / cyp ratio of 140:1 .
this suggests that allosteric activation 
 requires at least some degree of homomeric cyp  cyp interaction .
cyp complexes , at least in the dimeric form , 
 it is still not entirely clear what role these complexes may play in vivo in the detoxification and elimination of xenobiotic 
 agents , especially under conditions of cyp induction or endoplasmic 
 reticulum ( er ) stress .
one suggested physiological function basis 
 for the formation of cyp  cyp complexes is their ability to 
 prompt a hypothetical  functional allosteric mechanism  

future studies with relevant in vivo model systems 
 may shed some light in this area .
the identification of the existence 
 of homomeric cyp complexes 
 has led to interest in the possibility of heteromeric cyp complexes 
 as well . using the same methodology that revealed homomeric interactions 
 between individual cyp2c2 molecules , kemper and colleagues identified 
 additional heteromeric interactions between cyp2c2 and cyp2e1 .
this implied that differences in homo- and 
 heteromeric oligomerization state between isoforms may help explain 
 some of the relative differences seen in activity .
indeed , this was 
 found to be the case in complexes formed between cyp1a2 and cyp2b4 
 in reconstituted systems . in a reconstituted 
 system containing cpr , lipid , and
both cyp1a2 and cyp2b4 enzymes , 
 a small increase in catalytic activity for the cyp2b4 substrate benzphetamine 
 was detected .
however , activity against another cyp2b4 substrate , 
 7-pentoxyresorufin - o - dealkylation ( prod ) , was dramatically 
 inhibited .
inhibition of prod was also 
 dependent on cpr levels , with the inhibitory effect being more pronounced 
 at subsaturating cpr concentrations , suggesting competition for reductase 
 binding between the two isoforms . in a follow - up study , backes and 
 colleagues determined that it was likely that a ternary complex containing 
 cpr , cyp1a2 , and cyp2b4 was formed , for cyp2b4 activity was dramatically 
 reduced at subsaturating reductase concentrations .
this result suggested an alternative model where , under 
 substoichiometric conditions , cpr is specifically bound to cyp1a2 
 and at higher cpr concentrations , reductase binding to cyp1a2 becomes 
 saturated , resulting in the formation of a quaternary complex in which 
 cpr binds to both cyp1a2 and cyp2b4 enzymes . in a more recent study examining the interaction between cyp2c9 
 and cyp2c19 in reconstituted systems , kupfer and
colleagues found 
 evidence of a metabolic interaction between even these highly related 
 cyp isoforms .
previously , using in vitro systems reconstituted with purified enzyme , cyp2c19 
 was the most active isoform for methoxychlor - o - demethylation .
however , in hlm , cyp2c9 performed the bulk of methoxychlor - o - demethylation .
interestingly , while an equimolar ratio 
 of cpr to cyp2c9 supported maximal rates of methoxychlor - o - demethylation , the rate of methoxychlor - o - demethylation 
 by cyp2c19 was not fully saturated in the same system ; even when reconstituted 
 with a 9-fold molar excess of cpr , cyp2c19 methoxychlor - o - demethylation and s - mephenytoin hydroxylation kinetics 
 were not fully saturated ( as compared with that in hlm ) .
however , 
 when a binary reconstitution system was prepared by mixing cyp2c9 
 and cyp2c19 enzymes , methoxychlor - o - demethylation 
 and s - mephenytoin hydroxylation by cyp2c19 were inhibited 
 dramatically , with the amount of inhibition dependent on the amount 
 of cpr and substrate used , yet increasing concentrations of cpr were 
 generally able to overcome the inhibitory effects . by contrast , in 
 the incubation containing only cyp2c9 , diclofenac hydroxylation was 
 activated by the presence of cyp2c19 , thus demonstrating that cyp 
cyp 
 interactions can modulate the catalytic rates of a variety of oxidation 
 reactions but that this modulation is highly dependent on the substrate 
 undergoing metabolism , with some substrates activating metabolism 
 and others inhibiting it .
surprisingly , 
 subramanian and colleagues found that while addition 
 of cyp2d6 to a reconstituted incubation of cyp2c9 and ( s)-flurbiprofen increased the ks by 20-fold , 
 it had no effect on the km , but it decreased 
 the vmax by 50% .
one possible explanation for this discrepancy is that affinity constants 
 determined by heme spin state ( i.e. , optical spectrum ) perturbation 
 may mask total ligand binding because they measure only substrate 
 interactions that perturb the heme iron .
it is possible that , in the presence of an additional cyp isoform , 
 the spin state shift may be less complete , thereby not reflecting 
 the effects of additional ligand binding .
the authors additionally noted 
 that the effects on metabolism were dependent on the order of addition 
 of the enzymatic constituents , with the greatest rate enhancements 
 observed when cpr was added before the second cyp , again suggesting 
 a competition for reductase binding . in yet another study , yamazaki et al .
discovered similar types of 
 enzymatic stimulation between cyp1a2 and other isoforms , including 
 cyp3a4 , cyp2c9 , and cyp2d6 , which indicate a more generalizable phenomenon 
 among the hepatic cyps .
as more 
 substantial biophysical and functional evidence has accrued 
 demonstrating heteromeric interaction between individual cyp isoforms , 
 attention has shifted to structurally defining the mechanism of this 
 interaction .
two different studies conducted in 2010 by the backes 
 and tracy laboratories were instructive in this regard .
the backes 
 group set out to answer the question of whether two different cyp 
 isoforms need to be located in the same membrane to functionally interact , 
 thereby potentially excluding long - range allosteric effects .
their experimental system consisted of incorporating 
 cyp1a2 and cyp2b4 into vesicles with cpr individually and together 
 as a cyp  cyp complex . if the two cyps functionally interacted 
 in the same membrane , then metabolic stimulation ( or inhibition ) would 
 be observed only when both cyps were present in the same vesicle , 
 and a mixture of both the individual cyp1a2cpr and cyp2b4cpr 
 vesicles would simply produce an additive effect .
indeed , this is 
 exactly what the authors observed prior to confirming this functional 
 interaction between these two cyps with cross - linking and coimmunoprecipitation 
 experiments .
work from the tracy lab examined the effect of 
 the hydrophobic 
 n - terminal anchor sequence on mediating the interaction between cyp2c9 
 and cyp3a4 . using a reconstituted enzyme 
 system containing both cyp2c9 and cyp3a4 ,
the authors demonstrated 
 that cyp2c9 mediated ( s)-naproxen metabolism was 
 inhibited as much as 80% by the presence of cyp3a4 , while the km remained unchanged .
the degree of inhibition 
 was directly proportional to the cyp3a4 concentration and indirectly 
 proportional to the cpr concentration , suggesting a competition for 
 reductase .
oddly , cyp2c9 did not seem to alter cyp3a4-mediated testosterone 
 metabolism . in order to study the role of the hydrophobic n - terminal 
 anchor sequence ,
the experiments were performed with both n - terminally 
 truncated and full - length cyp isoforms .
there was no evidence for 
 metabolic inhibition when the full - length cyp3a4 and cyp2c9 isoforms 
 were incubated in the presence of the truncated form of the other 
 cyp isoform , but evidence for inhibition was found only when both 
 full length forms were present .
this indicated that interaction between 
 the hydrophobic n - termini of the two cyp isoforms was required for 
 functional inhibition .
it is possible that , in this context , the hydrophobic 
 n - termini may orient the cyps in the membrane . finally , the authors 
 conducted coimmunoprecipitation studies to demonstrate that the two 
 cyps were directly interacting in the membrane .
it has now been 
 firmly established that cyp s form both 
 homomeric and heteromeric complexes with functional consequences . 
 the difficult work of defining how these complexes interact structurally 
 and what their in vivo roles may be now lies ahead .
of all the protein  protein interactions in which cyps have 
 been documented to participate , perhaps the most enigmatic is that 
 with nonenzymatic proteins , including the progesterone receptor membrane 
 component 1 ( pgrmc1 ) and bovine and 
 human serum albumin ( bsa / hsa ) .
although 
 these proteins do not provide reducing equivalents to the cyp catalytic 
 cycle , they are still capable of modulating cyp - mediated metabolism . 

presently , these mechanisms remain to be fully elucidated ; intriguingly , 
 they may involve direct delivery of a drug substrate to the particular 
 cyp .
below , we provide a brief summary of what is currently known 
 regarding these cyp protein  protein interactions . despite 
 its rather precise identification ,
the progesterone receptor membrane 
 component 1 is a somewhat misleading name , as it neither binds progesterone 
 directly nor does it share any homology with progesterone receptors .
in contrast , pgrmc1 shares a high degree of structural homology 
 with cytochrome b5 .
pgrmc1 has been 
 demonstrated to either increase or decrease the rate of cyp - mediated 
 metabolism , depending on the cyp 
pgrmc1 also seems to play a role in the development 
 of certain cancers , as recent reports suggest that it may be the inscrutable 
 
 receptor , a known biomarker of tumor cell 
 proliferation . in mammals , there exist two 
 additional pgrmc1 family isoforms , prgmc2/dg6 and neudesin , although it 
 is not known if they also interact with cyp enzymes .
prgmc1 is highly 
 expressed in the human liver and typically colocalized in the smooth 
 endoplasmic reticulum ( ser ) with cyp proteins .
it has been shown to 
 interact directly with cyp enzymes , including cyp3a4 , cyp2c9 , cyp7a1 , 
 cyp21a2 , and cyp51 .
pgrmc1 activates 
 progesterone oxidation mediated by cyp21a2 and is essential for cyp51 activity in the cholesterol synthesis 
 pathway .
min and colleagues reported 
 that the coexpression of pgrmc1 with cyp21a1 enhanced progesterone 
 21-hydroxylation in cos-7 cells , while a heme - deficient pgrmc1 mutant 
 had no effect . in an experiment with 
 the yeast homologue , dap1 , hugues et al .
showed that both cyp51a1 
 and cyp61a1 can be positively regulated by the presence of dap1 .
remarkably , prgmc1 has been revealed 
 to exhibit isoform - selective effects in its interaction with cyps .
oda found that pgrmc1 generally reduced cyp activities , either through 
 decreased vmax and km values ( in the case of cyp3a4 ) or simply through a decreased vmax ( in the case of cyp2c9 ) ; however , in the 
 case of cyp2e1 , it had no effect .
coimmunoprecipitation 
 studies suggested that the mechanism behind the differential effects 
 may be differences in binding affinity between pgrmc1 and individual 
 cyp isoforms .
szczesna - skorupa and kemper found similar results in 
 their studies of the interactions of pgrmc1 with cyp2c2 , cyp2c8 , and 
 cyp3a4 in transfected hepg2 cells . in 
 each case , pgrmc1 downregulated cyp activities , which could be rescued 
 by increased expression of cpr .
pgrmc1 was found to have a high affinity 
 for cpr , but only in the absence of cyp2c2 , indicating competition 
 between pgrmc1 and cyp2c2 for binding to cpr .
in contrast to what 
 was observed with the drug - metabolizing cyp isoforms , cyp51 activity 
 was reduced with decreasing levels of pgrmc1 , indicating that a separate 
 mechanism may be in place for the interaction of pgrmc1 with cyps 
 involved in steroid synthesis .
the mechanism of cyp activation 
 or inhibition by pgrmc1 is highly 
 debatable ; however , it is conceivable that it may involve drug delivery 
 or sequestration , as a hydrophobic binding pocket has been identified 
 on pgrmc1 . in any case , much work remains 
 to elucidate the structural and functional details of the cyp 
human 
 serum albumin ( hsa ) is a 65 kda monomeric , slightly basic globular 
 protein that circulates as a major component of blood plasma and is 
 the most abundant protein in the human body .
hsa functions primarily as a carrier for 
 steroids , fatty acids , and thyroid hormone .
additionally , it plays 
 a role in stabilizing osmotic pressure by regulating extracellular 
 fluid volume .
hsa binds a large variety of drugs and xenobiotic agents 
 and has a major impact on their pharmacokinetics and disposition . under normal physiological circumstances , hsa 

however , 
 it is synthesized in the liver and is present in the ser of hepatocytes , so it is conceivable that hsa could come into direct contact with 
 cyp isoforms .
moreover , the extent of drug or toxin protein 
 binding has long been known to effect in vitro - to - in vivo extrapolations of disposition .
therefore , in an effort to improve in vitro - to - in vivo predictions , researchers began to add either hsa 
 or bovine serum albumin ( bsa , a cheaper and more well - characterized 
 substitute ) to both microsomal incubations and recombinant baculovirus - expressed 
 enzymes .
this accomplished the desired 
 effect of improving pharmacokinetic predictions by increasing the in vitro intrinsic clearance rate , clint ( vmax / km ) to more 
 closely match what is seen in vivo .
this effect is 
 often mediated through a decrease in the substrate km , and it has been observed for a number of different 
 cyp isoform / drug combinations , including cyp2c8 , cyp2c9 , and cyp1a2 .
however , for some cyp isoforms , including cyp2c19 and cyp2d6 , 
 bsa was found to be inhibitory .
established that the degree of activation or inhibition observed was 
 dependent on the concentration of bsa used , with lower concentrations 
 activating and higher concentrations being inhibitory .
in addition , bsa s cyp activation / inhibition 
 profile was demonstrated to deviate from that observed with hsa , indicating 
 the importance of the albumin source for comparison studies and in vitro - to - in vivo extrapolations .
a number of theories have been proposed to 
 account for the effect seen with albumin , including direct drug delivery , 
 allosterically induced conformational change , and inhibitory long - chain 
 unsaturated fatty acid sequestration .
although no single theory fits 
 the data precisely , recent evidence seems to lend credence to long - chain 
 unsaturated fatty acid sequestration .
of course , because none of these theories are mutually 
 exclusive , it may be that multiple mechanisms are involved rather 
 than a single predominant mechanism with any particular drug / cyp isoform . 

lastly , other proteins related to albumin , such as human intestinal 
 fatty acid binding protein ( ifabp ) , have also been demonstrated to 
 improve in vitro - to - in vivo predictions 
 based on microsomal incubations , suggesting 
 a more generalizable phenomena with this class of proteins .
in addition to clearly defining the particular mechanisms of albumin  cyp 
 interaction , future work in this area will also need to delineate 
 exactly which members of this protein family interact with individual 
 cyp isoforms .
protein 
 interaction will no doubt further enhance our ability to correlate in vitro metabolism to that observed in vivo .
the cyp pathway of oxidative transformation of drugs and other 
 xenobiotics is critical for the elimination of toxicity in the host 
 organism .
proper functioning of this pathway relies on effective formation 
 of protein  protein complexes among the cyps , their electron 
 transfer partners , and other proteins that can influence their function . 

mutations in either the cyp or an interacting protein that effect 
 the structure or stability of the cyp or its protein partner can lead 
 to deficiencies or alternative pathways of biotransformation , which 
 in turn may produce toxic metabolites . therefore , a comprehensive 
 understanding of the many different types and functions of protein 
protein 
 interactions that cyps undergo is necessary to predict and treat such 
 toxicities . new techniques
protein interactions 
 that are beginning to be developed will be a boon for research in 
 this area . additionally , from a pharmacological point of view
, modulating 
 protein  protein interactions may provide an avenue for therapeutic 
 intervention .
conceivably , 
 cyps might also interact with phase ii , or drug conjugating , enzymes 
 such as glucuronidases , sulfotransferases , or gluthione - s - transferases to modulate their activity as well .
protein interactions 
 increases , we will not only improve our ability to predict and prevent 
 toxic cyp 
drug interactions but also gain a new appreciation 
 for the many unique attributes of this versatile enzyme family .
 through their unique oxidative chemistry , 
 cytochrome p450 monooxygenases 
 ( cyps ) catalyze the elimination of most drugs and toxins from the 
 human body . protein  
 protein interactions play a critical role 
 in this process . 
 historically , the study of cyp  protein interactions 
 has focused on their electron transfer partners and allosteric mediators , 
 cytochrome p450 reductase and cytochrome b5 . 
 however , cyps can bind 
 other proteins that also affect cyp function . 
 some examples include 
 the progesterone receptor membrane component 1 , damage resistance 
 protein 1 , human and bovine serum albumin , and intestinal fatty acid 
 binding protein , in addition to other cyp isoforms . 
 furthermore , disruption 
 of these interactions can lead to altered paths of metabolism and 
 the production of toxic metabolites . in this review , 
 we summarize 
 the available evidence for cyp protein  protein interactions 
 from the literature and offer a discussion of the potential impact 
 of future studies aimed at characterizing noncanonical protein  
 protein 
 interactions with cyp enzymes . 
Introduction
Cytochrome P450 Interactions
with Cytochrome
P450 Reductase and Cytochrome b5
Cytochrome
P450 Interactions with Other CYP
Isoforms
Cytochrome P450 Interactions with Other Proteins
Conclusions and Future Perspectives