DRUG METABOLISM REVIEWS, 31(1), 195–203 (1999)

FLAVODOXIN AS A MODEL FOR THEP450-INTERACTING DOMAIN OFNADPH CYTOCHROME P450REDUCTASE*

CHRISTOPHER M. JENKINS† andMICHAEL R. WATERMAN‡

Department of BiochemistryVanderbilt University School of MedicineNashville, Tennessee 37232-0146

I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

II. PURIFICATION AND IDENTIFICATION OF THE E. COLIP450 REDUCTASE SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

III. COMPARISON OF THE FLAVODOXIN/FLAVODOXINREDUCTASE SYSTEM WITH NADPH–CYTOCHROME P450REDUCTASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

IV. ADDITIONAL PROPERTIES OF FLAVODOXIN . . . . . . . . . . . . . . . 201

* This paper was refereed by Linda J. Roman, Ph.D., Department of Biochem-istry, University of Texas Health Sciences Center, San Antonio, TX 78284.

† Current address: Department of Bioorganic Chemistry & Molecular Pharma-cology, Washington University, 660 South Euclid Avenue, St. Louis, MO 63110.

‡ To whom correspondence should be sent. Fax: (615) 322-4349.

195

Copyright 1999 by Marcel Dekker, Inc. www.dekker.com

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V. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

I. INTRODUCTION

Heterologous protein expression systems have proven particularly useful inthe study of P450 enzymes. These systems have permitted the determination ofenzymatic activities and substrate specificities of different P450s as well as mak-ing available large quantities of these enzymes for structure/function analysis.Different expression systems utilized for such purposes include animal cells [1,2],yeast [3], baculovirus [4], and bacteria [5]. Bovine 17α-hydroxylase/17,20-lyasecytochrome P450 (P450c17) has been expressed in all four systems and providesa good comparison among them [6]. Each system has certain advantages, but thebacterial expression system produces the largest amount of P450c17 and thereforeis the most useful in producing large quantities of the purified enzyme for detailedbiochemical analysis. Incorporation of a histidine tag to the carboxy-terminal endof the P450 protein facilitates its purification without alteration of enzymaticactivities [7]. Levels of P450 expression in excess of 1 µmol/L of culture havebeen achieved using bacterial expression systems [8].

High-level bacterial expression of many different microsomal and mitochon-drial P450s has been accomplished in laboratories throughout the world usingdifferent expression vectors and expression strategies, all of which have evolvedfrom the original scheme developed by Barnes et al. [5]. These P450s are local-ized to the inner bacterial membrane facing the cytoplasm [5]. One deficiencyof bacterial expression systems compared to those in animal cells, yeast, or insectcells is the absence of NADPH cytochrome P450 reductase required for supportof P450 activities [9]. Therefore, strategies have been developed for expressionof fusion proteins between P450 and P450 reductase in order to generate func-tional P450s within intact bacteria [10]. However, Barnes et al. demonstrated inintact E. coli that P450c17 could convert externally added progesterone to 17α-hydroxyprogesterone, showing that a reductase system which could support theactivity of microsomal P450s was present in Escherichia coli [5]. There is nosuch system in bacteria which will support the activity of mitochondrial P450s.In this article, we describe the characterization of the bacterial system whichsupports the activity of recombinant microsomal P450s and compare the pro-perties of this system with those of eukaryotic NADPH cytochrome P450 reduc-tase.

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FLAVODOXIN VERSUS P450 REDUCTASE 197

II. PURIFICATION AND IDENTIFICATION OF THE E. COLIP450 REDUCTASE SYSTEM

The original work by Barnes et al. showed that the E. coli P450c17 reductasewas cytosolic and heat-labile [5]. Fractionation of E. coli cytosol demonstratedthat this is a two-component reductase system [11]. One component isolated froma high-salt (150–500 mM KCl) elution fraction from diethylaminoethyl (DEAE)cellulose is a yellow-orange protein having a molecular weight of about 20 kDaand an absorbance spectrum resembling bacterial flavodoxins which are solubleflavin mononucleotide (FMN)-containing proteins. Upon purification, this proteinwas identified as E. coli flavodoxin by amino-terminal sequence analysis [11].The second component was derived from a low-salt DEAE cellulose fraction(25–150 mM KCl) and by spectral analysis, and a subsequent amino-terminalsequence determination was found to be E. coli flavodoxin reductase, a 29-kDaprotein [11]. Flavodoxin reductase is an NAD(P)H-binding, flavin adenine dinu-cleotide (FAD)-containing protein which can transfer electrons to flavodoxin.Reconstitution of P450c17 activity with these two purified E. coli proteins wasmuch more efficient when NADPH was used as source of reducing equivalentsrather than NADH. Because flavodoxin is an FMN-binding protein similar to theFMN-binding domain of P450 reductase, it was predicted to interact with P450s.By optical methods, its binding constant to P450c17 was estimated to be approxi-mately 1.0 µM [11].

Porter and Kasper noted in 1986 that the sequence of NADPH cytochromeP450 reductase could be separated into two domains, one at the amino-terminuswhich is FMN binding and has an homology to flavodoxin and one at the carboxy-terminus which binds FAD and NADPH and resembles flavodoxin reductase [9].Thus, in one sense, they predicted that flavodoxin and flavodoxin reductase mightsupport microsomal P450 activities. Flavodoxin/flavodoxin reductase is the fifthbiochemical system identified by which P450s can be reduced. The five classesof cytochrome P450 redox systems are seen in Fig. 1.

III. COMPARISON OF THE FLAVODOXIN/FLAVODOXINREDUCTASE SYSTEM WITH NADPH–CYTOCHROME

P450 REDUCTASE

Having established similarities between flavodoxin/flavodoxin reductase andNADPH–cytochrome P450 reductase in cofactor binding, enzymatic activity, andinteractions with microsomal P450s, studies were carried out to determinewhether this E. coli reductase system could be used as a model for NADPH–cytochrome P450 reductase. Using a flavodoxin affinity resin, several different

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FIG. 1. Five known classes of electron-transfer systems for members of theP450 superfamily. Class 5, the flavodoxin/flavodoxin reductase system, is the onlyclass which has not been shown to function in reduction of endogenous P450s.

eukaryotic P450s were found to bind to flavodoxin (Table 1). There are twosurprising results contained in Table 1 [12]. First, P450scc (cholesterol side-chaincleavage cytochrome P450) binds flavodoxin. This is a mitochondrial P450 whoseactivity cannot be supported by the flavodoxin/flavodoxin reductase system eventhough the binding constant of flavodoxin to this protein is similar to that inmicrosomal P450s. Furthermore, mutant forms of P450scc which bind the mito-chondrial P450 iron sulfur electron-transfer protein adrenodoxin poorly [14] showa decrease in flavodoxin binding. Because there is a change in flavodoxin bindingin these mutants, it is suggested that the flavodoxin binding sites and the adreno-doxin binding sites on P450scc overlap [12]. However, the binding of flavodoxinto the P450scc mutants remains much stronger than adrenodoxin binding, indicat-ing that the mutated lysine residues, which play major roles in adrenodoxin bind-ing to P450scc, play less important roles in flavodoxin binding to P450scc. Sec-ond, the P450 domain of P450BM-3 does not bind to flavodoxin even though it

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FLAVODOXIN VERSUS P450 REDUCTASE 199

binds the C-terminal domain of P450BM-3, which is very closely related toNADPH–cytochrome P450 reductase. Thus, the bacterial flavodoxin associatesfunctionally with many and probably all microsomal P450s but does not associatewith the P450 domain of the bacterial P450, P450BM-3. The fact that flavodoxinbinds to all microsomal P450s tested indicates its resemblance to the FMN do-main of NADPH–cytochrome P450 reductase, which can also interact with allmicrosomal P450s. The interaction between the P450 and reductase domains ofP450BM-3 is somehow different than those between microsomal proteins.

A further comparison of flavodoxin with P450 reductase was possible by theuse of Anabaena flavodoxin mutants produced by Carlos Gomez-Moreno andhis colleagues [15]. Carrying out site-directed mutagenesis in the FMN-binding(flavodoxin-like) domain of rat NADPH–cytochrome P450 reductase, Shen andKasper identified two distinct clusters of negatively charged amino acids [16].One cluster is required for cytochrome c reduction (cluster II) and the other forP450 reduction (cluster I). Mutagenesis of two acidic residues in Anabaena fla-vodoxin (D144A and E145A) significantly decreased bovine P450c17 activities,with D144A causing an 85% decrease and E145A a 40% decrease [15]. Themutants bind to P450c17 with equal or even greater affinity than the unmodified,wild-type flavodoxin, and P450-dependent H2O2/superoxide production was notincreased using these mutant forms of flavodoxin. Therefore, these mutations inflavodoxin do not affect binding to P450c17 or electron coupling between theflavodoxin mutants and P450c17. In addition, these mutations actually increasecytochrome c reductase activity. The acidic residues at 144 and 145 in Anabaenaflavodoxin align with the acidic residues in cluster I of rat NADPH–cytochromeP450 reductase, which influence P450 activities but do not influence cytochromec reduction (Fig. 2). This is further evidence that flavodoxin strongly resembles

TABLE 1

Interactions of Proteins with Flavodoxin–Sepharose

Binding No binding

P450c17 2 substrate P450BM-3P450c17 1 progesterone Heme domain of P450BM-3P4501A2 3β-HSDb

P4502B1 HemoglobinP4502D6a

Cytochrome c

a Data from Ref. 13.b 3β-Hydroxysteroid dehydrogenase.Source: Data from Ref. 12.

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FIG. 2. Amino acid sequence alignment among Anabaena Fld, E. coli Fld,P450BM-3-reductase domain (P450BMR), and rat P450 reductase (CPR). β5aand β5b refer to β-sheet regions in the Anabaena Fld crystal structure. Symbols(∆) above the Anabaena sequence represent mutagenesis sites reported in ourwork [15] and those below in the rat CPR sequence are reported by Shen andKasper [16]. Acidic amino acid clusters I (P450) and II (cyt c) are indicated andunderlined.

the FMN-binding domain of NADPH–cytochrome P450 reductase. Recently, thethree-dimensional structure of rat liver NADPH–cytochrome P450 reductase hasbeen determined [17]. The FMN-binding domain is found to be structurally verysimilar to flavodoxins, whereas the FAD and NADPH binding domain is verysimilar in structure to flavodoxin reductase.

The results above indicate that the soluble prokaryotic flavodoxin/flavodoxinreductase system is a potentially informative model of the eukaryotic, membrane-bound NADPH–cytochrome P450 reductase. Differences between these twoP450 reductase systems are, however, also evident [18]. First, the maximal activ-ity of P450c17 that can be achieved with an excess of the E. coli flavodoxin/flavodoxin reductase system (5–6 nmol 17α-hydroxyprogesterone/min/nmolP450c17) is 10-fold less than the optimal P450c17 turnover number achievedwith NADPH–cytochrome P450 reductase. Second, there is a difference in theFMN redox states between the two P450 reductase systems. A one-electron-reduced flavodoxin reduces P450c17, whereas the two-electron reduced form ofNADPH cytochrome P450 reductase serves the same role. Finally, theflavodoxin/P450c17 complex is almost completely disrupted by moderate ionicstrength (,100 mM NaCl), whereas the P450 reductase/P450c17 complex is onlypartially disrupted by higher ionic strength (,300 mM NaCl).

Clearly, flavodoxin/flavodoxin reductase is a good soluble protein model formembrane-bound NADPH–cytochrome P450 reductase. Perhaps in some bacte-rial systems it will be found that P450 activities are supported by flavodoxin/flavodoxin reductase. For example, soluble P450s of unknown function have re-cently been described in Anabaena [19]. Perhaps the endogenous activities ofthese enzymes will be found to be supported by Anabaena flavodoxin/flavodoxinreductase.

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FLAVODOXIN VERSUS P450 REDUCTASE 201

IV. ADDITIONAL PROPERTIES OF FLAVODOXIN

Overexpression of flavodoxin in E. coli provided the opportunity to easilyobtain relatively large quantities of this protein [11]. Because of the tight bindingof purified P450c17 to purified flavodoxin cited earlier, we wondered whether itmight be practical to use a flavodoxin affinity column to purify microsomal P450sfrom cells. As a test system, a flavodoxin Sepharose column was used to purifyP450s from bovine adrenocortical microsomes. Figure 3 shows the elution oftotal P450 proteins from such a column. Sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) analysis of the eluted proteins showed threemajor protein bands [12]. Based on N-terminal amino acid sequence analysis,two of these proteins were identified as P450c17 and P450c21, which are thetwo expected P450s in bovine adrenocortical microsomes. Interestingly, a thirdmajor steroidogenic microsomal protein, 3β-hydroxysteroid dehydrogenase (3β-HSD), was purified along with these P450s. It was found that 3β-HSD does notbind to flavodoxin [12] (Table 1), indicating that there may be a steroidogeniccomplex present in adrenocortical microsomes consisting of P450c17, P450c21,and 3β-HSD. Such a complex, if present, would permit efficient shuttling ofintermediates in the adrenocortical steroidogenic pathway from one active siteto another. These results indicate that flavodoxin Sepharose will be useful inthe purification of P450s from microsomal samples from animal cells and from

FIG. 3. Ionic-strength-dependent elution profile of bovine adrenocorticalmicrosomal P450s from a flavodoxin (Fld) Sepharose column. P450 was quanti-fied from reduced CO/reduced difference spectra. The specific content of thepeak fraction indicated is 2 nmol P450/mg protein.

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heterologous P450 expression systems. This latter use for flavodoxin Sepharosein P450 purification from an E. coli expression system has been reported [18].

We are attempting to take advantage of the tight binding of flavodoxin tomicrosomal P450s in two additional types of experiments. In efforts to crystallizemicrosomal P450s, we are trying to crystallize P450/flavodoxin complexes. Thiscomplex formation may stabilize the P450 and should also change the solubilityproperties of the P450, both of which may enhance the possibilities of crystalliz-ing these membrane-bound proteins. Furthermore, these studies could lead tohigh-resolution structural data of a functional P450/flavodoxin complex. In addi-tion, we are trying to localize the flavodoxin binding sites on P450c17 usingchemical modification of amino acids plus and minus the presence of flavodoxin.This should give excellent insight into P450c17 residues involved in the interac-tion with NADPH–cytochrome P450 reductase.

V. CONCLUSION

The P450 reductase system in E. coli has been found to be a two-componentflavodoxin/flavodoxin reductase system. This reductase system normally playsessential roles in the function of endogenous E. coli enzymes, including methio-nine synthase, anaerobic ribonucleotide reductase, biotin synthase, and pyruvateformate lyase. Upon overexpression of recombinant microsomal P450s, flavo-doxin/flavodoxin reductase is captured for the support of P450 activities. Wehave found that flavodoxin serves as a very good model for the FMN-bindingdomain of NADPH–cytochrome P450 reductase and we believe that this solubleprotein reductase system will be very useful in evaluating many of the propertiesof interactions of the eukaryotic membrane-bound P450 reductase with micro-somal P450s. Of particular importance may be studies leading to the crystalliza-tion of P450s with flavodoxins. The tertiary structure of several flavodoxins areknown [18] and the interaction with P450s is quite strong. Thus, flavodoxin bind-ing should render eukaryotic P450s more soluble, thereby enhancing their crystal-lization and optimizing opportunities to determine the structural details of theP450/reductase interaction.

ACKNOWLEDGMENTS

This work was supported in part by USPHS Grants GM37942 and ES00267and C.M.J. was supported in part by T32 ES07028.

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FLAVODOXIN VERSUS P450 REDUCTASE 203

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