The α-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Biochimica et Biophysica Acta 1674 (2004) 319 – 326 http://www.elsevier.com/locate/bba

The a-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions Scott B. Mulrooneya,1, David R. Meinhardtb, Lucy Waskellb,c,* a

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA b Department of Anesthesiology, University of Michigan, Ann Arbor, MI 48109, USA c Department of Anesthesia, University of Michigan, VA Medical Center, Research Service 129, 2215 Fuller Rd. Ann Arbor, MI 48105, USA Received 23 April 2004; received in revised form 2 July 2004; accepted 2 August 2004 Available online 23 August 2004

Abstract Cytochrome b 5 (cyt b 5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an a-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b 5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b 5. Insertion of alanine into an a-helix causes all amino acids at its carboxyl terminus to be rotated by 1008. The ability of the alanine insertion mutants of cyt b 5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b 5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic ahelix of cyt b 5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions. D 2004 Elsevier B.V. All rights reserved. Keywords: Cytochrome b 5; Cytochrome P450 2B4; Mutagenesis; Membrane protein; Protein–protein interaction

1. Introduction The cytochromes (cyts) P450 are a superfamily of ubiquitous mixed-function oxidases that are found in high concentration in mammalian hepatocytes where they are responsible for the metabolism of the vast majority of drugs and xenobiotics consumed by man as well as the biosynthesis and degradation of a number of endogenous compounds [1]. Hepatic microsomal cyts P450 are anchored to the membrane by an amino-terminal hydro-

* Corresponding author. Department of Anesthesia, University of Michigan, VA Medical Center, Research Service 129, 2215 Fuller Rd. Ann Arbor, MI 48105, USA. Tel.: +1 734 769 7100x5858; fax: +1 734 213 6985. E-mail address: [email protected] (L. Waskell). 1 Present address: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.001

phobic domain and in some cases possibly by an additional internal membrane binding region [2,3]. Cyt b 5 is bound to the membrane by a carboxyl terminal hydrophobic a-helical domain consisting of 23 amino acid residues. It is involved primarily in lipid biosynthesis where it donates electrons for specific reactions [4,5]. In microsomes and purified reconstituted mixed function oxidase systems, the 133-amino-acid amphipathic form of cyt b 5 can also bind to and provide electrons for catalysis by selected cyts P450 in the presence of some but not all substrates [6,7]. For example, the detergent-solubilized cyt b 5 markedly stimulates the metabolism of the anesthetic, methoxyflurane, by cyt P450 2B4 [8,9] and is required for the 17,20-lyase activity, but not the 17 a-hydroxylase activity, of CYP 17 (cytochrome P450 17 a-hydroxylase and 17,20 lyase) [10]. In an attempt to elucidate the mechanism by which cyt b 5 enhances the metabolism of selected substrates by specific

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cyts P450, we have elected to investigate the ability of cyt b 5 to stimulate the metabolism of the volatile anesthetic, methoxyflurane, by cyt P450 2B4 in a purified reconstituted system. It has recently been demonstrated that catalysis by cyt P450 2B4 proceeds significantly faster and more efficiently in a cyt P450 2B4–cyt b 5 complex than in a cyt P450 2B4–cyt P450 reductase complex [11]. The nature and structure of the cyt P450 2B4–cyt b 5 complex are therefore critical to the understanding of the role of cyt b 5 in catalysis by cyt P450 2B4. Cyt P450 2B4 and cyt b 5 are amphipathic membrane-bound proteins. Formation of a functional complex between the two proteins in aqueous solution requires both the intact cytosolic heme domains and the membrane-spanning domains. Since in aqueous solution amphipathic detergents and proteins aggregate via hydrophobic interactions between their hydrophobic domains and electrostatic forces between their soluble domains [12,13], it has been assumed that in an aqueous solution of the cyt P450–cyt b 5 complex the hydrophobic domains of cyt P450 2B4 and cyt b 5 are in contact while electron transfer occurs between the interacting heme domains. It has been known for many years that the soluble form of cyt b 5 (a form lacking the membrane-binding hydrophobic domain) does not bind to or stimulate catalysis by cyt P450 [14,15]. The amphipathic cyt b 5 protein with its hydrophobic transmembrane a-helical domain is required for full stimulation of cyt P450 activity. However, a mutant cytochrome b 5 with one half of the membrane anchor supports cyt P450 catalysis at 20% of the rate of the wild type [15]. In aqueous solution, amphipathic cyt b 5 exists as a octamer while cyt P450 is hexameric [16,17]. When the respective membrane anchors of cyt b 5 and cyt P450 are removed, cyt b 5 no longer aggregates and minimal aggregation of the cyt P450 2B4 is observed at low ionic strength [18]. In view of these experimental results, it has been assumed that the hydrophobic membrane anchors of cyt b 5 and cyt P450 interact and facilitate the formation of a complex whose hemecontaining domains are competent in electron transfer. At present it cannot be ruled out that the membrane anchor of cyt b5 functions to relieve a self-inhibitory conformation of cyt P450 2B4, although this is considered unlikely. In addition to studies involving interactions of the membranebinding domains of cyt b 5 and cyt P450, other research has focused on how the heme-containing hydrophilic domains interact. For example, by using a homology model of cyt P450 2B4 in conjunction with alanine-scanning mutagenesis studies, the binding site for cyt b 5 on the heme domain of cyt P450 2B4 was localized to a depression on the proximal surface near the axial cysteine where the heme comes closest to the surface [8]. These studies showed that the binding site for cyt P450 reductase partially overlapped the cyt b 5 binding site. It is not known whether the ability of cyt b 5 to stimulate metabolism of only selected forms of cyt P450 is due to an interaction between specific amino acids in the hydrophobic domains of cyt P450 2B4 and cyt b 5. If interactions

between specific side chains of the cyt b 5 and cyt P450 hydrophobic domains are required for binding, then disruption of the specific helix–helix packing would destroy that specific interface while leaving any nonspecific hydrophobic interaction relatively intact. An alternative explanation is that the requirement for the membrane-spanning domain of cyt b 5 is mediated simply by nonspecific hydrophobic forces between these lipophilic domains. To determine whether specific or nonspecific interactions between the membrane anchors of cyt b 5 and cyt P450 2B4 play a role in their interaction, alanine was inserted at six different positions in the membrane-binding domain of cyt b 5. Insertion of an alanine into an a-helix causes all amino acids distal to the insertion to undergo a 1008 rotation and would be expected to disrupt any amino acidspecific helix–helix interactions between the two hydrophobic domains [19]. Expression, purification and characterization of the six alanine insertion mutants of cyt b 5 demonstrate that in aqueous solution in a purified reconstituted system lacking membranes, the hydrophobic domain of cyt b 5 interacts with cyt P450 2B4 via nonspecific interactions.

2. Experimental procedures 2.1. Materials Tergitol NP-10, y-aminolevulinic acid, hemin chloride, carbenicillin, sodium hydrosulfite (dithionite) and DEAE Sepharose fast-flow were purchased from Sigma (St. Louis, MO); sodium deoxycholate was from Fisher Biotech (Hampton, NH); Complete Mini-Protease Inhibitor tablets were purchased from Roche Applied Sciences (Indianopolis, IN). Methoxyflurane was purchased from Abbott Laboratories (Abbott Park, IL). Unless otherwise stated, all molecular biological methods were performed according to standard protocols [20]. Since the membrane anchor of cyt b 5 is easily removed by proteolysis, all buffer solutions were filter-sterilized into autoclaved bottles and only sterile glassware or plasticware came in contact with the preparation. All spectra were recorded on a Cary 1 spectrophotometer (Varian). 2.2. Site-directed mutagenesis Mutagenesis was performed using plasmid pLW01b5mem which contains the rabbit cyt b 5 gene downstream of a T7 promotor [8]. The QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA) was used according to the manufacturer’s instructions. The oligonucleotides for the six alanine insertion mutant proteins are shown in Table 1. The correct sequence of the mutated cyt b 5 genes was verified by automated DNA sequencing at the University of Michigan Biomedical Research Core Facility.

S.B. Mulrooney et al. / Biochimica et Biophysica Acta 1674 (2004) 319–326 Table 1 Oligonucleotides used for mutagenesis of cyt b 5

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Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL) using bovine serum albumin as the standard. SDS-polyacrylamide gels were stained either with GelCode or with SilverSNAP using a silver stain kit (Pierce) according to the manufacturer’s instructions.

ethanol. After each addition of heme, the spectrum was recorded. A difference spectrum was calculated using the equation: difference spectrum = (spectrum after addition spectrum before addition). After the first several aliquots of heme had been added, the resulting difference spectra resembled the spectrum of cyt b 5 with a peak at 412 nm and a minimal absorbance at 385 nm. The titration was considered complete when the peak of the difference spectrum shifted to 410 nm and a noticeable increase in absorbance at 385 nm due to accumulation of excess Trisliganded heme occurred (see Fig. 1) [22]. Once the amount of heme required to reconstitute the apo cyt b 5 in a 2-ml sample of the cell culture is known, it is straightforward to determine the amount of heme required to reconstitute the apoprotein in the 500-ml cell culture. The cyt b 5 concentration in the lysate was calculated from a reduced minus oxidized spectrum by measuring the difference between the absorbance at 424 and 409 nm and using an extinction coefficient of 185 mM 1 cm 1 [23].

2.4. Expression of cyt b 5

2.6. Cyt b 5 purification

Expression of cyt b 5 was achieved using plasmid pLW01-b5mem which contains the complete gene for the amphipathic cyt b 5 cloned into the high copy T7 expression plasmid pLW01 [8]. Plasmid pLW01-b5mem or its mutated derivative was transformed into E. coli strain C41(DE3) for large-scale expression [21]. Culture growth, membrane isolation, and purification of cyt b 5 were performed as previously described [22].

The protein was purified as previously described with minor modifications [22]. Cells from the 500-ml culture were thawed and resuspended in 25–30-ml Buffer A. After adding two Complete Mini-Protease Inhibitor tablets (Roche Applied Sciences), the cells were lysed by sonication. The apo cyt b 5 was reconstituted by adding a 10% molar excess of heme based on the small-scale cyt b 5 determination performed above. This excess heme is removed during the purification process. The sonicated cells containing the reconstituted cyt b 5 were centrifuged at 3000g for 15 min at 4 8C and the supernatant containing the membrane fraction was then centrifuged at 109,000g for 1 h at 4 8C. The dark red membrane pellets were resuspended in ~30 ml of Buffer B with the aid of a brief 30-s sonication pulse. The preparation was then diluted to a total protein concentration of 4 mg/ml and stirred at 4 8C for a total of 4 h, and subsequently centrifuged at 109,000g for 1 h at 4 8C. The dark red supernatant containing the cyt b 5 was retained. A DEAE-Sepharose column was equilibrated with Buffer C and run at room temperature to prevent the formation of a Na deoxycholate gel at high salt concentrations. After loading the crude preparations onto a 2.5-cm dia.16-cm DEAE-Sepharose column, the cyt b 5 was recovered with a single-step elution using 0.4 M NaCl, 20 mM Tris, 1 mM EDTA, pH 8 at 25 8C, 0.4% Na deoxycholate (buffer D). Fractions that contained red color were combined, concentrated to i20 ml using an Amicon YM-10 ultrafiltration stirred cell, and chromatographed on a 5-cm dia.62-cm column of Superdex-75 which had been equilibrated with 20 mM NaPO4, 1 mM EDTA, 0.4% Na deoxycholate, pH 7.6. Fractions with the highest 412/280 nm absorbance ratios were combined and loaded onto a second DEAE-Separose column (2.516 cm) equilibrated

Mutant

Sequencea

Ala Ala Ala Ala Ala Ala

5V-ccaccgtcgattccGCGaattccagctggtggac-3V 5V-ccaattccagctggGCGtggaccaactgggtg-3V 5V-ctggtggaccaacGCGtgggtgatccccgcc-3V 5V-gtgatccccgccGCGatctccgccctgatc-3V 5V-gccatctccgccctgatcGCGgtggcactgatg-3V 5V-ccctgatcgtggcactgGCGatgtatcgcctctac-3V

a

104/105 108/109 111/112 116/117 120/121 123/124

Inserted alanine codon is shown in upper case.

2.3. Protein analysis

2.5. Reconstitution of the apo cyt b 5 in the E. coli lysate Because of the high level of expression, most of the cyt b 5 is expressed as the apoprotein which is readily reconstituted by addition of exogenous heme to the lysed cells. It is important that no more than a small excess of heme be added to the preparation in order to avoid interference by the free heme in later experiments. For this reason, the amount of heme required for reconstitution of the apoprotein in the 500 ml of cell culture was determined by first measuring how much heme was required to reconstitute the apo cyt b 5 in a 2-ml sample. A 2-ml aliquot of the original 500-ml culture was centrifuged at 10,000g for 1 min at room temperature. The cell pellet was resuspended in 1 ml of cold 10 mM Na/KPO4, 1 mM EDTA, pH 7, 1% Tergitol NP-10 (Buffer B). The cells were sonicated with a Vibra Cell sonicator (Sonic Materials, Danbury, CT). Two 30-s pulses at 40% power with a 50-W maximum setting were delivered with cooling on ice between pulses. The sonicated cells were diluted 20-fold into 20 mM Tris, 1 mM EDTA, pH 8 at 25 8C, 0.4% Na deoxycholate (Buffer C) and an absorbance spectrum from 350 to 600 nm was recorded. The apo cyt b 5 was reconstituted by titrating it with 1-Al aliquots of a filtered 0.7 mM solution of hemin Cl in 0.1 N NaOH and 50%

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Fig. 1. A bhelical netQ representation of the hydrophobic C-terminal tail of cyt b 5 and a typical alanine insertion mutant. (A) The wild-type sequence, and (B) a representative example of the helix resulting from insertion of an alanine (in box) between Ala116 and Ile117. This diagram follows the convention used by Ausubel et al. [20].

with Buffer C. The cyt b 5 was eluted with a 300-ml linear gradient of 0 to 0.4 M NaCl in 20 mM Tris, 1 mM EDTA, pH 8 at 25 8C, 0.4% Na deoxycholate. The deoxycholate was removed on a Sephadex G25 column (1100 cm) preequilibrated with 10 mM KPO4 buffer pH 8.0 and 1 mM EDTA. The preparations of cyt b 5 were N99% pure as judged by SDS-PAGE analysis (data not shown). The specific content of the cyt b 5 was determined as previously described [22]. 2.7. Determination of methoxyflurane metabolism by cyt P450 The metabolism of methoxyflurane (CCl2HCF2OCH3) by cyt P450 was determined by measuring the amount of fluoride ion produced [9]. Reactions containing cyt P450 2B4, cyt P450 reductase, dilauroylphosphatidylcholine (DLPC), and an NADPH generating system were prepared as previously described [8]. Cyt P450 2B4 and cyt P450 reductase were a generous gift from Dr. Larry Gruenke. Fluoride ion was measured with an Orion fluoride electrode (Orion 9609BN) using a commercial fluoride ion standard solution (Thermo Orion, Beverly, MA). The buffer used in determining fluoride ion concentration was prepared by

dissolving 50 g of NaOH in ~250-ml water, adding 142-ml glacial acetic acid, and bringing the final volume to 500 ml (pH 5–5.5). For each sample for fluoride determination, a 5% volume of this acetate buffer was added before taking readings. Standard and unknown samples for fluoride measurements were placed in a small Teflon cup designed to fit around the electrode tip. 2.8. Determination of the equilibrium dissociation constant (K d) of the cyt b 5 – cyt P450 complex Binding of cyt b 5 to cyt P450 was measured by observing the change in spin state of cyt P450 upon addition of cyt b 5 [8]. The change in spin state from low spin to high spin is known as a Type I spectral change and is a result of the conversion of the hexacoordinated low spin heme to a pentacoordinate heme. A solution containing 5.3 AM cyt P450 2B4, 0.8 mM DLPC, 0.1 M potassium phosphate, pH 7.4 and 20% glycerol was prepared and incubated at room temperature for 1 h. The DLPC stock solution was prepared by mixing 5 mg of the solid in 1 ml of water and placing it in a plastic tube immersed in a sonication water bath. The mixture was sonicated for 1 h with the temperature maintained at 25–308C. Following

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centifugation at 12,000g for 5 min, the supernatant containing the lipid vesicles was added to the cyt P450containing solution. A saturated solution of methoxyflurane in 0.1 M potassium phosphate, pH 7.4 and 20% glycerol was added to the cyt P450-containing solution to obtain a final concentration of 0.3 AM cyt P450 and 53 AM DLPC. One side of a dual chamber cuvette was filled with 1.0 ml of the 0.3 AM cyt P450 solution. The other chamber was loaded with 1.0 ml of a cyt b 5 solution in a buffer of 0.1 M potassium phosphate, pH 7.4, 20% glycerol which was saturated with methoxyflurane. After equilibrating at 25 8C for several minutes, the absorbance at 385 and 420 nm was recorded. The solutions in the two chambers were then mixed by inversion several times and the absorbance was monitored over a period of ~10 min until it stabilized. The final concentration of cyt P450 2B4 was 0.15 AM while the cyt b 5 concentration varied (0, 0.1, 0.2, 0.6, and 1.0 AM). Controls were performed to check for nonspecific protein binding to the cuvette surfaces by using cyt b 5 or cyt P450 at the concentrations used in the experiment in one chamber and buffer in the second. The total absorbance changes due to cyt b 5 binding (A 420 before mixing A 420 after mixing) (A 385 before mixing A 385 after mixing) were fit to a binding curve as previously described [8]. 2.9. Tryptophan fluorescence The tryptophan fluorescence of a 2 AM solution of wildtype and mutant cyts b 5 was measured in 20 mM sodium phosphate, 1 mM EDTA, pH 7 containing 0.4% sodium deoxycholate following excitation of the samples at 293 nm. The emission spectra were recorded between 300 and 450 nm with a Shimadzu RF5000U Spectrofluorimeter at 25 8C using 5-nm excitation and emission slits. 2.10. Kinetics of the binding of cyt b 5 to cyt P450 2B4 Stock solutions of 98 AM cyt P450 and 5 mg/ml (8 mM) DLPC were mixed and diluted to give final concentrations of 5.0 AM and 0.3 mM, respectively, in a buffer of 0.1 M KPO4, 20% glycerol, pH 7.4 (final molar ratio of cyt P450/ DLPC, 1:60). The buffer solution had been previously saturated with methoxyflurane by adding several drops of this compound and mixing for several minutes. Saturation was indicated by a small amount of excess organic layer remaining at the bottom of the buffer solution. The kinetics of binding between cyt b 5 and cyt P450 2B4 was measured in a Hi-Tech SF-40 stopped-flow spectrophotometer at 30 8C in the single-wavelength mode at 385 nm after rapid mixing of equal amounts of the above cyt P450-containing solution with a methoxyflurane-saturated solution of 0.1 M KPO4, 20% glycerol, pH 7.4 containing 25 AM cyt b 5 (wild-type or mutants). The data from fitting three to seven absorbance traces were averaged to calculate the rate constant and amplitude of the first phase of this multiphasic reaction.

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3. Results 3.1. Alanine-insertion scanning mutagenesis of the membrane anchor of rabbit cyt b 5 The work presented here was undertaken to address the question of whether the presumed interaction between the hydrophobic domains of cyt b 5 and cyt P450 2B4 is nonspecific, or if specific side chain contacts are required. A previous study from this laboratory reported that substitution of the hydrophobic membrane anchor with a continuous sequence of 22 leucines resulted in a protein which was unable to stimulate catalysis by cyt P450 2B4. It was concluded that this protein so tightly self-associated, it likely did not have an opportunity to bind to cyt P450 [15]. Since these drastic changes in the membrane anchor resulted in experimental data with significant uncertainties, it was decided to make more conservative and presumably readily interpretable changes. Rather than individually mutating each of the 26 residues in the N-terminal a-helical domain of cyt b 5, it was decided to insert an alanine residue at six points between S104/N105, W108/W109, N111/W112, A116/I117, L120/I121, and A123/L124 along the a-helix of the membrane anchor of cyt b 5. In general, insertion mutagenesis is expeditious and should be more disruptive to helix–helix interactions than replacement mutagenesis and thus better suited for initial screening for functionally and structurally important residues. Examples include, but are not limited to, lactose permease [24] and glycophorin A [19]. The insertion of alanine leaves the sequence of the segments on either side of the insertion point intact, but will displace residues on the C-terminus side of the mutation by 1008 relative to those on the N-terminal side of the insertion. The rotation of residues in the a-helix of the membranespanning domain of cyt b 5 is illustrated in a bhelical netQ diagram in Fig. 1. If specific helix–helix contacts are required, through a precise stereochemical fit and/or by ionic attraction by closely positioned side chains at the interface of the cyt b 5 and cyt P450 2B4 hydrophobic domains, then disruption of the contact surface by rotation of the helix distal to the inserted alanine would be expected to decrease the affinity of cyt b 5 for cyt P450 2B4. If insertion of alanine at any of the six positions caused a significant change in binding characteristics to cyt P450 2B4, then more intense mutagenesis studies centered around that insertion point could be performed. If, however, the helix–helix interaction is nonspecific in nature, disruption of the cyt b 5 helix by insertion of an alanine residue should leave the cyt b 5 –cyt P450 2B4 binding relatively unchanged. All six alanine insertion mutants were generated on the first attempt (two out of two candidates of each mutagenesis reaction). Recent studies in this laboratory demonstrated that E. coli strain C41(DE3) grown at 37 8C gave significantly higher and more consistent yields of recombinant cyt b 5 [22]. Similar results were obtained when this strain was

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used for expression of the alanine insertion mutants. The cyt b 5 was purified from the membrane fraction of E. coli and the final pure preparation revealed a single band on SDSPAGE. The specific content of the purified proteins is shown in Table 2. Mutations in the C-terminal hydrophobic domain did not alter the ability of mutant cyt b 5 to bind heme nor modify the ability of the mutants to incorporate into the bacterial membranes. 3.2. Characterization of the affinity of the alanine-insertion mutants of cyt b 5 for cyt P450 2B4 under steady-state and presteady-state conditions The ability of wild-type and mutant cyt b 5 to bind to cyt P450 2B4 was measured under both steady-state and presteady-state conditions. The K ds of the various cyt b 5– cyt P450 complexes which were determined from the steady-state binding studies are shown in Table 2. They reveal that the binding of the mutant cyts b 5 is not significantly different from that of the wild-type protein. The K d for the wild type is 0.09 AM. The kinetics of the binding of cyt b 5 to cyt P450 was measured by stoppedflow spectrophotometry by rapidly mixing both proteins and following the formation of the high spin form of cyt P450 2B4. Under these conditions the kinetics of binding is multiphasic, which is likely partially secondary to selfaggregation of the individual proteins. Table 2 provides data which indicate that the rate constants for the initial binding phase of the mutants and wild-type cyts b 5 are similar as are the overall kinetic traces. 3.3. Ability of alanine insertion mutants of cyt b 5 to stimulate methoxyflurane metabolism by cyt P450 2B4 The ability of cyt P450 2B4 to metabolize methoxyflurane is enhanced four- to fivefold by the presence of cyt b 5. Each of the mutants was tested for its ability to stimulate catalysis by cyt P450 2B4. This assay measures the ability of ferrous cyt b 5 to bind to and reduce the

oxyferrous form of cyt P450 2B4 whereas the previous assays determined the affinity of ferric cyt b 5 for ferric cyt P450 2B4 under steady-state and presteady-state conditions. The results are summarized in Table 2. The data reveal that methoxyflurane metabolism is stimulated by the mutant cyts b 5 to a slightly greater extent than by the native cyt b 5, with insertions in the distal part of the tail having, on average, a somewhat more pronounced effect. The 4.3-fold stimulation observed for wild-type cyt b 5 is similar to previously determined values [8]. Although the relative stimulation of methoxyflurane metabolism by wild-type cyt b 5 is similar to previous reports, it is worth noting that the absolute values shown here are much smaller due to the fact that only one of the two possible metabolic pathways of methoxyflurane leads to the formation of fluoride ion whereas the earlier study reported activity in terms of total reaction product. Cyt b 5 stimulates both pathways to the same extent [25]. 3.4. The tryptophan environment in wild-type and mutant cyts b 5 Rabbit cyt b 5 contains four tryptophan residues, one in the hydrophilic heme binding domain (Trp 22) and three (Trp 108, 109, 112) in the hydrophobic membrane-spanning C-terminal domain. Trp 22 is not fluorescent in the native protein [26] but becomes fluorescent when cyt b 5 is denatured and unfolded [27]. It has been suggested that Trp 112 does not emit but undergoes non-radiative energy transfer to Trp 108. Hence, Trp 108 and 109 are the most important fluorophores in the membrane anchor of cyt b 5 and their fluorescence has been used to probe the interaction between the membrane anchor and lipid bilayers [28–31]. The emission spectrum of tryptophan is sensitive to the polarity of its environment and reflects the average exposure of a fluorescent moiety to the aqueous phase. In a hydrophobic environment the emission maximum is shifted to shorter wavelengths whereas a polar environment results in emission at longer wavelengths [27,32].

Table 2 Summary of the properties of wild-type and alanine insertion mutants of cyt b 5 Cyt b 5

– Wild type Ala 104/105 Ala 108/109 Ala 111/112 Ala 116/117 Ala 120/121 Ala 123/124 a

Specific content of cyt b 5 (nmol heme/mg protein)

MF metabolism (nmol F min 1 nmol cyt P450 1FS.D.)

Ratio of MF metabolism (Fcyt b 5)a

K d, cyt b 5–cyt P450 complex (AMFS.D.)

Phase 1b k 1 (s )

– 50.3 48.3 40.6 50.1 44.0 36.1 42.5

0.17F0.02 0.73F0.07 0.87F0.04 0.90F0.13 1.17F0.19 1.04F0.13 1.26F0.10 1.17F0.23

– 4.3 5.1 5.3 6.9 6.1 7.4 6.9

– 0.090F0.027 0.049F0.015 0.080F0.022 0.084F0.065 0.044F0.022 0.064F0.019 0.046F0.028

– 9.28F1.44 4.65F0.41 4.52F0.03 6.01F0.43 6.49F0.51 12.33F4.71 5.72F0.24

1

A (%)

kmax (nm)c

Peak width (nm)d

– 53 33 60 41 28 62 49

– 338 339 341 341 337 340 340

– 57 61 61 59 58 57 58

Ratio of methoxyflurane metabolism in the presence and absence of cyt b 5. The rate constant and amplitude for the first phase of the association of cyt b 5 and cyt P450 were determined by stopped-flow spectrophotometry as described in Section 2. A is the amplitude of the phase with the rate constant of k. c k maximum of fluorescence emission. d Width in nm of the emission peak at 1/2 maximum intensity. b

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In an effort to determine if insertion of alanine at different positions in the membrane anchor and especially between Trp 108 and 109 and Asn-111 and Trp-112 would significantly alter the nature of the environment of these residues, the fluorescence spectrum of the native and mutant proteins was determined. The data in Table 2 demonstrate that the wavelength of the emission maximum for the wildtype cyt b 5 was 338 nm compared to 341 nm for the Ala 108/109 and 111/112 mutant proteins. These results suggest that when the tryptophans are adjacent to a small residue such as alanine, they have increased exposure to the solvent. The emission maximum of free tryptophan under similar conditions is 354 nm in agreement with the value in the literature [32]. Thus, the tryptophans in the protein are in a considerably more hydrophobic milieu compared to tryptophan free in solution. The width of the fluorescence peak at one-half of the emission maximum for the various cyts b 5 is provided in Table 2. The full width of the emission peak at one half maximum intensity for tryptophan free in solution is 65 nm compared to 57 nm for the wild-type cyt b 5, indicating that the tryptophans in cyt b5 are more restricted in their motion than the free tryptophan [33]. It also appears that the tryptophans in the Ala 104/105 and 108/109 insertion mutants are more mobile than those in the wildtype protein.

4. Discussion Interprotein binding via transmembrane helices occurs through either specific or nonspecific interactions. One of the best-studied examples of specific membrane interactions is glycophorin A, which undergoes dimerization both in membrane and in aqueous solution through association of a single 23-residue-long transmembrane helix [34]. The helix–helix interface forms a ridge and groove surface and, along with seven specific amino acid side chains, forms a very specific packing. Changing any of these side chains by mutagenesis even with conservative substitutions results in disruption of the dimerization. In contrast to these specific helix–helix binding effects, there can also be nonspecific interactions which are most notable when membrane proteins contain extramembraneous domains that themselves have a significant interaction [35]. One example of this is the bacterial aspartate chemoreceptor, Tar, which has been examined by saturation cysteine mutagenesis/disulfide cross-linking studies [36]. The Tar receptor forms a dimer through interaction of both its membrane-spanning a-helices and periplasmic domains. It is unlikely that the interaction between the a-helical membrane spanning domains would be sufficient to drive the dimerization. Rather, the periplasmic domains interact and this juxtaposes the helices such that they can bind in a relatively loose, nonspecific manner. For dimerization to occur in a membrane, the van der Waal’s interaction between the membrane-spanning helices should be stronger than that between the helices and lipid.

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In the case of cyt b 5 and cyt P450 2B4, previous studies from this laboratory have established the participation of specific amino acids on the hydrophilic domains of both proteins where binding takes place [8]. Yet the affinity of these hydrophilic domains alone is not enough to promote a productive interaction [9]. It has therefore been suggested that the hydrophobic C-terminal domain of cyt b 5 serves to provide proper spatial orientation and juxtaposition of the binding sites on the heme domains of cyt b 5 and cyt P450 2B4. A similar conclusion was reached in a study of CYP17 (P-45017a, 17a-hydroxylase-17,20-lyase) [10]. Thus, the hydrophobic domains of cyt b 5 and cyt P450 2B4, through mainly nonspecific interactions, increase the mutual affinity of the two heme domains and it is likely that the specific interactions between the heme domains constrain the membrane anchors to form weak side-by-side interactions. Another factor that may also contribute binding energy for the hydrophobic domains is the electrostatic interaction between the complementary helix dipoles of the N-terminal and C-terminal domains of cyt P450 and cyt b 5, respectively. In a membrane, binding can be enhanced by 1– 3 kcal by the electrostatic attraction between oppositely charged helix dipoles [37]. Nonspecific binding of the hydrophobic domains of cyt b 5 and cyt P450 may allow for changes in interhelical binding which may be necessary to convert an initial encounter complex between cyt b 5 and cyt P450 2B4 which is not yet capable of electron transfer into a complex competent in electron transfer. There is an alternative explanation for the mode of action of the membrane anchor of cyt b 5. It might bind a part of the cyt P450 molecule other than the membrane anchor to form a functionally competent electron transfer complex. At the present time, there is no evidence to support this alternative possibility. 4.1. Conclusions In summary, this report has addressed the question of whether specific side chain interactions in the hydrophobic a-helical membrane-binding C-terminal domain of cyt b 5 are required for binding to cyt P450. This was examined by inserting alanine at six different positions along the a-helix of cyt b 5 to change the register of the amino acids in the membrane spanning domain. Characterization of the mutant proteins indicated they (1) stimulated the cyt P450catalyzed metabolism of methoxyflurane, and (2) bound to cyt P450 2B4 under presteady-state and steady-state conditions to the same extent as the wild-type protein. Insertion of alanine into the membrane anchor of cyt b 5 did not result in a significant loss of its ability to bind cyt P450 and support catalysis by this mixed function oxidase. These results were interpreted to indicate that the membranespanning domain of cyt b 5 binds to cyt P450 2B4 by nonspecific interactions and not by unique structurally complementary interfaces.

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Acknowledgement This work was supported by an NIH Grant (GM35533) and a VA Merit Review Grant to L.W.

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