ARCHIVES
Vol.
OF BIOCHEMISTRY
290, No. 2, November
AND
BIOPHYSICS
1, pp. 277-284,
1991
Identification and Characterization of an NADPHCytochrome P450 Reductase Derived Peptide Involved in Binding to Cytochrome P450’ Steven Department
Received
G. Nadler2a3 and Henry W. Strobe14 of Biochemistry and Molecular Biology, University
March
27, 1991, and in revised
form
June
Academic
Press,
Inc.
NADPH”-cytochrome P450 reductase and cytochrome P450 are enzymes involved in the microsomal mixed function oxidation of lipids, drugs, and various other compounds. The reductase is a flavoprotein and the only mammalian protein known to contain 1 mol each of FMN and FAD per mole of enzyme (1, 2). The function of the reductase is to catalyze the reduction of the physiological acceptor, cytochrome P450 (3). However, it can also reduce other proteins such as cytochrome b5 (4), cytochrome c (l), and heme oxygenase (5). Cytochrome P450 reductase 0003.9861/91 $3.00 Copyright c) 1991 by Academic Press, All rights of reproduction in any form
School, Houston,
Texas 77025
12, 1991
The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (l-ethyl-3-[3(dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the K, and an 80% decrease in kJK,,, (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450. c> 1991
of Texas Medical
has at least two domains, a hydrophobic membrane binding domain and a hydrophilic domain. The membrane binding domain is within the first 55-60 N-terminal amino acids (6). When the membrane binding domain is removed by protease treatment, the solubilized reductase can no longer reduce cytochrome P450 (7). Thus, the membrane binding region may serve to increase the local concentration of the reductase by anchoring it to the membrane, or it may play a specific role in binding to cytochrome P450. This membrane binding domain has been isolated and purified. Whereas Gum and Strobe1 (6) have shown that there was no effect on substrate hydroxylation when the membrane binding peptide was added to reductasecytochrome P450 vesicles, Black et al. (8) reported inhibition of electron transfer from reductase to cytochrome P450 when a similar experiment was performed. The discrepancy between these observations may have been due to the fact that the peptide was purified by different ’ This research was supported by Grant AU-1067 from the Robert A. Welch Foundation and Grant CA37148 from the National Cancer Institute, DHHW. ’ Recipient of a predoctoral fellowship from the Rosalie B. Hite Foundation for Cancer Research. Some of the data in this paper are taken from the thesis project of S.G.N. submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Biomedical Sciences of the University of Texas Health Science Center at Houston. 3 Present address: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510. 4 Author to whom correspondence and reprint requests should be addressed at Department of Biochemistry and Molecular Biology, the University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77225. ’ Abbreviations used: EDC, 1-ethyl-3-(3.(dimethylamino)propyl]carbodiimide; HPLC, high-performance liquid chromatography; Tris, tris(hydroxymethyl)aminomethane; NADPH, nicotinamide adenine dinucleotide phosphate; FAD, Aavin-adenine dinucleotide; FMN, tlavin mononucleotide; Cyt P450 PB-b, cytochrome P450 IIBl; Cyt P45Oc, cytochrome P450 IAl, TFA, trifluoroacetic acid; SDS: sodium dodecyl sulfate; TPCK, L-1-P-tosylamino-2phenylethyl chloromethyl ketone; PVDF, polyvinylidene fluoride; DLPC, dilauroylphosphatidylcholine; PMSF, phenylmethylsulfonyl fluoride. 275
Inc. reserved.
278
NADLER
AND
methods in the two studies. The hydrophilic domain of the reductase contains the two flavins and is involved in the catalytic function of the enzyme. Evidence suggests that two electrons are transferred, in two one-electron steps, from NADPH to FAD to FMN to cytochrome P450 (2, 9, 10-12). The cytochromes P450 are heme-containing, integral membrane proteins. The crystal structure for a soluble form of this protein, P450 cam, is known (13). Nelson and Strobe1 (14) have proposed that the membrane-bound forms of cytochrome P450 are similar in structure to P450 cam, except for the first 66 N-terminal amino acids. They propose that this region may serve to anchor the cytochrome P450 molecule in the membrane, although a recent study suggests only the first 21 N-terminal amino acids are within the membrane (15). Presumably, the hydrophilic domains of both the reductase and cytochrome P450 bind to form a catalytically competent complex. Studying the interaction between reductase and cytochrome P450 is important not only in understanding the mechanism of action of the cytochrome P450 system, but also of other redox proteins in general. It is still not clear whether stable reductase-cytochrome P450 complexes exist or if transient complexes form due to random collisions upon lateral diffusion in the membrane. There is evidence to support each of these possibilities (16, 17). In either case, since there are severalfold more molecules of the various forms of cytochrome P450 per molecule of reductase in the endoplasmic reticulum membrane, binding between reductase and cytochrome must be very efficient and specific for catalysis to occur effectively. We have previously shown that electrostatic interactions play a role in the binding of the two proteins (18). Specifically, carboxyl residues on the reductase are involved in binding to cytochrome P450. This is not surprising since the reductase is highly acidic, having 102 carboxyl residues out of a total 678 amino acids. Apparently, the negative charges interact with positive charges on cytochrome P450. Lysine 384 and the N-terminus of cytochrome P450IIB4 (P450LM,) have been identified as residues involved in the interaction with the reductase (19,20). Our goal in these studies was to identify the specific amino acids on cytochrome P450 reductase which are involved in binding to cytochrome P450 PB-b. The water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), with the nucleophile methylamine, was used to modify cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. This reagent has been used to identify various other binding domains of redox proteins, including adrenodoxin-adrenodoxin reductase (21), cytochrome b5cytochrome b5 reductase (22), cytochrome c-cytochrome c peroxidase (23), and cytochrome P450 reductase-cytochrome c (24). In this paper, we demonstrate that modification of a limited number of carboxyl groups in cytochrome P450 reductase causes a significant inhibition in
STROBEL
the interaction with cytochrome P450 PB-b. When the modification was performed in the presence of the cytochrome, there was no inhibition of binding between the modified reductase and cytochrome P450. We have also identified a trypsin-generated peptide of the reductase which contains the majority of the methylamine label. Amino acid sequencing and analysis suggest that one or more of the carboxyl groups of ASP-113, -118, -121, and -129, or GLU-115, -116, and -127, is involved in the interaction with cytochrome P450 PB-b. MATERIALS
AND
METHODS
Materials EDC, TPCK-trypsin, and dilauroyl phosphatidylcholine were obtained from Sigma. Methylamine HCl was from Aldrich. [‘*C]Methylamine was obtained from Amersham. All solvents were HPLC grade or better.
Methods Protein purification. Cytochrome P450 reductase was purified using previously published procedures of Dignam and Strobe1 (25) and Yasukochi and Masters (26), except 2’5’-ADP-agarose was used as the affinity resin. The reductase was washed extensively while bound to the affinity resin with 50 mM KPi, 20% glycerol, and 0.1% cholate, pH 7.5, to remove the nonionic detergent Renex 690. Cytochrome P450 PB-b (P450 IIBl) was purified using the procedure of Ryan et al. (27) with minor modifications. Renex 690 was used as the nonionic detergent. Hydroxylapatite column chromatography was performed after the DE52 column. Renex was removed as described above while the cytochrome was bound to the hydroxylapatite column. The specific activity of cytochrome P450 reductase was typically 35-50 amol cyt c reduced/min/ mg protein, assayed using the procedure of Dignam and Strobe1 (25). The specific content of cytochrome P450 PB-b was 12 nmol/mgprotein. EDC modification of cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. A total of 10 nmol each of cytochrome P450 reductase and cytochrome P450 PB-b was inserted into vesicles containing 200 ag DLPC, in a final volume of 1 ml, using the cholate dialysis technique (18). The sample was then treated in 8 mM NaPi, pH 6.8, containing 1 mM benzphetamine, with 5 mM EDC and 200 mM methylamine at 22°C for 1 h. Benzphetamine was added to enhance the interaction between the two proteins. The reaction was quenched with 0.1 M ammonium acetate. The sample was then diluted 25-fold with 0.1 M Tris-Cl and 0.1% Renex 690, pH 8.4, and loaded on a DEAE A-25 column at 4”C, equilibrated with the same buffer. The resin was washed with approximately 20 bed volumes of 0.1 M Tris-Cl and 0.5% sodium cholate, pH 8.4, to wash off both the cytochrome and Renex. The reductase was eluted from the column with 0.5 M NaCl in the cholate buffer. PMSF was added to 0.2 mM to prevent proteolysis. The sample was then dialyzed for 12 h against 5 mM NaPi, pH 7.5. Prior to dialysis, 200 ag/ml DLPC was added to the protein to form phospholipid vesicles. The reductase was then modified with [‘*C]methylamine as follows. A total of 50 pM reductase was reacted with 5 mM EDC and 200 mM [Wlmethylamine (specific activity, 0.43 mCi/nmol) in 8 mM NaPi, pH 6.8, for 1 h at 22°C. The reaction was quenched with 0.2 M ammonium acetate. Unlabeled methylamine was added to 1 M in the dialysis tubing, before dialysis, to displace any noncovalent [Wlmethylamine which was bound to the protein. The sample was dialyzed extensively against 0.1 M ammonium bicarbonate, pH 8.0, for 36 h until there were no counts above background remaining in the dialysis buffer. Typically, 60% of the protein was recovered after this modification procedure. The rate of modification of carboxyl groups appeared to be mainly dependent on the concentration of EDC and methylamine, but not on the concentration of carboxyl groups (data not shown).
PEPTIDE
DERIVED
FROM
NADPH-CYTOCHROME
Ttypsin digestion. Typically, 1 mg of the EDC-modified reductase was digested with a 1:25 (w/w) amount of TPCK-trypsin for 48 h at 37’C. The reaction was run in 0.1 M ammonium bicarbonate, pH 8.0, containing 8 M urea. The reaction was quenched by boiling the sample for 5 min and then either loading the sample directly on the HPLC or freezing at -20°C. Purification of tryptic peptides. Approximately 10 nmol of tryptic peptides was separated on a HPLC system using a Vydac C-18 reverse phase column (150 X 4.6 mm, 5 p). A Waters 6000A and an M-45 pump were used with a Waters 660 gradient programmer to form the wateracetonitrile gradient. Chromatography was performed at room temperature at a flow rate of 1 ml/min. A gradient from O-37.5% B in the first 60 min, 37.5-75% B in 60-90 min, and 75-100% B in 90-105 min was run. Buffer A consisted of 0.1% trifluoroacetic acid (TFA) in water. Buffer B was composed of 0.1% TFA, 20% water, and 79.9% acetonitrile. Peptides were detected at 214 nm using a Waters 440 variable wavelength detector. Fractions corresponding to peaks of absorbance were collected. An aliquot of each fraction was counted for radioactivity on a Packard 3255 scintillation counter. The major radioactive peak was then dried down under nitrogen to remove the acetonitrile and repurified on an Aquapore C-8 reverse phase column. The same buffer system was used, except a 0 to 60% buffer B gradient over 60 min was used to elute the peptides. The flow rate was 1 ml/min and 214-nm peaks were again collected and aliquots checked for radioactivity. Amino acid analysis was perAmino acid analysis and sequencing. formed on a Beckman 121 M amino acid analyzer. The sample was hydrolyzed in V~CUO in 6 N HCl at 110°C for 18 h. Norleucine was included as an internal standard. Tryptophan was not determined. The peptide was sequenced on an Applied Biosystems Model 470 A gas phase sequencer which was connected directly to a Model 120A HPLC system. Approximately 200 pmol was applied to a PVDF filter and sequenced. Benzphetamine demethylation assays were performed Kinetic assays. using the fluorometric procedure of Waxman and Walsh (28). The assays were run with a constant reductase concentration of 0.04 PM. The cytochrome P450 PB-b concentration was varied from 0.0125 to 0.25 FM. NADPH and benzphetamine were present at 600 and 1 mM, respectively. The assay mixture was incubated for 8 min during which the time course of product formation is linear with time. Fluorescence was detected using a Perkin-Elmer LS-5 spectrofluorometer. Details of the assay are described in Nadler and Strobe1 (18).
P450
0
279
REDUCTASE
12
3
4
5
6
7
8
4hr
8hr
9
Time (Hours) Ffoc SminlSmin
30min lhr
2hr
3hr
MW (x10-3)
116 97.4 66.2
-
42.7
-
FIG. 1. Time course of the incorporation of [i4C]methylamine into the reductase molecule. (top) 50 pM of reductase was treated with 200 mM [i4C]methylamine and 5 mM EDC in 8 mM NaP,, pH 6.8, at 22OC, for the indicated times. The reaction was quenched with 0.2 M ammonium acetate, pH 7.5. The sample was then dialyzed extensively for 48 h against 10 mM KPi, 0.5 M NaCl, and 0.5% cholate, pH 7.5. (bottom) To check for intermolecular crosslinking, a 5-pg aliquot of reductase at each time point was removed, quenched with ammonium acetate, and run on a 7.5% SDS polyacrylamide gel.
RESULTS
Time Course of Incorporation of [14C]Methylamine The amount of incorporation of radioactive methylamine, using the carbodiimide EDC, into the unprotected reductase was determined. As seen in Fig. 1 (top), the incorporation of methylamine appears to approach saturation after approximately 8 h, with the incorporation of 6 mol of methylamine per mole of reductase. Therefore, under these conditions, only a small percentage of the total number of carboxyl residues is free to react with the carbodiimide. A sample at each time point was quenched and run on an SDS-polyacrylamide gel to check for intermolecular crosslinking. As seenin Fig. 1 (bottom), there does not appear to be any crosslinking until 3 h. We, therefore, performed all the incubations of the subsequent studies for 2 h or less. Presumably, the intermolecular crosslink occurs due to competition between lysine residues and methylamine for the EDC-activated carboxyl. Since there are 102 carboxyl residues on the reductase, only 6% of the carboxyl residues are modified with this procedure.
Kinetic Effects of EDC Modification Cytochrome P450 reductase was modified first in the presence of cytochrome P450 PB-b, and then the cytochrome was removed and the reductase was again modified. The first and second modifications are termed protected and unprotected reductase, respectively. The first step of the modification should alter those residues on the reductase which are not involved in binding to cytochrome P450. The proteins are present during the modification at a concentration approximately 80 times the binding constant. Assuming a Kd of 0.1 PM, although this may be lower due to the low salt concentration during the modification reaction, approximately 89% of the protein molecules are present as the reductase-cytochrome complex. The majority of the residues involved in the binding of the two proteins should, therefore, be in close proximity and inaccessible to the modifying reagents. The second step of the modification would then alter the amino acids which are most likely to be involved in the interaction
280
NADLER
AND
with cytochrome P450. The possibility also exists that the first modification induces a conformational change in the reductase which makes residues more reactive although they are not involved in binding to the cytochrome P450. In Fig. 2, the K,,, and V,,,,, between modified cytochrome P450 reductase and cytochrome P450 were determined, as described previously. Saturating concentrations of NADPH and benzphetamine were used at a constant reductase concentration, while varying cytochrome P450 concentration. Using this procedure, a measure of the interaction between the two proteins could be determined. The control sample was treated similarly to the modified sample, except EDC was omitted. The kinetic constants of the control were virtually identical to those of the native protein. The kinetic constants from the data in Fig. 2 (top) were determined using a double reciprocal plot (Fig. 2, bottom) and are shown in Table I. The protected reductase, interestingly, has a slightly decreased K,,, compared to the control. The V,,, of the protected reductase is also decreased. This may be due to modification in or near the NADPH and FAD binding site, since these regions should be exposed to the aqueous environment and are probably not protected by the cytochrome P450. NADPH has been previously shown to donate its electrons
STROBEL TABLE
K?l Sample Control EDC-modified, EDC-modified,
PB-b]
(PM)
40
0
l/[P:50
Pi!b]
,,IM)
FIG. 2. Kinetic effect of EDC modification of cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. (top) Benzphetamine demethylation was determined as described under Materials and Methods, at a constant reductase concentration of 0.04 pM and varying cytochrome P450 PB-b concentration. (0) Control, sham treated, (0) reductase, protected with P450 PB-b; (A.) reductase, unprotected. (bottom) Double reciprocal plot of the data presented above. (0) Control, sham treated; (0) reductase, protected; (A) reductase, unprotected.
protected unprotected
(PM) 0.097 0.056 0.231
V max (nmol/min) 2.86 1.39 1.31
L/Km (min-‘) 737 621 141
Note. The reductase was modified as described under Materials and Methods. Data were from Fig. 2. The kinetic constants were determined from a plot of l/[P450 PB-b]r,, vs 1 V and are the mean of at least two determinations.
to FAD (10). The unprotected reductase yielded a fourfold increase in K,,, compared to the protected reductase and a twofold increase compared to the control. However, there was no change in the V,,, of the unprotected reductase compared to the protected sample. The k,,JK,, catalytic efficiency, was decreased 20% for the protected sample and 80% for the unprotected sample. The significant decrease in k,,,/K, of the unprotected reductase compared to the protected sample appears to be mainly due to a K,,, effect. We are, therefore, confident that the second modification of the differential modification procedure is specific for labeling residues on the reductase involved in the binding to cytochrome P450. It is also important to note that the significant inhibition of the K,, as well as the catalytic efficiency, is a result of modification of only a limited number of the 102 carboxyl residues. Differential
[P450
I
Kinetic Effect on the Ability of Cytochrome P450 Reductase to Interact with Cytochrome P450 PB-b
Modification
In order to identify the residues on the reductase which were modified with EDC, and are most likely involved in binding to cytochrome P450, we chose to use the differential modification procedure described above. The reductase was first modified in the presence of cytochrome P450 PB-b and the substrate benzphetamine, with EDC plus unlabeled methylamine. The reductase was then separated from the cytochrome P450. As shown in Fig. 3, the DEAE A-25 column effectively removed the modified reductase from the cytochrome. The reductase was then modified with EDC plus [14C]methylamine. Those residues which are labeled with 14Care most likely involved in binding to cytochrome P450. In a separate experiment where the reductase was modified in the presence of cytochrome P450 with EDC and [14C]methylamine, there was 0.68 mol of methylamine incorporated per mole of reductase after 1 h. This is less than the approximately 1.5 mol of methylamine incorporated per mole of reductase in the absence of cytochrome P450 seen in Fig. 1. This suggeststhat cytochrome P450 is protecting certain residues from modification. When the modification was
PEPTIDE A -
DERIVED
0 --x
---
_
,“_
--
NADPH-CYTOCHROME
c .~
REDUCTASE
--‘
FROM
, d
.:
FIG. 3. SDS polyacrylamide gel of EDC-modified P450 PB-b and reductase. A 7.5% SDS polyacrylamide gel of reductase which was modified in the presence and absence of cytochrome P450 and separated on a DEAE A-25 column. (A) 5 pg each of P450 reductase and cytochrome P450 PB-b; (B) 5 pg of reductase after the first EDC modification and separation of the A-25 column; (C) 5 pg of reductase after the second EDC modification.
performed as described above, however, using unlabeled methylamine in the first protection step, and [14C]methylamine in the second unprotected step, approximately 3.2 mol of methylamine per mole of reductase was incorporated after 1 h. This is a greater number of carboxyls modified than would be expected if the reductase was modified alone, as seen in Fig. 1. It would appear that the carboxyls on the reductase which are not protected by cytochrome P450 are slower to react with EDC and methylamine than those which are protected by the cytochrome P450. Although there are a total of 3.9 mol of methylamine incorporated after the differential modification procedure, compared to approximately 3 mol incorporated when the reductase was modified alone, this difference is not very significant when the experimental error of approximately 10% is taken into account. The small amount of additional modification, however, may indicate that there was a cytochrome-P450-induced conformational change of the reductase leading to modification of these exposed groups. In a previous publication we have addressed the possibility of conformational changes of the reductase following EDC modification and concluded that there was no significant conformational change of the reductase upon EDC modification (18). Identification of Methylamine-Labeled on the Reductase
Carboxyl Groups
The reductase which was first modified with EDC and unlabeled methylamine in the presence of cytochrome P450 and subsequently with [14C]methylamine, was sub-
P450
281
REDUCTASE
jected to trypsin digestion under denaturing conditions. The resulting peptides were separated by HPLC on a Vydac C-18 reverse phase column, using the conditions described under Materials and Methods. A typical HPLC chromatogram is shown in Fig. 4. An aliquot of each peak was counted for radioactivity. Approximately 95% of the radioactivity which was loaded on the column was recovered in the fractions. Due to the inherent difficulty in obtaining protein which had a large amount of radioactivity (since a high concentration of methylamine was required causing a low specific radioactivity) we repeated the modification and C-18 chromatography three times to be sure that the labeling of the peak fraction was reproducible. The peak identified with an arrow in Fig. 4 (top), termed Peak I, contained approximately 40% of the total radioactivity which eluted from the column. The percentage of radioactivity was determined by dividing the radioactivity in Peak I by the sum of the radioactivity in the approximately 150 fractions (volume corrected).
20 1000
$Yj 800 cl &
600
gJ 400 5 u
200 0
I 20
4.0
Lo
Time
(minutes)
Time
60 (minutes)
40
io
lb0
'
160
FIG. 4. HPLC chromatogram of [“Clmethylamine-modified tryptic peptides. (t.op) Tryptic peptides of EDC-modified reductase were separated on a high-pressure Vydac C-18 reverse phase column. Approximately 5 nmol was loaded on the column. The peptides were eluted using a gradient of O-37.5% B in the first 60 min, 37.5-75% B in 60-90 min, and 75-100% B in 90-105 min, with a flow rate of 1.0 ml/min. The buffers used are described under Materials and Methods. The absorbance at 214 nm was detected with a full scale of 2.0 A. (bottom) Radioactivity associated with HPLC-purified peptides. A 25% aliquot of each fraction was removed and counted for radioactivity.
282
NADLER
AND
3 f t w zi 8 5 e 8 9 20
40
60
60
100
120
Time (minutes) FIG. 5. Repurification of Peak I. Peak purified on an Aquapore C-8 reverse phase contains >90% of the radioactivity loaded conditions are as described under Materials bance at 214 nm was detected with a full
I identified in Fig. 4 was recolumn. The peak identified on the column. The gradient and Methods. The absorscale of 0.125 A.
Although the chromatograms were slightly different in the three experiments, Peak I always migrated at a similar position. In the lower portion of Fig. 4, it can be seen that the radioactivity associated with Peak I is the only major peak of radioactivity present. The remaining 60% of the radioactivity is most likely distributed in limited amounts into a number of residues, since the other approximately 150 fractions contained lessthan 1% of the radioactivity.‘j The peak at approximately 37 min appears to be FAD/ FMN. Peak I, which contained the major portion of radioactivity, was repurified on a C-8 reverse phase column. The labeled peak is indicated by an arrow in Fig. 5. This peak on the C-8 column contained >90% of the radioactivity. The unusually late migration of this peptide on both the C-18 and the C-8 column may be due to the modification of charged residues, rendering them more nonpolar which would cause more retention on the reverse phase column. The Peak I peptide was then submitted to amino acid analysis. Amino Acid Sequencing and Analysis of Peak I In order to identify the EDC-modified amino acids, Peak I was subjected to amino acid sequencing and analysis. Approximately 200 pmol of Peak I was loaded on a PVDF filter and sequenced by gas phase automated Edman degradation. We sequenced eleven residues and obtained the sequence:
STROBEL
Due to the high background (often present in gas phase sequencing) and small amount of amino acid releasedfrom the Edman degradation, we could not accurately determine the first two residues. However, the amino acid sequence which was determined corresponds to residues 109-119 of rat liver NADPH-cytochrome P450 reductase, except for the residue at position 8 of the peptide. In the sequence determined by Porter and Kasper (29), this residue was a glutamic acid, whereas we have detected an aspartic acid. This may be due to an incorrect sequence determined by Porter and Kasper, or it may represent a site of modification of glutamic acid which caused a shift in the mobility of the Pth-glutamic acid derivative on the HPLC. The repetitive yield of sequencing was approximately 94%. The sequence which we determined is presumably a portion of the tryptic peptide corresponding to residues 109-130 of the reductase. Within these 21 amino acids are 7 carboxyl containing amino acids. These are Asp-113, Glu-115, and -116, Asp-118 and -121, Glu127, and Asp-129. Due to the small amount of peptide and the very low radioactivity, we were unable to determine which specific amino acid(s) was modified. Since the peptide contained approximately 40% of the radioactivity, this would correspond to approximately 1.3 mol of carboxyl groups modified (based on a total of 3.2 mol modified in the labeling step). This modification may be on discrete amino acids or possibly over all seven residues. We also performed amino acid analysis on the peptide. The analysis shown in Table II best matches the predicted composition of residues 109-130 in comparison to other tryptic peptides. The composition also matches residues
TABLE Amino
Amino
119.
6 On some chromatographic runs there was a peak which contained 10% of the radioactivity; however, the presence of this peak and its retention time were not reproducible and may have represented incomplete trypsin digestion, or incomplete chemical modification.
II of Peak
GUY
0.9 (1)
Ala Val Ile Leu
1.6 (2)
Arg LYS
I
Moles of amino acid per mole of peptide 2.5 (4)
Phe His
Glu-Asp-Tyr-Asp-Leu
Composition
Asx Thr Ser Glx Pro
Tyr
109 X-X-Ser-Ala-Asp-Pro-
acid
Acid
1.6 (0) 3.7 (3) 3.8 (3) 0.9 (2)
0.9 (0) 0.9 (1) 1.8 (3) 0.7 (1)
0.0 (0) 0.8 (0) 0.0 (0) 1.2 (1)
Note. Amino acid analysis was performed as described under Methods. The data are the average of three analyses. Tryptophan was not determined. Glycine was corrected for background contamination. The value in parentheses represents the number of residues determined from residues 109-130 of cytochrome P450 reductase.
PEPTIDE
DERIVED
FROM
NADPH-CYTOCHROME
P450
REDUCTASE
283
the possibility that other amino acids on the reductase, besides those which we were able to modify, are also involved in the interaction with cytochrome P450. The role of other amino acids is suggestedby the fact that we only obtained a 4-fold increase in Km, whereas we had previCONCLUSION ously observed (35) a 30-fold increase in K,,, when the salt Protein-protein interactions play a central role in the concentration was raised 3-fold. On the other hand, the regulation of metabolic processes. Previous studies have salt effects were studied by altering the ionic strength in shown the importance of charged amino acids in the in- the reconstituted assay mixture, thus affecting both reductase and cytochrome P450. teraction between cytochrome P450 reductase and cytochrome P450 (18). Makower et al. (19) have shown that The study presented in this paper will aid in our unmodification of lysine residues on cytochrome P45OLM, derstanding of the cytochrome P450 system in general. leads to a decreased rate of reduction by cytochrome P450 Further studies are in progress to use site-directed mutagenesis to identify the specific amino acids and deterreductase. Tamburini and Schenkman (30) and Bernhardt et al. (31) have also shown that modification of carboxyl mine their contribution to binding. residues on the reductase causes a decrease in the ability to reduce cytochrome P450. In a previous study, we have ACKNOWLEDGMENTS shown that carboxyl group modification of the reductase The expert technical assistance of Anne Bernhard is gratefully apdecreasesthe ability of the reductase to interact with both preciated. We also thank Christopher Chin of the University of Texas cytochrome P450 PB-b and P45Oc (18). This effect was and Katherine Stone of the Yale University Protein and Nucleic Acid seen with both benzphetamine and ethoxycoumarin as Chemistry Facility for performing the amino acid analysis and sequencing. substrates. In this paper we have attempted to identify the specific carboxyl residues on the reductase involved in the interREFERENCES action with cytochrome P450 PB-b. The water-soluble 1. Dignam, J. D., and Strobel, H. W. (1975) Biochem. Biophys. Res. carbodiimide EDC was used with the labeled nucleophile Commun. 63,845-852. methylamine to modify carboxyl residues. A differential 2. Iyanagi, T., and Mason, H. S. (1973) Biochemistry 12, 2297-2308. modification technique which has been used to study the 3. Strobel, H. W., Lu, A. Y. H., Heidema, J., and Coon, M. J. (1970) interaction of cytochrome c and cytochrome c peroxidase J. Biol. Chem. 245, 4851-4854. (23), as well as other proteins, was used in our study. 4. Enoch, H. G., and Strittmatter, P. (1979) J. Biol. Chem. 254,89768981. Table I clearly shows that the residues modified in the 5. Yoshida, T., Noguchi, M., and Kikuchi, G. (1980) J. Biol. Chem. second versus the first step of the procedure specifically 255, 4408-4420. lead to an increased Km, as opposed to a V,,,,, change. 6. Gum, J. R., and Strobel, H. W. (1981) J. Biol. Chem. 256, 7478Presumably, only some of these modified residues are ac7486. tually involved in the increased Km. I. Black, S. D., and Coon, M. J. (1982) J. Biol. Chem. 257, 5929The evidence presented suggeststhat the carboxyl res5938. idues on cytochrome P450 reductase which are important 8. Black, S. D., French, J. S., Williams, C. H., Jr., and Coon, M. J. for binding to cytochrome P450 are within amino acid (1979) Biochem. Biophys. Res. Commun. 91, 1528-1535. residues 109-130. We are unable to pinpoint which of the 9. Vermilion, J. L., Ballou, D. P., Massey, V., and Coon, M. J. (1981) seven negatively charged amino acids in this region conJ. Biol. Chem. 256, 266-277. tribute to the interaction. The carboxyl containing amino 10. Kurzban, G. P., and Strobel, H. W. (1986) J. Biol. Chem. 261, acids in this region are aspartic acid -113, -118, -121, and 782447830. -129, and glutamic acid -115, -116, and -127. Interestingly, 11. Iyanagi, T., Makino, N., and Mason, H. S. (1974) Biochemistry 13, 1801-1810. these amino acids are within the predicted flavin mononucleotide (FMN) binding domain (32). This is consistent 12. Yasukochi, Y., Peterson, J. A., and Masters, B. S. S. (1979) J. Biol. Chem. 254, 7097-7104. with previous studies which show that electron efflux from 13. Poulos, T. (1988) Pharmacol. Res. 5, 67-75. the reductase is from the FMN prosthetic group. In ad14. Nelson, D. R., and Strobel, H. W. (1988) J. Biol. Chem. 260,6038dition, two acidic amino acids, aspartic acid -113 and 6050. -118 are conserved in all cytochrome P450 reductase pro15. Vergeres, G., Kasper, W. H., and Richter, C. (1989) Biochemistry teins for which sequencesare known (33,34). Presumably, 28, 3650-3655. the amino acids which we have identified serve to anchor 16. Miwa, G. T., and Lu, A. Y. H. (1984) Arch. Biochem. Biophys. 234, the proposed FMN domain on the reductase to surface161-166. accessible positively charged amino acids on cytochrome 17. Wagner, S. L., Dean, W. L., and Gray, R. D. (1984) J. Biol. Chem. P450, thereby orienting the electron donating FMN region 259, 2380-2395. with a putative acceptor site in the cytochrome P450 mol- 18. Nadler, S. G., and Strobel, H. W. (1988) Arch. Biochem. Biophys. ecule for reduction of the heme iron. We cannot rule out 261.418-429.
109-130 much better than residues 109-167, suggesting that the peptide ends at residue 130 and not at the second tryptic site.
284
NADLER
19. Makower, K. (1984)
A., Bernhardt, R., Rabe, H., Janig, G. R., and Ruckpaul, Biomed. Biochim. Acta 43, 1333-1341.
20. Blanck, J., Smettan, Ruckpaul, K. (1984)
G., Ristan, O., Ingleman-Sundberg, Eur. J. Biochem. 144,509-513.
21. Geren, L. M., O’Brien, P., Stonehuerner, J. Biol. Chem. 259, 2155-2160. 22. Dailey, 5396.
H. A., and Strittmatter,
23. Bechtold, 5200.
R., and Bosshard,
24. Nisimoto,
Y. (1986)
25. Dignam, 1123. 26. Yasukochi, 5337-5344.
AND
J. Biol.
J. D., and Strobel, Y., and Masters,
P. (1979) H. R. (1985) Chem.
266,
H. W. (1977)
J., and Millett, J. Biol.
M.,
and
F. (1984)
Chem.
254,5388-
J. Biol. Chem.
260,5190-
14,232-14,239. Biochemistry
B. S. S. (1976)
J. Biol.
16, 1116Chem.
251,
STROBEL 27. Ryan, D. E., Thomas, P. E., Korzeniowski, D., and Levin, W. (1979) J. Biol. Chem. 254, 13651374. 28. Waxman, D. J., and Walsh, C. (1983) Biochemistry 22,4846-4955. 29. Porter, J. D., and Kasper, C. B. (1985) Proc. N&l. Acad. Sci. USA 82,973-977. 30. Tamburini, P. P., and Schenkman, J. B. (1986) Mol. Pharmacol. 30, 178-185. 31. Bernhardt, R., Pommerening, K., and Ruckpaul, K. (1987) Biochem.
Int. 14,823-832. 32. Porter, J. D., and Kasper, C. B. (1986) Biochemistry 25,1682-1687. 33. Sutter, T. R., Sangyard, D., and Loper, J. C. (1990) J. Biol. Chem. 265, 16,428-16,436. 34. Murakami, H., Yabusaki, Y., Sakaki, J., Shibata, M., and Ohkawa, H. (1987) DNA 6, 1899197. 35. Strobel, H. W., Nadler, S. G., and Nelson, D. R. (1989) Drug Metab. Reu. 20(2-4), 519-533.
Vol.
OF BIOCHEMISTRY
290, No. 2, November
AND
BIOPHYSICS
1, pp. 277-284,
1991
Identification and Characterization of an NADPHCytochrome P450 Reductase Derived Peptide Involved in Binding to Cytochrome P450’ Steven Department
Received
G. Nadler2a3 and Henry W. Strobe14 of Biochemistry and Molecular Biology, University
March
27, 1991, and in revised
form
June
Academic
Press,
Inc.
NADPH”-cytochrome P450 reductase and cytochrome P450 are enzymes involved in the microsomal mixed function oxidation of lipids, drugs, and various other compounds. The reductase is a flavoprotein and the only mammalian protein known to contain 1 mol each of FMN and FAD per mole of enzyme (1, 2). The function of the reductase is to catalyze the reduction of the physiological acceptor, cytochrome P450 (3). However, it can also reduce other proteins such as cytochrome b5 (4), cytochrome c (l), and heme oxygenase (5). Cytochrome P450 reductase 0003.9861/91 $3.00 Copyright c) 1991 by Academic Press, All rights of reproduction in any form
School, Houston,
Texas 77025
12, 1991
The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (l-ethyl-3-[3(dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the K, and an 80% decrease in kJK,,, (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450. c> 1991
of Texas Medical
has at least two domains, a hydrophobic membrane binding domain and a hydrophilic domain. The membrane binding domain is within the first 55-60 N-terminal amino acids (6). When the membrane binding domain is removed by protease treatment, the solubilized reductase can no longer reduce cytochrome P450 (7). Thus, the membrane binding region may serve to increase the local concentration of the reductase by anchoring it to the membrane, or it may play a specific role in binding to cytochrome P450. This membrane binding domain has been isolated and purified. Whereas Gum and Strobe1 (6) have shown that there was no effect on substrate hydroxylation when the membrane binding peptide was added to reductasecytochrome P450 vesicles, Black et al. (8) reported inhibition of electron transfer from reductase to cytochrome P450 when a similar experiment was performed. The discrepancy between these observations may have been due to the fact that the peptide was purified by different ’ This research was supported by Grant AU-1067 from the Robert A. Welch Foundation and Grant CA37148 from the National Cancer Institute, DHHW. ’ Recipient of a predoctoral fellowship from the Rosalie B. Hite Foundation for Cancer Research. Some of the data in this paper are taken from the thesis project of S.G.N. submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Biomedical Sciences of the University of Texas Health Science Center at Houston. 3 Present address: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510. 4 Author to whom correspondence and reprint requests should be addressed at Department of Biochemistry and Molecular Biology, the University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77225. ’ Abbreviations used: EDC, 1-ethyl-3-(3.(dimethylamino)propyl]carbodiimide; HPLC, high-performance liquid chromatography; Tris, tris(hydroxymethyl)aminomethane; NADPH, nicotinamide adenine dinucleotide phosphate; FAD, Aavin-adenine dinucleotide; FMN, tlavin mononucleotide; Cyt P450 PB-b, cytochrome P450 IIBl; Cyt P45Oc, cytochrome P450 IAl, TFA, trifluoroacetic acid; SDS: sodium dodecyl sulfate; TPCK, L-1-P-tosylamino-2phenylethyl chloromethyl ketone; PVDF, polyvinylidene fluoride; DLPC, dilauroylphosphatidylcholine; PMSF, phenylmethylsulfonyl fluoride. 275
Inc. reserved.
278
NADLER
AND
methods in the two studies. The hydrophilic domain of the reductase contains the two flavins and is involved in the catalytic function of the enzyme. Evidence suggests that two electrons are transferred, in two one-electron steps, from NADPH to FAD to FMN to cytochrome P450 (2, 9, 10-12). The cytochromes P450 are heme-containing, integral membrane proteins. The crystal structure for a soluble form of this protein, P450 cam, is known (13). Nelson and Strobe1 (14) have proposed that the membrane-bound forms of cytochrome P450 are similar in structure to P450 cam, except for the first 66 N-terminal amino acids. They propose that this region may serve to anchor the cytochrome P450 molecule in the membrane, although a recent study suggests only the first 21 N-terminal amino acids are within the membrane (15). Presumably, the hydrophilic domains of both the reductase and cytochrome P450 bind to form a catalytically competent complex. Studying the interaction between reductase and cytochrome P450 is important not only in understanding the mechanism of action of the cytochrome P450 system, but also of other redox proteins in general. It is still not clear whether stable reductase-cytochrome P450 complexes exist or if transient complexes form due to random collisions upon lateral diffusion in the membrane. There is evidence to support each of these possibilities (16, 17). In either case, since there are severalfold more molecules of the various forms of cytochrome P450 per molecule of reductase in the endoplasmic reticulum membrane, binding between reductase and cytochrome must be very efficient and specific for catalysis to occur effectively. We have previously shown that electrostatic interactions play a role in the binding of the two proteins (18). Specifically, carboxyl residues on the reductase are involved in binding to cytochrome P450. This is not surprising since the reductase is highly acidic, having 102 carboxyl residues out of a total 678 amino acids. Apparently, the negative charges interact with positive charges on cytochrome P450. Lysine 384 and the N-terminus of cytochrome P450IIB4 (P450LM,) have been identified as residues involved in the interaction with the reductase (19,20). Our goal in these studies was to identify the specific amino acids on cytochrome P450 reductase which are involved in binding to cytochrome P450 PB-b. The water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), with the nucleophile methylamine, was used to modify cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. This reagent has been used to identify various other binding domains of redox proteins, including adrenodoxin-adrenodoxin reductase (21), cytochrome b5cytochrome b5 reductase (22), cytochrome c-cytochrome c peroxidase (23), and cytochrome P450 reductase-cytochrome c (24). In this paper, we demonstrate that modification of a limited number of carboxyl groups in cytochrome P450 reductase causes a significant inhibition in
STROBEL
the interaction with cytochrome P450 PB-b. When the modification was performed in the presence of the cytochrome, there was no inhibition of binding between the modified reductase and cytochrome P450. We have also identified a trypsin-generated peptide of the reductase which contains the majority of the methylamine label. Amino acid sequencing and analysis suggest that one or more of the carboxyl groups of ASP-113, -118, -121, and -129, or GLU-115, -116, and -127, is involved in the interaction with cytochrome P450 PB-b. MATERIALS
AND
METHODS
Materials EDC, TPCK-trypsin, and dilauroyl phosphatidylcholine were obtained from Sigma. Methylamine HCl was from Aldrich. [‘*C]Methylamine was obtained from Amersham. All solvents were HPLC grade or better.
Methods Protein purification. Cytochrome P450 reductase was purified using previously published procedures of Dignam and Strobe1 (25) and Yasukochi and Masters (26), except 2’5’-ADP-agarose was used as the affinity resin. The reductase was washed extensively while bound to the affinity resin with 50 mM KPi, 20% glycerol, and 0.1% cholate, pH 7.5, to remove the nonionic detergent Renex 690. Cytochrome P450 PB-b (P450 IIBl) was purified using the procedure of Ryan et al. (27) with minor modifications. Renex 690 was used as the nonionic detergent. Hydroxylapatite column chromatography was performed after the DE52 column. Renex was removed as described above while the cytochrome was bound to the hydroxylapatite column. The specific activity of cytochrome P450 reductase was typically 35-50 amol cyt c reduced/min/ mg protein, assayed using the procedure of Dignam and Strobe1 (25). The specific content of cytochrome P450 PB-b was 12 nmol/mgprotein. EDC modification of cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. A total of 10 nmol each of cytochrome P450 reductase and cytochrome P450 PB-b was inserted into vesicles containing 200 ag DLPC, in a final volume of 1 ml, using the cholate dialysis technique (18). The sample was then treated in 8 mM NaPi, pH 6.8, containing 1 mM benzphetamine, with 5 mM EDC and 200 mM methylamine at 22°C for 1 h. Benzphetamine was added to enhance the interaction between the two proteins. The reaction was quenched with 0.1 M ammonium acetate. The sample was then diluted 25-fold with 0.1 M Tris-Cl and 0.1% Renex 690, pH 8.4, and loaded on a DEAE A-25 column at 4”C, equilibrated with the same buffer. The resin was washed with approximately 20 bed volumes of 0.1 M Tris-Cl and 0.5% sodium cholate, pH 8.4, to wash off both the cytochrome and Renex. The reductase was eluted from the column with 0.5 M NaCl in the cholate buffer. PMSF was added to 0.2 mM to prevent proteolysis. The sample was then dialyzed for 12 h against 5 mM NaPi, pH 7.5. Prior to dialysis, 200 ag/ml DLPC was added to the protein to form phospholipid vesicles. The reductase was then modified with [‘*C]methylamine as follows. A total of 50 pM reductase was reacted with 5 mM EDC and 200 mM [Wlmethylamine (specific activity, 0.43 mCi/nmol) in 8 mM NaPi, pH 6.8, for 1 h at 22°C. The reaction was quenched with 0.2 M ammonium acetate. Unlabeled methylamine was added to 1 M in the dialysis tubing, before dialysis, to displace any noncovalent [Wlmethylamine which was bound to the protein. The sample was dialyzed extensively against 0.1 M ammonium bicarbonate, pH 8.0, for 36 h until there were no counts above background remaining in the dialysis buffer. Typically, 60% of the protein was recovered after this modification procedure. The rate of modification of carboxyl groups appeared to be mainly dependent on the concentration of EDC and methylamine, but not on the concentration of carboxyl groups (data not shown).
PEPTIDE
DERIVED
FROM
NADPH-CYTOCHROME
Ttypsin digestion. Typically, 1 mg of the EDC-modified reductase was digested with a 1:25 (w/w) amount of TPCK-trypsin for 48 h at 37’C. The reaction was run in 0.1 M ammonium bicarbonate, pH 8.0, containing 8 M urea. The reaction was quenched by boiling the sample for 5 min and then either loading the sample directly on the HPLC or freezing at -20°C. Purification of tryptic peptides. Approximately 10 nmol of tryptic peptides was separated on a HPLC system using a Vydac C-18 reverse phase column (150 X 4.6 mm, 5 p). A Waters 6000A and an M-45 pump were used with a Waters 660 gradient programmer to form the wateracetonitrile gradient. Chromatography was performed at room temperature at a flow rate of 1 ml/min. A gradient from O-37.5% B in the first 60 min, 37.5-75% B in 60-90 min, and 75-100% B in 90-105 min was run. Buffer A consisted of 0.1% trifluoroacetic acid (TFA) in water. Buffer B was composed of 0.1% TFA, 20% water, and 79.9% acetonitrile. Peptides were detected at 214 nm using a Waters 440 variable wavelength detector. Fractions corresponding to peaks of absorbance were collected. An aliquot of each fraction was counted for radioactivity on a Packard 3255 scintillation counter. The major radioactive peak was then dried down under nitrogen to remove the acetonitrile and repurified on an Aquapore C-8 reverse phase column. The same buffer system was used, except a 0 to 60% buffer B gradient over 60 min was used to elute the peptides. The flow rate was 1 ml/min and 214-nm peaks were again collected and aliquots checked for radioactivity. Amino acid analysis was perAmino acid analysis and sequencing. formed on a Beckman 121 M amino acid analyzer. The sample was hydrolyzed in V~CUO in 6 N HCl at 110°C for 18 h. Norleucine was included as an internal standard. Tryptophan was not determined. The peptide was sequenced on an Applied Biosystems Model 470 A gas phase sequencer which was connected directly to a Model 120A HPLC system. Approximately 200 pmol was applied to a PVDF filter and sequenced. Benzphetamine demethylation assays were performed Kinetic assays. using the fluorometric procedure of Waxman and Walsh (28). The assays were run with a constant reductase concentration of 0.04 PM. The cytochrome P450 PB-b concentration was varied from 0.0125 to 0.25 FM. NADPH and benzphetamine were present at 600 and 1 mM, respectively. The assay mixture was incubated for 8 min during which the time course of product formation is linear with time. Fluorescence was detected using a Perkin-Elmer LS-5 spectrofluorometer. Details of the assay are described in Nadler and Strobe1 (18).
P450
0
279
REDUCTASE
12
3
4
5
6
7
8
4hr
8hr
9
Time (Hours) Ffoc SminlSmin
30min lhr
2hr
3hr
MW (x10-3)
116 97.4 66.2
-
42.7
-
FIG. 1. Time course of the incorporation of [i4C]methylamine into the reductase molecule. (top) 50 pM of reductase was treated with 200 mM [i4C]methylamine and 5 mM EDC in 8 mM NaP,, pH 6.8, at 22OC, for the indicated times. The reaction was quenched with 0.2 M ammonium acetate, pH 7.5. The sample was then dialyzed extensively for 48 h against 10 mM KPi, 0.5 M NaCl, and 0.5% cholate, pH 7.5. (bottom) To check for intermolecular crosslinking, a 5-pg aliquot of reductase at each time point was removed, quenched with ammonium acetate, and run on a 7.5% SDS polyacrylamide gel.
RESULTS
Time Course of Incorporation of [14C]Methylamine The amount of incorporation of radioactive methylamine, using the carbodiimide EDC, into the unprotected reductase was determined. As seen in Fig. 1 (top), the incorporation of methylamine appears to approach saturation after approximately 8 h, with the incorporation of 6 mol of methylamine per mole of reductase. Therefore, under these conditions, only a small percentage of the total number of carboxyl residues is free to react with the carbodiimide. A sample at each time point was quenched and run on an SDS-polyacrylamide gel to check for intermolecular crosslinking. As seenin Fig. 1 (bottom), there does not appear to be any crosslinking until 3 h. We, therefore, performed all the incubations of the subsequent studies for 2 h or less. Presumably, the intermolecular crosslink occurs due to competition between lysine residues and methylamine for the EDC-activated carboxyl. Since there are 102 carboxyl residues on the reductase, only 6% of the carboxyl residues are modified with this procedure.
Kinetic Effects of EDC Modification Cytochrome P450 reductase was modified first in the presence of cytochrome P450 PB-b, and then the cytochrome was removed and the reductase was again modified. The first and second modifications are termed protected and unprotected reductase, respectively. The first step of the modification should alter those residues on the reductase which are not involved in binding to cytochrome P450. The proteins are present during the modification at a concentration approximately 80 times the binding constant. Assuming a Kd of 0.1 PM, although this may be lower due to the low salt concentration during the modification reaction, approximately 89% of the protein molecules are present as the reductase-cytochrome complex. The majority of the residues involved in the binding of the two proteins should, therefore, be in close proximity and inaccessible to the modifying reagents. The second step of the modification would then alter the amino acids which are most likely to be involved in the interaction
280
NADLER
AND
with cytochrome P450. The possibility also exists that the first modification induces a conformational change in the reductase which makes residues more reactive although they are not involved in binding to the cytochrome P450. In Fig. 2, the K,,, and V,,,,, between modified cytochrome P450 reductase and cytochrome P450 were determined, as described previously. Saturating concentrations of NADPH and benzphetamine were used at a constant reductase concentration, while varying cytochrome P450 concentration. Using this procedure, a measure of the interaction between the two proteins could be determined. The control sample was treated similarly to the modified sample, except EDC was omitted. The kinetic constants of the control were virtually identical to those of the native protein. The kinetic constants from the data in Fig. 2 (top) were determined using a double reciprocal plot (Fig. 2, bottom) and are shown in Table I. The protected reductase, interestingly, has a slightly decreased K,,, compared to the control. The V,,, of the protected reductase is also decreased. This may be due to modification in or near the NADPH and FAD binding site, since these regions should be exposed to the aqueous environment and are probably not protected by the cytochrome P450. NADPH has been previously shown to donate its electrons
STROBEL TABLE
K?l Sample Control EDC-modified, EDC-modified,
PB-b]
(PM)
40
0
l/[P:50
Pi!b]
,,IM)
FIG. 2. Kinetic effect of EDC modification of cytochrome P450 reductase in the presence and absence of cytochrome P450 PB-b. (top) Benzphetamine demethylation was determined as described under Materials and Methods, at a constant reductase concentration of 0.04 pM and varying cytochrome P450 PB-b concentration. (0) Control, sham treated, (0) reductase, protected with P450 PB-b; (A.) reductase, unprotected. (bottom) Double reciprocal plot of the data presented above. (0) Control, sham treated; (0) reductase, protected; (A) reductase, unprotected.
protected unprotected
(PM) 0.097 0.056 0.231
V max (nmol/min) 2.86 1.39 1.31
L/Km (min-‘) 737 621 141
Note. The reductase was modified as described under Materials and Methods. Data were from Fig. 2. The kinetic constants were determined from a plot of l/[P450 PB-b]r,, vs 1 V and are the mean of at least two determinations.
to FAD (10). The unprotected reductase yielded a fourfold increase in K,,, compared to the protected reductase and a twofold increase compared to the control. However, there was no change in the V,,, of the unprotected reductase compared to the protected sample. The k,,JK,, catalytic efficiency, was decreased 20% for the protected sample and 80% for the unprotected sample. The significant decrease in k,,,/K, of the unprotected reductase compared to the protected sample appears to be mainly due to a K,,, effect. We are, therefore, confident that the second modification of the differential modification procedure is specific for labeling residues on the reductase involved in the binding to cytochrome P450. It is also important to note that the significant inhibition of the K,, as well as the catalytic efficiency, is a result of modification of only a limited number of the 102 carboxyl residues. Differential
[P450
I
Kinetic Effect on the Ability of Cytochrome P450 Reductase to Interact with Cytochrome P450 PB-b
Modification
In order to identify the residues on the reductase which were modified with EDC, and are most likely involved in binding to cytochrome P450, we chose to use the differential modification procedure described above. The reductase was first modified in the presence of cytochrome P450 PB-b and the substrate benzphetamine, with EDC plus unlabeled methylamine. The reductase was then separated from the cytochrome P450. As shown in Fig. 3, the DEAE A-25 column effectively removed the modified reductase from the cytochrome. The reductase was then modified with EDC plus [14C]methylamine. Those residues which are labeled with 14Care most likely involved in binding to cytochrome P450. In a separate experiment where the reductase was modified in the presence of cytochrome P450 with EDC and [14C]methylamine, there was 0.68 mol of methylamine incorporated per mole of reductase after 1 h. This is less than the approximately 1.5 mol of methylamine incorporated per mole of reductase in the absence of cytochrome P450 seen in Fig. 1. This suggeststhat cytochrome P450 is protecting certain residues from modification. When the modification was
PEPTIDE A -
DERIVED
0 --x
---
_
,“_
--
NADPH-CYTOCHROME
c .~
REDUCTASE
--‘
FROM
, d
.:
FIG. 3. SDS polyacrylamide gel of EDC-modified P450 PB-b and reductase. A 7.5% SDS polyacrylamide gel of reductase which was modified in the presence and absence of cytochrome P450 and separated on a DEAE A-25 column. (A) 5 pg each of P450 reductase and cytochrome P450 PB-b; (B) 5 pg of reductase after the first EDC modification and separation of the A-25 column; (C) 5 pg of reductase after the second EDC modification.
performed as described above, however, using unlabeled methylamine in the first protection step, and [14C]methylamine in the second unprotected step, approximately 3.2 mol of methylamine per mole of reductase was incorporated after 1 h. This is a greater number of carboxyls modified than would be expected if the reductase was modified alone, as seen in Fig. 1. It would appear that the carboxyls on the reductase which are not protected by cytochrome P450 are slower to react with EDC and methylamine than those which are protected by the cytochrome P450. Although there are a total of 3.9 mol of methylamine incorporated after the differential modification procedure, compared to approximately 3 mol incorporated when the reductase was modified alone, this difference is not very significant when the experimental error of approximately 10% is taken into account. The small amount of additional modification, however, may indicate that there was a cytochrome-P450-induced conformational change of the reductase leading to modification of these exposed groups. In a previous publication we have addressed the possibility of conformational changes of the reductase following EDC modification and concluded that there was no significant conformational change of the reductase upon EDC modification (18). Identification of Methylamine-Labeled on the Reductase
Carboxyl Groups
The reductase which was first modified with EDC and unlabeled methylamine in the presence of cytochrome P450 and subsequently with [14C]methylamine, was sub-
P450
281
REDUCTASE
jected to trypsin digestion under denaturing conditions. The resulting peptides were separated by HPLC on a Vydac C-18 reverse phase column, using the conditions described under Materials and Methods. A typical HPLC chromatogram is shown in Fig. 4. An aliquot of each peak was counted for radioactivity. Approximately 95% of the radioactivity which was loaded on the column was recovered in the fractions. Due to the inherent difficulty in obtaining protein which had a large amount of radioactivity (since a high concentration of methylamine was required causing a low specific radioactivity) we repeated the modification and C-18 chromatography three times to be sure that the labeling of the peak fraction was reproducible. The peak identified with an arrow in Fig. 4 (top), termed Peak I, contained approximately 40% of the total radioactivity which eluted from the column. The percentage of radioactivity was determined by dividing the radioactivity in Peak I by the sum of the radioactivity in the approximately 150 fractions (volume corrected).
20 1000
$Yj 800 cl &
600
gJ 400 5 u
200 0
I 20
4.0
Lo
Time
(minutes)
Time
60 (minutes)
40
io
lb0
'
160
FIG. 4. HPLC chromatogram of [“Clmethylamine-modified tryptic peptides. (t.op) Tryptic peptides of EDC-modified reductase were separated on a high-pressure Vydac C-18 reverse phase column. Approximately 5 nmol was loaded on the column. The peptides were eluted using a gradient of O-37.5% B in the first 60 min, 37.5-75% B in 60-90 min, and 75-100% B in 90-105 min, with a flow rate of 1.0 ml/min. The buffers used are described under Materials and Methods. The absorbance at 214 nm was detected with a full scale of 2.0 A. (bottom) Radioactivity associated with HPLC-purified peptides. A 25% aliquot of each fraction was removed and counted for radioactivity.
282
NADLER
AND
3 f t w zi 8 5 e 8 9 20
40
60
60
100
120
Time (minutes) FIG. 5. Repurification of Peak I. Peak purified on an Aquapore C-8 reverse phase contains >90% of the radioactivity loaded conditions are as described under Materials bance at 214 nm was detected with a full
I identified in Fig. 4 was recolumn. The peak identified on the column. The gradient and Methods. The absorscale of 0.125 A.
Although the chromatograms were slightly different in the three experiments, Peak I always migrated at a similar position. In the lower portion of Fig. 4, it can be seen that the radioactivity associated with Peak I is the only major peak of radioactivity present. The remaining 60% of the radioactivity is most likely distributed in limited amounts into a number of residues, since the other approximately 150 fractions contained lessthan 1% of the radioactivity.‘j The peak at approximately 37 min appears to be FAD/ FMN. Peak I, which contained the major portion of radioactivity, was repurified on a C-8 reverse phase column. The labeled peak is indicated by an arrow in Fig. 5. This peak on the C-8 column contained >90% of the radioactivity. The unusually late migration of this peptide on both the C-18 and the C-8 column may be due to the modification of charged residues, rendering them more nonpolar which would cause more retention on the reverse phase column. The Peak I peptide was then submitted to amino acid analysis. Amino Acid Sequencing and Analysis of Peak I In order to identify the EDC-modified amino acids, Peak I was subjected to amino acid sequencing and analysis. Approximately 200 pmol of Peak I was loaded on a PVDF filter and sequenced by gas phase automated Edman degradation. We sequenced eleven residues and obtained the sequence:
STROBEL
Due to the high background (often present in gas phase sequencing) and small amount of amino acid releasedfrom the Edman degradation, we could not accurately determine the first two residues. However, the amino acid sequence which was determined corresponds to residues 109-119 of rat liver NADPH-cytochrome P450 reductase, except for the residue at position 8 of the peptide. In the sequence determined by Porter and Kasper (29), this residue was a glutamic acid, whereas we have detected an aspartic acid. This may be due to an incorrect sequence determined by Porter and Kasper, or it may represent a site of modification of glutamic acid which caused a shift in the mobility of the Pth-glutamic acid derivative on the HPLC. The repetitive yield of sequencing was approximately 94%. The sequence which we determined is presumably a portion of the tryptic peptide corresponding to residues 109-130 of the reductase. Within these 21 amino acids are 7 carboxyl containing amino acids. These are Asp-113, Glu-115, and -116, Asp-118 and -121, Glu127, and Asp-129. Due to the small amount of peptide and the very low radioactivity, we were unable to determine which specific amino acid(s) was modified. Since the peptide contained approximately 40% of the radioactivity, this would correspond to approximately 1.3 mol of carboxyl groups modified (based on a total of 3.2 mol modified in the labeling step). This modification may be on discrete amino acids or possibly over all seven residues. We also performed amino acid analysis on the peptide. The analysis shown in Table II best matches the predicted composition of residues 109-130 in comparison to other tryptic peptides. The composition also matches residues
TABLE Amino
Amino
119.
6 On some chromatographic runs there was a peak which contained 10% of the radioactivity; however, the presence of this peak and its retention time were not reproducible and may have represented incomplete trypsin digestion, or incomplete chemical modification.
II of Peak
GUY
0.9 (1)
Ala Val Ile Leu
1.6 (2)
Arg LYS
I
Moles of amino acid per mole of peptide 2.5 (4)
Phe His
Glu-Asp-Tyr-Asp-Leu
Composition
Asx Thr Ser Glx Pro
Tyr
109 X-X-Ser-Ala-Asp-Pro-
acid
Acid
1.6 (0) 3.7 (3) 3.8 (3) 0.9 (2)
0.9 (0) 0.9 (1) 1.8 (3) 0.7 (1)
0.0 (0) 0.8 (0) 0.0 (0) 1.2 (1)
Note. Amino acid analysis was performed as described under Methods. The data are the average of three analyses. Tryptophan was not determined. Glycine was corrected for background contamination. The value in parentheses represents the number of residues determined from residues 109-130 of cytochrome P450 reductase.
PEPTIDE
DERIVED
FROM
NADPH-CYTOCHROME
P450
REDUCTASE
283
the possibility that other amino acids on the reductase, besides those which we were able to modify, are also involved in the interaction with cytochrome P450. The role of other amino acids is suggestedby the fact that we only obtained a 4-fold increase in Km, whereas we had previCONCLUSION ously observed (35) a 30-fold increase in K,,, when the salt Protein-protein interactions play a central role in the concentration was raised 3-fold. On the other hand, the regulation of metabolic processes. Previous studies have salt effects were studied by altering the ionic strength in shown the importance of charged amino acids in the in- the reconstituted assay mixture, thus affecting both reductase and cytochrome P450. teraction between cytochrome P450 reductase and cytochrome P450 (18). Makower et al. (19) have shown that The study presented in this paper will aid in our unmodification of lysine residues on cytochrome P45OLM, derstanding of the cytochrome P450 system in general. leads to a decreased rate of reduction by cytochrome P450 Further studies are in progress to use site-directed mutagenesis to identify the specific amino acids and deterreductase. Tamburini and Schenkman (30) and Bernhardt et al. (31) have also shown that modification of carboxyl mine their contribution to binding. residues on the reductase causes a decrease in the ability to reduce cytochrome P450. In a previous study, we have ACKNOWLEDGMENTS shown that carboxyl group modification of the reductase The expert technical assistance of Anne Bernhard is gratefully apdecreasesthe ability of the reductase to interact with both preciated. We also thank Christopher Chin of the University of Texas cytochrome P450 PB-b and P45Oc (18). This effect was and Katherine Stone of the Yale University Protein and Nucleic Acid seen with both benzphetamine and ethoxycoumarin as Chemistry Facility for performing the amino acid analysis and sequencing. substrates. In this paper we have attempted to identify the specific carboxyl residues on the reductase involved in the interREFERENCES action with cytochrome P450 PB-b. The water-soluble 1. Dignam, J. D., and Strobel, H. W. (1975) Biochem. Biophys. Res. carbodiimide EDC was used with the labeled nucleophile Commun. 63,845-852. methylamine to modify carboxyl residues. A differential 2. Iyanagi, T., and Mason, H. S. (1973) Biochemistry 12, 2297-2308. modification technique which has been used to study the 3. Strobel, H. W., Lu, A. Y. H., Heidema, J., and Coon, M. J. (1970) interaction of cytochrome c and cytochrome c peroxidase J. Biol. Chem. 245, 4851-4854. (23), as well as other proteins, was used in our study. 4. Enoch, H. G., and Strittmatter, P. (1979) J. Biol. Chem. 254,89768981. Table I clearly shows that the residues modified in the 5. Yoshida, T., Noguchi, M., and Kikuchi, G. (1980) J. Biol. Chem. second versus the first step of the procedure specifically 255, 4408-4420. lead to an increased Km, as opposed to a V,,,,, change. 6. Gum, J. R., and Strobel, H. W. (1981) J. Biol. Chem. 256, 7478Presumably, only some of these modified residues are ac7486. tually involved in the increased Km. I. Black, S. D., and Coon, M. J. (1982) J. Biol. Chem. 257, 5929The evidence presented suggeststhat the carboxyl res5938. idues on cytochrome P450 reductase which are important 8. Black, S. D., French, J. S., Williams, C. H., Jr., and Coon, M. J. for binding to cytochrome P450 are within amino acid (1979) Biochem. Biophys. Res. Commun. 91, 1528-1535. residues 109-130. We are unable to pinpoint which of the 9. Vermilion, J. L., Ballou, D. P., Massey, V., and Coon, M. J. (1981) seven negatively charged amino acids in this region conJ. Biol. Chem. 256, 266-277. tribute to the interaction. The carboxyl containing amino 10. Kurzban, G. P., and Strobel, H. W. (1986) J. Biol. Chem. 261, acids in this region are aspartic acid -113, -118, -121, and 782447830. -129, and glutamic acid -115, -116, and -127. Interestingly, 11. Iyanagi, T., Makino, N., and Mason, H. S. (1974) Biochemistry 13, 1801-1810. these amino acids are within the predicted flavin mononucleotide (FMN) binding domain (32). This is consistent 12. Yasukochi, Y., Peterson, J. A., and Masters, B. S. S. (1979) J. Biol. Chem. 254, 7097-7104. with previous studies which show that electron efflux from 13. Poulos, T. (1988) Pharmacol. Res. 5, 67-75. the reductase is from the FMN prosthetic group. In ad14. Nelson, D. R., and Strobel, H. W. (1988) J. Biol. Chem. 260,6038dition, two acidic amino acids, aspartic acid -113 and 6050. -118 are conserved in all cytochrome P450 reductase pro15. Vergeres, G., Kasper, W. H., and Richter, C. (1989) Biochemistry teins for which sequencesare known (33,34). Presumably, 28, 3650-3655. the amino acids which we have identified serve to anchor 16. Miwa, G. T., and Lu, A. Y. H. (1984) Arch. Biochem. Biophys. 234, the proposed FMN domain on the reductase to surface161-166. accessible positively charged amino acids on cytochrome 17. Wagner, S. L., Dean, W. L., and Gray, R. D. (1984) J. Biol. Chem. P450, thereby orienting the electron donating FMN region 259, 2380-2395. with a putative acceptor site in the cytochrome P450 mol- 18. Nadler, S. G., and Strobel, H. W. (1988) Arch. Biochem. Biophys. ecule for reduction of the heme iron. We cannot rule out 261.418-429.
109-130 much better than residues 109-167, suggesting that the peptide ends at residue 130 and not at the second tryptic site.
284
NADLER
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