Highly homologous cytochromes P-450 and b5: a model to study protein-protein interactions in a reconstituted monooxygenase system

6

Biochimica et Biophysica Acta, 1122 (1992) 6-14 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05,00

BBAPRO 34241

Highly homologous cytochromes P-450 and bs: a model to study protein-protein interactions in a reconstituted monooxygenase system Paavo Honkakoski

a

Annika Linnala-Kankkunen a n d M a t t i A. L a n g a,1

b, S e r g e i

A. U s a n o v c

a Department of Pharmacology and Toxicology, Unicersity of Kuopio, Kuopio (Finland), h Department of Biochemistry and Biotechnology, Unicersity of Kuopio, Kuopio (Finland) and c hzstitate of Bioorganic Chemistry, BSSR Academy of Sciences, Minsk (USSR)

(Received 20 November 1991) (Revised manuscript received 13 February 1992)

Key words: Protein-protein interaction; Cytochrome:/'-450 isozyme Cytochrome b 5 from mouse and rat liver formed a type I spectral complex with two murine cytochrome P-450 isozymes, the P450Coh and P450PBI. Mouse b 5 stimulated the reactions catalyzed by reconstituted P450Coh and an equimolar amount of b5 to P450Coh was needed for maximal effect. In contrast, rat b5 inhibited P450Coh-mediated reactions progressively starting from 1:1 ratio of b 5 to P-450. Neither b 5 had any effect on reactions catalyzed by P45015a, an isozyme highly homologous to P450Coh, but with a point mutation (Arg-129 ~ Ser) at site considered important for P-450-b 5 interactions. In case of P450PBI, neither b 5 protein had any effect on the associated activities at b 5 : / - 4 5 0 ratios below 1, and a progressive inhibition occurred when b5 :/'-450 ratio was above 1. The results were similar with either rat or mouse liver NADPH-cytochrome P-450 reductase used in reconstitution demonstrating that the critical differences take place in P-450-b 5 interactions. Kinetic and spectral experiments revealed that the stimulator3, and inhibitory effects of b 5 on the enzymatic reactions were due to corresponding changes in the reaction velocity, and that b 5 does not compete with the flavoprotein nor with the substrate for binding to P-450. These results indicate that the high spin shift of P-450 does not necessarily correlate with enhanced reaction rates. Also, the increase in the coupling efficiency of P450PBI may result from the increased affinity for substrate in the presence of b 5. Sequenation of mouse b 5 peptides generated with proteinases revealed three amino acid changes between the mouse and rat b 5, two of which appeared at the hydrophobic domain necessary for the P-450-b5 interaction. This could explain the species specificity of b s proteins in supporting the P-450-mediated reactions. This is the first time functionally important differences in the interaction of highly homologous cytochromes/'-450 and b 5 have been demonstrated. Isozymes P45015a and P450Coh, and mouse and rat b 5 could serve as an excellent model for further studies on the nature and significance of P-450-b 5 interactions.

Introduction Microsomal cytochrome b5 is a membrane-bound protein consisting of a hydrophilic N-terminal hemecontaining moiety and a hydrophobic C-terminal membrane-anchoring segment [1,2]. b 5 is a component of the acyl CoA desaturase complex and participates also

I Present address: IARC, WHO, 150 cours Albert-Thomas, 69732 Lyon, France. P-450 nomenclature: a new nomenclature for P-450 isozymes has been proposed [44]. This report deals with mouse liver isozymes P450Coh (P4502a-5), P45015a (P4502a-4) and P450PBI (most probably P4502b-10), which are referred to in this article by their trivial names.

in reactions catalyzed by some P-450 enzymes (for review see Ref. 3). The role of b 5 in P-450-mediated reactions has been studied with antibodies, reconstituted systems, by Abbreviations: BZDM, benzphetamine N-demethylase; b5, cytochrome bs; DLPC, dilauroyl phosphatidylcholine; CNBr, cyanogen bromide; COH, coumarin 7-hydroxylase; DTT, dithiothreitol; ECDE, 7-ethoxycoumarin O-deethylase; HPLC, high-performance liquid chromatography; NADPH, nicotinamide adenine dinueleotide phosphate (reduced); PROD, 7-pentoxyresorufin O-dealkylase, PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride; P-450, cyv~chrome/'-450; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gd electrophoresis; TFA, trifluoroacetic acid; T15aOH, testosterone 15a-hydroxylase. Correspondence: P. Honkakoski, Department of Pharmacology and Toxicology, University of Kuopio, POB 1627, 70211 Kuopio, Finland.

reincorporation of b 5 into microsomes, and recently, by eDNA expression [4-10]. The effects have been either inhibitory, negligible or stimulatory depending on the po450 isozyme and the substrate used and on the method of investigation [11-13]. The role of b5, although not clearly understood, is proposed to include, e.g., promotion of substrate binding, direct electron transfer to P-450, enhanceffaent of active oxygen utilization by preventing H20 2 production, and an effectot (non-electron transfer) function on P-450 [3,9]. Cytochrome b 5 can induce type I spectral changes of P-450 [14] resulting from an increased content of highspin P-450. The degree of spectral shift seems to correlate with the enhancement of the catalytic rate, at least with some isozymes [7]. Other studies have implicated that high-spin P-450 can be reduced more quickly [15] leading to enhanced reaction rates, although other views have been expressed [16]. Chemical modification experiments [17,18] have clearly shown the importance of b 5 carboxylate groups for complexation with cytochrome P-450. However, the /-450 amino acids carrying the opposite charge have not been identified, although P450cam contains a cluster of basic amino acids (Arg-72, -112, -364 and Lys-344) possibly involved in complexation [19]. b 5 is also able to protect certain P-450s from cAMP-dependent phosphorylation at Ser in sequence Arg-Arg-X-Ser near position 130 suggesting that site for b 5 binding is at or close to this motif [20,21]. However, the hydrophobic domain of b 5 seems to be essential for the interaction as well [3]. The aim of this study was to gain further understanding on the interaction of b 5 and P-450 isozymes. For this purpose, two highly homologous, yet distinct species of b 5 from mouse and rat liver were used in reconstitution experiments with two highly homologous P-450 isozymes, P450Coh and PZt5015a. Additionally, a third P-450 isozyme from 2B family was used. With this model we were able to show that the formation of a functional complex is highly b 5 and P-450 isozyme specific, and may depend on only few amino acids in each protein. Materials and Methods

Chemicals. All chromatographic matrices were from Pharmacia (Uppsala, Sweden). Sodium eholate was from Serva (Heidelberg, Germany), sodium deoxycholate was bought from Fluka (Buchs, Switzerland) and Emulgen 911 was purchased from Kao Atlas (Tokyo, Japan). DLPC, CHAPS and 7-ethoxyeoumarin were from Sigma (St. Louis, MO, USA). Coumarin, 7-hydroxycoumadn and resorufin were bought from Aldrich (Milwaukee, Wl, USA), 7-pentoxyresorufin was from Pierce (Rockford, OR, USA). BenzphetamineHC! was a kind gift from Upjohn Company (Kalamazoo,

MI, USA). All other chemicals were of the highest grade commerially available. Protein and peptide purification. P450Coh and P450PBI were purified to electrophoretic homogeneity as described [22,23]. The proteins were dialyzed against 50 mM phosphate buffer (pH 7.4) containing 20% glycerol and 20/.tM EDTA. The physicochemical and catalytic properties of the isozymes have been described [23]. P45015a, closely related to P450Coh, was partially purified from livers of D2 mice by using octyl-Sepharose and hydroxyapatite. This isozyme is the major catalyst of testosterone 15a-hydroxylation [24,25]. Liver microsomal cytochrome b 5 was purified from both Wistar rat and D2 mouse. Microsomes (about 2 g protein) were solubilized by 0.5% Emulgen 911 and 0.5% cholate in 10 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM DTT, 0.1 mM EDTA and 20% glycerol, and centrifuged at 100000×g for 60 rain. The supernatant was applied on a DEAE-Sepharose CL-6B column (1.6 × 25 cm) equilibriated with the solubilizing buffer. The column was washed with the solubilizing buffer containing first 150 mM NaCI until no cytochrome P-450 was detected in the eluate, and then NaCI concentration was raised to 250 raM. The fractions containing both cytochrome b 5 and NADPH-cytochrome P-450 reductase were eluted with solubilizing buffer plus 400 mM NaCI. The flavoprotein was removed by passing the sample through 2',5'ADP-Sepharose, and b:containing fractions were diluted with distilled water and applied on a DEAE-Sepharose column (1.6 x 8 era). The column was washed with 20 mM Tris-HCl buffer (pH 7.8) containing 0.4% cholate and 0.1 mM EDTA to remove non-ionic detergent, and with the same buffer containing additionally 150 mM NaCI until no absorbance at 280 nm was seen. The column was then washed with buffer containing 220 mM NaCl. The b 5 was eluted by a linear NaCI gradient from 220 mM to 500 mM in the equilibriation buffer. The best fractions (absorbance ratio of 413 to 280 nm greater than 1.5) were pooled. The sample was concentrated and the buffer was changed to 20 mM Tris-HCl (pH 8.0) containing 200 mM NaCI, 0.1 mM EDTA, 0.5% cholate and 1% deoxycholate by an Amicon YM-10 membrane. T h e 2-ml sample was applied on a Sephadex G-75 Superfine column (1.6 × 90 cm) and eluted with the same buffer at a flow rate of 10 ml/h. The fractions containing b5 with absorbance ratio of 413 to 280 nm greater than 2.9 were pooled, concentrated and dialyzed against 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 20/~M EDTA (reconstitution experiments) or against 0.1 M ammonium acetate (pH 7.5)(structural analysis). For amino acid sequencing, the cytochromes bs were further purified by reversed-phase HPLC (Vydae C 4 or C ~8 column 25 x 0.46 era) using a linear gradient of I0

to 70% of acetonitrile (HPLC S grade) in 0.1% TFA. Proteins were detected at 280 nm, and verified by SDS-PAGE and Coomassie blue staining. The isolated proteins were dried in vacuo and dissolved in proteinase digestion buffers recommended by the manufacturer (Boehringer). The mixtures (1:20 w/w enzyme to protein ratio)were incubated for 16 h at 37°C. One,tenth of the mixture was applied on an analytical HPLC column and developed with a linear gradient (0-50% for C4, 0-70% for Cms columns) of acetonitrile in 0;1% TFA. For isolation of the individual peptides, we used a gradient of 0-70% acetonitrile over 1 h and detection at 218 rim. The aminoterminal peptide was isolated using C4 and C]s chromatography, and treated with TFA to cleave the blocking group [2]. Peptides were sequenced by an 477A sequencer (Applied Biosystems). Resulting PTH-amino acid derivatives were analyzed by a 120A on-line HPLC system. Mouse and rat liver NADPH-cytochrome P-450 reductases were purified using DEAE-cellulose and affinity chromatography on 2',5'-ADP-Sepharose [26]. Catalytic assays. Reconstituted coumarin 7-hydroxylase and 7-ethoxycoumarin O-deethylase (100 pM substrate) were assayed using 1:6:300 molar ratio of P450Coh (typically 4-10 pmol), P-450 reductase and DLPC. Testosterone 15a-hydroxylation (100/zM substrate) was assayed using the same ratio of components and with 50 pmol of partially purified P45015a. The separation of testosterone metabolites was done according to Waxman et al. [27]. 7-pentoxyresorufin Odealkylation (2,/zM substrate) and benzphetamine Ndemethylation (1 mM substrate) were determined with 1 : 4: 300 ratio of P450PBI (typically 15-30 pmol),/'-450 reductase and DLPC [23]. The components were allowed to equilibriate 10 min before addition of cytochrome b s (specific amounts are detailed in the legends). After a 5 min preincubation, the reaction was started by the addition of NADPH (1.5 mM final concentration) and allowed to proceed at 37°C. The reaction products 7-hydroxycoumarin [28], resorufin [23] and formaldehyde [29] were measured according to methods cited. Blanks contained all components except P-450. Kinetic parameters were determined with 0.8-100 t~M final concentrations of coumarin and 7ethoxycoumarin and 62.5-1000/zM of benzphetamine with doubling the amounts of components and incubation volumes. 7-pentoxyresorufin concentration was varied from 0.25 to 2 I~M. NADPH consumption was" measured kinetically at 340 nm using e = 6.22 mM -~ em -~ and 7-ethoxycoumarin and benzphetamine as substrates. The amount of/-450 was 30-40 pmol, and the temperature 30"C. At the end of 5-min incubation, the reactions were stopped with trichloroacetic acid, and 7-hydroxycoumarin and formaldehyde were determined.

TABLE I

The parameters of spectral interaction between P-450 i~ozymes, substraws and cytochromes b 5 Difference spectra of 1 ~ M /'-450 in 50 mM potassium phosphate buffer, (pH 7.4) containing 20% glycerol and 300 # M DLPC were recorded from 350 to 450 nm at 300C. Substrate binding was done in 10 mm cuvettes, other recordings in 5 mm tandem cuvettes. Ligands were allowed to equilibriate for 3 min before scanniJ~g. Six to seven concentrations were used to calculate the K s, Kd, AAmax values and standard errors [14,32]. In case of b s and reductase binding, ligand depletion was taken into account, n.d., not calculated. Ks (#M)

AAmax (mA)

K d (/~M)

P450Coh (1 izM) +mouse b s + r a t bs

1.28_+0.12 0.87_+0.20

32_4-2 21_+2

0.2 +_0.06 0.43_+0.10

+ coumarin. no b 5 2/~M b 5

0.66_+0.04 0.55-+0.05

66_+2 49+1

n.d. n.d.

+ flavoprotein, no b 5 1/~M mouse b 5 1 t~M rat b s

1.24_+0.29 0.69_+0.32 0.91 _+0.12

15_+2 10+ 1 6_+1

0.37_+0.04 0.58_+0.19 0.39_+0.10

1.80+0.11 1.135:0.15

46_+2 34-+3

0.99+0.05 0.365:0.03

87_+ 1 36_+3

n.d. n.d.

P450PBI (1/~M) + m o u s e bs + r a t bs + benzphetamine, no b s 2 / z M b5

10.0 +0.4 5.4 5:0.2

Spectral studies. Difference spectra were recorded with a double beam spectrophotometer (Uvidec 610; Jasco, Kyoto, Japan) at 30°C as detailed in the Table I. The P-450-DLPC mixture was incubated for 10 min and pipetted to the front compartment of two tandem cuvettes (light path 5 mm). The other compartment contained an equal volume of buffer. After a baseline of equal absorbance was established, concentrated cytochrome b s was added in microliter quantities into the front compartment of the sample beam cuvette and to back compartment of the reference beam cuvette to eliminate the intrinsic absorption of b 5. Similar titrations were performed with coumarin added to P450Coh and benzphetamine added to P450PBI, and with NADPH-cytochrome P-450 reductase in the presence and absence of b 5. Affinity chromatography. Detergent-solubilized cytochrome b5 was immobilized on CNBr-activated Sepharose 4B as instructed by the manufacturer (Pharmacia). Partially purified P450Coh and P450PBI were dialyzed against 10 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT. Emulgen 911 was added to 0.2% concentration. In case of P450Coh, the buffer contained 100 ~M coumarin to stabilize the enzyme and to maintain it in high spin state. The amount of P-450

applied was 10-20% of the amount of matrix-bound cytochrome b 5. After application, the column was washed with dialyzing buffer and cytochrome P-450 was eluted by stepwise additions of KC! or NaCl. P450Coh was detected spectrally as a high spin form of /)-450 due to complexation with coumarin [22]. P450PBI was detected by reconstituting the 7-pentoxyresorufin O-dealkylation activity of the fractions as described above. Other methods. Cytochrome P-450 concentration was measured according to Omura and Sate [30]. Cytochrome b 5 was assayed with dithionite using E = 185 mM -I c m - i (AA(423-413 nm)) [3]. SDS-PAGE was performed according to Laemmli [31]. Kinetic and spectral binding parameters were calculated by using a nonlinear regression program [32].

Purification o f rat and mouse liver b 5. The established methods to purify cytochrome b 5 had to be modified for mouse b 5 preparations in order to separate a tightly associated protein. This was possible by adding 200 mM NaCI to the gel filtration step, which then resolved a 35-kDa contaminant. Fig. 1 shows that purified cytochromes b 5 are intact and electrophoretically homogeneous with apparent molecular masses of 16.5 kDa. The absolute spectra of oxidized and reduced forms of mouse b 5, which are indistinguishable from rat b 5 [3] are shown in Fig. 2. The molar absorp-

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Fig. 1. SDS-PAGE of the purified rat and mouse cytochromes b 5. Approx. 1/zg of mouse b 5 (lane 1)or rat b 5 (lane 2) and cytochrome c (2 #g, lane 3) were clectrophoresed in 12% acrylamidv gel according to Laemmli (1970), and stained with Coomassie blue. The molecular masses are indicated.

Fig. 2. The absolute spectra of mouse liver cytochrome b 5. The spectrum of oxidized ( ) and dithionite-reduced (- - -) mou~e liver cytochrome b 5 (3.5 p,M) was recorded in 50 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA.

tivities of the Soret peaks of the mouse and rat b 5 were 128 and 122 c m - l m M - ! (oxidized), and 188 and 186 cm-~ mM-~ (reduced), respectively• A high degree of purity of the preparation is evident from the high A4~3/A28o ratio. Both bss were devoid of detectable cytochrome /-450 and NADH- and NADPH-dependent cytochromc c reductase activity, and no 7-ethoxycoumarin O-deethylation in the preparations could be detected by adding NADPH-cytochrome /)-450 reductase (not shown). This indicates that the b 5 preparations were not contaminated by other components of the monooxygenase complex. Spectroscopic studies• Fig. 3 shows the spectral changes of P450Coh upon addition of cytochrome b 5 from rat and mouse• The result indicates a low-to-high spin shift of t h e / - 4 5 0 isozyme in response to complex formation with both bss. The equilibrium constant ( K S) and the maximal spectral change (AAron) were c a l c u l a t e d according to equation A A = A A m a x " [ b s ] / ( K ~ + [bs]) by the non-linear regression method of Duggleby [32]. These values were then used to calculate the concentrations of free and bound b 5 [14], assuming that /'-450 and b 5 form a bimolecular complex according to previous spectral [14] and crosslinking [17] studies and our catalytic results described below, and that the fraction of bound b 5 is directly proportional to spectral shift [14]. Dissociation constants (K d) of the P-450-b5 complex were calculated from these results (Table I)• Mouse b s tends to produce larger A A values than rat b s, indicating possibly a better fit of mouse than rat b s to P450Coh. Based on the dissociation constants, P450Coh seems to bind b s more tightly than P450PBI. Upon addition of b 5, the

10 Despite some differences, the ability of both bss to induce type I spectra is qualitatively similar. These results, however, demonstrate that cytochrome b 5 does not compete with the substrate or the flavoprotein for binding to P450Coh, and may therefore interact at distinct sites [18,33]. On the other hand, the affinity of benzphetamine to P450PBI seems to be enhanced by b 5. This indicates that different mouse isozymes may interact with their ligands in different ways. Catalytic studies. Fig. 4 presents the effect of rat and mouse cytochrome b 5 on reactions catalyzed by P450Coh (panel A), by P45015a (panel B), and by P450PBI (panel C). Coumarin 7-hydroxylation and 7ethoxycoumarin O-deethylation are stimulated by mouse b 5, up to 75 and 210%, respectively. The maximal stimulation was reached at b 5 to P-450 ratio of 1:1 and was maintained at this level when more cytochrome b 5 was added. In contrast, a progressive inhibition occurred with rat bs; coumarin 7-hydroxylation being inhibited less than 7-ethoxycoumarin O-deethylation. Interestingly, this inhibition took place only beyond the 1:1 ratio of b s to P-450. Contrary to the P450Coh, no activation of the P450PBl-mediated reactions by either b s was seen. Instead an up to 60% inhibition was found; notably though, even in this case the inhibition could only be observed above the 1:1 ratio of b5 to P-450. Surprisingly, neither b 5 had any effect on the reconstituted testosterone 15a-hydroxylation. This is interesting because this activity is catalyzed by P45015t~, highly homologous to P450Coh. The inhibitory effects found above the 1 : 1 ratio of b s to P-450 may be explained by diversion of electrons by excess b 5, or by competition between b 5 and P-450 for the cytochrome /-450 reductase, which can reduce both hemeproteins [34]. This competition apparently does not take place with P450Coh, since increasing the b s concentration beyond 1:1 ratio does not decrease the activity (Fig. 4A).

B

IA 4 4

0 U C g

4 4

370

430

370

430

Wavelength (nm) Fig. 3. The low-to-high spin shift of P450Coh upon addition of cytochrome bs from mouse (A) and rat liver (B). Cytochrome b 5 was added in microliter quantities to I `aM P-450 in 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 300 `aM DLPC in tandem cuvettes. Six to seven different concentrations between 0.25 and 4.0 ,aM were used to calculate the equilibrium constants presented in Table I. For clarity, additions of 0.25, 0.5, !.0 and 2.0 izM cytochrome b 5 are shown (traces 1 -4, respectively). The bar stands for 0.010 absorbance units.

affinity of P450Coh for coumarin did not change appreciably. In contrast, the affinity of benzphetamine for P450PBI was increased 2-fold in the presence of b 5. The addition of 7-pentoxyresorufin to P450PBI produced too small type I changes to reliably calculate the K, values. The binding constant of NADPH-cytochrome P-450 reductase for P450Coh was not much altered with the inclusion of either rat or mouse b 5. A

C

B

200 -

200

I 100 -

"---1 O" 012

4

8 Ratio

t

0

;

~)

of b5 to P450

4

8 0 1 2 in the r e c o n s t i t u ~ i o n

4

8

Fig. 4. The effect of mouse and rat cytoehrome b 5 on reactions catalyzed by P450Coh (A), P45015ot (B) or P450PBI (C). Mouse b 5 (white symbols) or rat b 5 (black symbols) was added to reconstituted systems and the catalytic rate was determined as described in Materials and Methods. Squares denote ECDE (A) or BZDM activity (C). Circles denote COH (A), T I 5 a O H (B) or PROD (C) activities. The turnover numbers for control incubations (100%) were 80, 18, 10, 124 and 3.1 nmol/min per nmol P-450 for ECDE, COH, TI5aOH, BZDM and PROD activities, respectively.

11

TABLE I1 The effect o f mouse and rat reductases on reconstituted P450Coh in the presence o f mouse and rat b5

250

Cytochrome P450Coh was reconstituted as described in Materials and Methods. Cylochrome b 5 was added at 2:1 ratio to /)-450.

-a

200"

Coumarin 7-hydroxylation ( n m o l / m i n per nmol P-450)

No b 5 added Mouse b s Rat b 5

mouse reductase

rat reductase

15.0 (100%) 29.6 (197%) 8.2 (55%)

17.0 (100%) 32.7 (192%) 10.6 (62%)

c o dl 100"~ . . . . . .

Q

>

Based on the above results, interaction between P-450 isozymes and b 5 seem to be highly specific. However, these results could be explained also by species-specific interactions between b 5 and cytochrome P-450 reductase, since the flavoprotein can reduce also b 5 [34]. To study this possibility, we used both rat and mouse flavoprotein to reconstitute the monooxygenase complex. As can be seen from Table II, rat and mouse flavoproteins support the reconstituted coumarin 7-hydroxylase equally well in the presence or absence of rat and mouse b 5. Therefore we conclude that the species differences seen in the reconstituted P450Coh-mediated reactions are only due to distinct interactions between rat and mouse b 5 w~th the P450Coh. Decreasing the amount of flavoprotcin leads to greater stimulation and inhibition by b 5 of P450Cohand P450PBI-catalyzed reactions, respectively (Fig. 5). The changes were similar but less dramatic than those observed by B6sterling et al. [6]. The mechanism of these enhanced effects is not clear, but presumably the proportion of bs-linked electron transfer is higher when the amount of flavoprotein is limiting. Kinetics of the P-450-mediated reactions. Table III shows that mouse b 5 increases and rat b 5 decreases the Vm~~ of the reconstituted P450Coh with either coumarin or 7-ethoxycoumarin as substrates. No essential changes in the K m could be found. Thus, b 5 does not affect substrate binding (in accordance with spectral binding data in Table I), but rather participates in the electron transfer. For P450PBI-mediated reactions, b 5 seems to affect mainly the Vm~,, although also a 30-50% decrease of the K m value was seen, supporting the spectral studies. Coupling of the P-450-mediated reactions in reconstituted systems. When P450Coh is used to reconstitute the monoo~genase complex it appears that neither b 5 is able to affect the coupling efficiency (Table IV) nor the affinity of substrate for P450Coh (Table III). This, together with the fact that mouse b 5 increases the Vma~ while rat b 5 decreases it suggests that (i) the position of substrate at the P450Coh active site is not affected

re

50"

0.33

1

3

9

Ratio of Fp to P450 Fig. 5. The effect of flavoprotein (Fp)/P-450 ratio on the catalytic rate of P450Coh and P450PBI in the presence of b 5. P450Coh-mediated ECDE (white symbols) and P450PBl-mediated BZDM (black symbols) were measured while varying the flavoprotein to P-450 ratio in the absence (dashed line) and presence of b 5 (circles; bs:P-450 ratio 2:1). Reconstitution was performed as described in Materials and Methods, and results from two separate experiments are shown.

by b s and (ii) mouse but not rat b 5 is able to stimulate the electron flow from flavoprotein to P450Coh. In any case, since the coupling efficiency is less than 100%, the system leaks electrons to hydrogen peroxide-producing pathways [35]. Interestingly, the reconstituted complex containing P450PBI seems to function differently. The coupling efficiency was increased from 62 to TABLE III The kinetic parameters o f P450Coh- and P450PBl-mediated reactions in the presence o f mouse and rat bs

Reconstitution and determination of activities was performed as described in Materials and Methods. Kinetic parameters were calculated [32]. Results from two independent experiments are shown. bs: P-450 ratio was 2:1 for P450Coh and 4:1 for P450PBI. n.d., not done. P450Coh

COH

ECDE

K m(/zM)

Vm~X(min-1)

K m(~zM)

Vm~~ ( m i n - I )

no b s mouse b s rat b 5

7, 10 8, 10 10, l l

18, 15 34, 28 12, 10

!0, i l 11, 14 13, n.d.

84, 82 187, 230 48, n.d.

P450PB!

PROD

no b s mouse b s rat b s

BZDM

K m (/zM)

l'~a ~ ( m i n - t )

Km (#M)

Vmax (min-i~

0.8, 0.9 0.8, i.0 0.5, 0.6

2.8, 2.9 2.1, 1.6 1. i, 1.0

83, 90 n.d. 43, 40

205, 178 n.d. 150, 125

12 TABLE IV The relationship between product formation and NADPH consumption in the presence o f mouse and rat b5 NADPH consumption and product formation were measured at 30°C in reconstituted systems described in Materials and Methods. When present, b s was added ratio of 2: l to P-450. Data from two independent measurements are shown, n.d., not done. Product NADPH Coupling efficiency formation consumption (% of product formed (rain- t) (rain- t) per NADPH consumed) P450Coh/ECDE no b 5 46. 48 mouse b 5 55 rat b 5 42 P450PBI/BZDM no b 5 102, 100 mouse b 5 n.d. rat b s 85, 89

84, 83

100 71 170, 158 n.d. 89, 99

55.58 55 60 60, 63 n.d. 87. 90

88% by b 5 with the simultaneous decrease in the K m (Tables I, III, IV). This suggests that the interaction of b 5 with P450PBI can affect the position of the substrate at the catalytic site and (therefore?) increase the yield of product, at the expense of hydrogen peroxide formation. Taken together, these data suggest that the contribution of b 5 to the function of monooxygenase complexes is P-450-isozyme-dependent. bs-P-450 interaction analyzed by affinity chromatography. Affinity chromatography on immobilized mouse b 5 shows that both P450Coh and P450PBI were bound on the column. Some of the hemeproteins were eluted while washing, but these fractions contained only small A

amounts of P450Coh or P450PBI. Both isozymes were eluted with salt concentration higher than 100 mM, which suggests that the interaction between cytochrome b 5 and /)-450 isozymes is of electrostatic nature. However, P450Coh does not bind to trypsinsolubilized b 5, which does not contain the membranespanning peptide [36]. This suggests that the electrostatic attraction between b 5 and P-450 is not sufficient for complex formation, and also hydrophobic interactions are essential [37,38], especially for enhanced catalysis [39]. Alternatively, cleavage of the membrane anchoring segment could change the conformation of the rest of b 5 protein rendering it unable to bind to P450Coh. Structural comparison of mouse and rat b5 and nature of interaction with P-450s. Because of essential differences in their ability to support the P450Cohcatalyzed reactions, we decided to compare the structures of mouse and rat b 5 in detail. Initial studies revealed that both b5s contained a blocked amino terminus. Therefore digestion with trypsin and endo Asp-N proteinases was carried out. Resulting peptides were separated on HPLC and sequenced. Only few differences in the HPLC chromatograms (Fig. 7) were detected indicating a close similarity between the two proteins. Sequencing the peptides revealed three amino acid differences between the two proteins (Asp-7 --, Glu at the N-terminus, and Ala-92 ~ Ser and Asp-97 ~ Thr at the beginning of the hydrophobic region; Table V). In spite of several attempts with HPLC, SDS-PAGE and blotting to PVDF membranes, we were unable to recover sufficient amounts of the membrane-spanning B

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20

30

Elution volume (ml) Fig. 6. Affinity chromatography of partially purified P450Coh (A) and P450PB] (B) on detergent-solubilized cytochrome b5 coupled to CNBr-activated Sepharose 4B. Partially purified P-450s (P450Coh, 65% high spin in the presence of coumarin; P450PBI, PROD turnover number 1.4 nmol/min per nmol P-450) were dialyzed extensively with 10 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM DTT, 0.1 mM EDTA. Emulgen 911 was added to 0.2% final concentration and in the case of P450Coh, 100 p.M coumarin was included in every buffer. The samples were applied on to affinity column equilibriatcd with dialyzing buffer plus 0.2% Emulgen 911. The columns were washed stepwise with the equilibriation buffer supplemented with indicated concentrations (raM) of NaCI. The black bars denote pools collected for further analysis. The elution of P450Coh was monitored by the ratio of high- to low-spin P-450 (percentage of high-spin P-450 indicated above black bars, A), and P450PB1 was detected by reconstituting the PROD activity (turnover numbers indicated above black bars, B).

13 A

I10 ' i

C

:10'

B

:10' :

D

10'

Fig. 7. The HPLC peptide maps of mouse b 5 (A, C) and rat b 5 (B, D) from digestion with endo-Asp N proteinase (A, B) and trypsin (C, D). About 20/zg of purified proteins was digested with proteinases. 90% of the reaction mixture was applied on a Vydac C~s column and developed with a 0-70% acetonitrile linear gradient in 0.1% TFA, and the peptides were detected at 218 nm. Asterisks show the extra peaks and peak shifts of mouse b s peptides as compared with rat b 5.

peptide for sequencing. Therefore we can not exclude the possibility that differences between rat and mouse proteins, contributing to their interaction with P-450, exist in this region. However, the extremely high ho-

TABLE V The comparison o f rat and mouse cytochrome b5 amino acid sequences

The numerals indicate amino acid residues starting from N-terminus (lane 1). Residues marked with uppercase letters denote MOUSE b 5 sequences (lane 2), and residues in lowercase letters denote rat b s residues (lane 3, [2]). Residues underlined indicate observed amino acid differences to rat b s (rat residue in parenthesis on lane 3). X indicates the blocked amino-terminal residue (acetylated alanine in rat bs). The seven N-terminal residues were deduced from overlapping peptides generated by TFA digestion. Note that residues 98-127 a r e lacking for mouse b 5. 1 lO 20 30 60 XEQSD KDVKY YTLEE IEKHK DSKST WVILH HKVYD LTKFL (e) 50 60 70 80 EEHPE GEEVL REQAD GDATE NFEDV GHSTD ARELS K T Y l l 90 100 110 120 GELHP DDRSK IAKPS ED(s) (t)tit t s e v n sswwt n w v i p a i s a L 130

vvatm

-LYR AED yr

mology observed may suggest that only few residues are critical for P-450 interaction. Further proof of the specific nature of interaction between b 5 and P-450 is given by the fact that P450Coh-catalyzed reactions are stimulated by mouse b 5 (or inhibited by rat b s) while P45015a-mediated activity remains unaffected. Our previous collaborating work has shown that P450Coh and P45015a differ by only 11 out of 494 amino acids, and display highly specific preference for hydroxylation of coumarin and testosterone, respectively [25]. It is obvious therefore, that one or more of these 11 residues regulate the interaction between b s and P-450. So far the specific residue(s) have not been identified, but it is very interesting to note that P450Coh has a sequence Arg-ArgPhe-Ser (residues 128-131), identical to rat P4502B1, which is known to be phosphorylated. The phosphorylation of this sequence inhibits the association of b 5 to P-450 and also diminishes the catalytic activity [40] suggesting that this site is identical or close to bs binding site [20]. In P45015a the second arginine is replaced by a serine, which makes it tempting to speculate that the sequence 128-131 is important in bs-P-450 interaction and that through this mutation (Arg-129 --, Ser) P45015a has lost its ability to associate with b 5 (or at least to receive electrons). The extension of the three-dimensional P450cam-b 5 model [19] to microsomai P-450s may be too early, because that model employed truncated b 5 lacking the membrane-spanning peptide essential for catalytic effects [37-39], and because studies from Negishi's group have cast some doubt on the correspondence of the P450cam and mammalian P-450 structures. Also the discrepancies in the spectral states between the purified [42] and microsomal [43] mouse P-450 mutants expressed in yeast remain unexplained, and may technically complicate these studies. Acknowledgments

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