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  • Int. J. Peptide Protein Rex 16, 1980, 83-96



    Center for Biochemistry, Justus Liebig-University of Giessen, Giessen, W . Germany

    Received 6 November, accepted for publication 20 November 1979

    Modification o f the protease solubilized NADPH-cytochrome P450 reductase (= NADPH-cytochrome c reductase) a t the critical S H group in the cosubstrate binding site affects K g A D p H but not V for the cytochrome c reduction. The increase of KEADpH is dependent on the size and the charge o f the substituent introduced. Substitution o f the cosubstrate site SH by the CN-, S2 O3 - and the (N-ethyl) succinimido group effects a 3-, 7- and 23-fold increase of K:ADpH, respectively. The critical SH group in the NADPH binding region can he specifi- cally radiolabeled by N-ethyl (2,3-'4CJ maleimide after preincubation o f the reductase with unlabeled NEM in the presence of 1 mM NADP+. The selective reaction at the essential cysteine in the cosubstrate site is demonstrated by pep- tide mapping of the thermolytic digest and urea SDS gel electrophoresis o f the cyanogen bromide fragments o f the reductase. Protease solubilized NADPH- cytochrome P450 reductase is inactivated by reagents directed to histidine, rrginine and lysine residues. NADP (H) ( 1 mMJ and 2I-AMP (1 mM) give effective protection only for the reaction of 1,2-~yclohexanedione (12 m M ) . The func- tional role of the basic amino acid residues for the cosuhstrate binding by the NADPH-cytochrome P450 reductase cannot be established therefore by the modification experiments described.

    The number of NADPH binding sites in the NADPH-cytochrome P450 reductase is determined to one sitelmol reductase by titration of the enzyme with NADP+ monitored by CD-spectroscopy.

    Key words; basic amino acid residues; CD-spectroscopy; cosubstrate binding site; modifi- cation of a critical SH-group.

    Abbreviations: Buffer A, 0.05 M potassium phos- phate/l mM Titriplex 111 pH = 7.5;Nbs2, 5,5'-dithiobis (2-nitrobenzoic acid); Nbs-, 2-thio-nitrobenzoate anion; Nbs-. l-carboxy-2-nitrophenyl-5-thio-; NEM, N-ethyl- maleimide; l 4 C-NEM, N-ethy1(2,3-l4 C)maleimide; p - CMB, p-chloromercuribenzoate; SDS, sodium dodecyl $ulfate; BrCN, cyanogen bromide.

    This paper forms part of the doctoral thesis of Mr. Tibor Lazar (Fachbereich Chemie Justus-Liebig- Universitat Gieoen 1979).

    NADPH-cytochrome c reductase is the flavoco- enzyme-containing fragment of the amphipathic membrane protein NADPH-cytochrome P-450 reductase (EC obtained by proteolytic digestion of liver microsomes. The enzymatic activity of the NADPH-cytochrome P 4 5 0 reductase is conserved in the hydrophilic domain NADPH-cytochrome c reductase with the exception that this fragment lacking the hydrophobic membrane anchoring peptide is

    0367-8377/80/060083-14 $02.00/0 0 1980 Munksgaard, Copenhagen 83

  • L LL \IPr R I T .4L

    unable to reduce cytochrome P450. As com- parative studies demonstrated, the kinetic parameters for the binding of the cosubstrate NADPH are identical in both forms of the enzyme.

    The hydrophilic domain NADPH-cytochrome c reductase contains. like the native membrane protein, six thiol groups/mol protein (Lazar et al., 1977; Knapp et al., 1977). Modification of the NADPH-cytochrome c reductase by 5 3 ' - dithiobis(2-nitrobenzoate) (= NBs2) (1 mM; + 4") causes a complete loss of the enzymatic activip by a slow reaction with three accessible SH-groups under the formation of the (S( 5-thio -2-nit r ob enzo at e))3 -red uct ase (( Nb s ) ~ - reductase)(Lazar er al., 1977). In the presence of cosubstrate or its analogues (NADP+, 2'-AMP) one cysteinyl residue is protected against the attack of 5,5'dithiobis(2-nitrobenzoate) (Nbs,). NADPH-cytochrome c reductase substituted at the two other accessible cysteinyl residues with the Nbs-group ( ( N ~ S ) ~ -reductase) shows the specific activity of the unmodified enzyme and is completely inactivated by renewed treatment with Nbs, under the formation of the (Nbs),- reductase. Lazar er al. (1977) obtained kinetic evidence for a concurrent reaction between the irreversible inhibitor Nbsz and the cosubstrate analogues (NADP+, 2'-AMP) for the Same site of the enzyme, suggesting that there is an 'essential' cysteine residue at or near the NADPH-binding site of the NADPH-cytochrome c reductase.

    In this paper we present direct evidence that the modification at this 'essential' cysteinyl residue affects the attachment of the cosubstrate NADP (H) by steric hindrance and electrostatic interactions of the groups introduced with the NADPH-binding site.

    Blockage of the essential cysteinyl residue with the Nbs-group proved to be an unsuitable method for the specific introduction of a label stable under the conditions of structural studies. The observation that this special SH-group shows an increased reactivity in the (Nbs),- reductase opens a new route for the specific labeling of the NADPH-cytochrome c reductase.

    A more detailed characterization of the NADPH-binding site in the NADPH-cytochrome c reductase is intended by studying the effects of histidine and tyrosine specific reagents and

    the modification of basic amino acid residues on the NADPH-cytochrome c reductase activity.


    Enzymes, substrates, coenzymes and inhibitors were obtained from commercial sources at the highest purity quality available and were used without further purification. Sephadex G-50 and G-150, 2',5'-ADP-Sepharo= 4B and Octyl- Sepharose CL4B were products of Deutsche Pharmacia GmbH (Freiburg, F.R.G.). Potassium (14 C)cyanide (61 mCi/mmol), standardized (14C) labeled toluene (1.118 x lo6 d.p.m./g toluene) and N-ethyl(2 ,3-'4C)maleimide (8.4 mCi/mmol) were delivered from Amersham Buchler GmbH (Braunschweig, F.R.G . The premixed scintillation cocktail Riasolve' was a product of W. Zinsser Scintillators (Frankfurt, F.R.G.). Thin layer sheets Polygram CEL 300 and Polygram SIL G (Macherey and Nagel, F.R.G.) were used for peptide mapping.


    Analytical methods Assay of NADPH-cytochrome c reductase ac- tivity, spectrophotometric determination of the NADPH-cytochrome c reductase, estimation of the number of sulfhydryl groups and the flavin content in the enzyme was performed as reported earlier (Lazar er al., 1977).

    Polyacrylatnide gel electrophoresis Purity of the enzyme preparations was moni- tored by disc eiectrophoresis in a medium pore polyacrylamids gel (total monomer concen- tration: 7.5% (w/v); degree of cross linkage: 2.6%; pH of separation: 9.5). Sodium dodecyl sulfate disc electrophoresis in gels polymerized from 10% (w/v) acrylamide were performed as described by Weber & Osborne (1969).

    Amino acid analysis Samples for amino acid analysis were hydrolyzed 22 or 77 h at 105" with 6.7 N HCl in an evacu- ated sealed tube and analyzed with a Beckman Model 120 automatic amino acid analyzer equipped with high sensitivity cuvets and recorders.

    Established techniques were used for the determination of the tryptophan content by



    N-bromosuccinimide (Spande & Witkop, 1967) and the N-terminal amino acid of the reductase (Gray, 1967).

    Peptide mapping Digestion of the reductase (250-500 pg) with thermolysin was carried out at 50" for 8 h in 0.5% NH4HC03/1 mM CaCl, using an enzyme to substrate ratio = 1 : l O O (wlw). Peptide mapping of the digests was done as described by Chen (1976) using Polygram SIL G thin layer plates. The spots were visualized by fluorescamine (Brosius, 1978). Radioautogra- phies of the peptide maps with Polaroid film Type 766 were performed using a 0-camera LB 290 A (Laboratorium Prof. Berthold, Wildbad, F.R.G.).

    Purification of NADPH-cytochrorne c reductase The isolation of the enzyme has been described previously (Lazar etal., 1977). Final purification of the reductase was achieved by adsorption on a 2',5'-ADP-Sepharose 4B column (1.4 x 20 cm) equilibrated with 50 mM potassium phosphate/ 1 mM Titriplex 111, pH = 7.5 (= buffer A) and eluted b y a linear gradient, 0-2 mM 2'-AMP in buffer A.

    Preparation of ( N ~ S ) ~ -reductase Reductase ( 1 4 ~ ~ ) was incubated with 1 mM Nbs, in buffer A for 2 4 h at +4". After this time the modified enzyme was separated from excess reagent b y gel filtration through a Sephadex G-50 column (2.5 x 30cm) using buffer A as an eluant.

    Preparation of ( S - ~ y a n o ) ~ -reductase by treatment o f (NbsJ3-reductase with K I4CN (NBs),-reductase ( 1 4 . 7 ~ ~ ) was treated with a 50nM solution of K14CN (spec. radioact.: 0.3 mCi/mmol) in 0.1 M potassium phosphate/ 1 nibf Titriplex 111, pH = 7.5. During the reac- tion aliquots were removed for the determi- nation of the enzymatic activity and the amount of 5-thio-2-nitrobenzoate anion liberated/mol protein. After a reaction time of about 60min the sample was applied t o a Sephadex G-50 column (2.5 x 30cm). Both equilibration and elution were performed with 0.1 M potassium phosphate/l mM Titriplex 111, pH = 7.5. Elution was continuously monitored by measuring the

    absorbancies at 280 and 412nm. The protein eluates were collected and concentrated by ultrafiltration. The radioactivity was determined by mixing 50 and loop1 of the sample with l O m l of Riasolve and counting in a Nuclear Chicago Mark I1 scintillation counter. Counting efficiency was determined by addition of lOpl of l4 C-labeled toluene standard t o each counting vial.

    Cleavage of (S-cyano) 3-reducrase The solution of (S-cyano), -reductase ( 1 0 ~ ~ ) in 0.1 M potassium phosphatell mbf Titriplex 111, p H = 7.5 containing SOmbi KCN was brought to pH = 9.0 by addition of 2 N NaOH. After addition of 20% (w/v) sodium dodecyl sulfate in 0.05 M borate buffer, pH = 8.5 up t o a final concentration of 1% (w/v), the enzyme solution was incubated at 37" for 20 h (Kuehl et al., 1976). In some experiments the reaction mixture contained 0.1 1 mM p-CMB (molar ratio enzyme : reagent = 1 : 12).

    Cyanogen bromide cleavage of NADPH-cytochrome c reductase S-carboxymethylation of NADPH-cytochrome c reductase was performed following the proce- dure of Kuhn e t al. (1 974). 0.03 pniol carboxy- methylated protein was dissolved in 3 ml of 70% formic acid and a 2000-fold excess of cyanogen bromide was added. The reaction was allowed to proceed at room temperature in the dark for 16 h. The reaction mixture was freeze- dried and the lyophilized material redissolved in 1 ml 0.01 bf H 3 P 0 4 / l % (w/v) SDS/8 M urea/ 1% (w/v) 0-niercaptoethanol (pH = 6.8). The CNBr-peptides were separated by urea-SDS gel electrophoresis (1 2.5% polyacrylamide; bisacrylamide : acrylamide = 1 : 10 (w/w); gel length: 7.5 cm)(Swank & Munkres. 1971). The gels were stained with Comassie brilliant blue R-250. The gels were sliced into 1-mm fractions, dissolved in 0.2ml of Soluene@ overnight at 25", and counted after addition of 2 n d Riasolve.

    Modification o f arginine residues by 2.3-butanedione, 1,2-c~~clohexanedione NADPH-Cytochrome c reductase was incubated with either 1,2-cyclohexanedione or phenyl- glyoxal in 0.1 M borate buffer pH = 8.2 k 0.2



    (Yankeelov. 1972). The final enzyme concen- tration was 1 7 ~ ~ . During the course of the reactions aliquots were removed for assays of enzyme activity.

    Ph o tooxida tion of NA DPH-cy ro chrome c reductase Test solution in a jacketed reaction vessel con- nected to a circulating water bath was illumin- ated with a 500 W lamp placed 20 cm from the sample. The emitted light was focused by a lens. The irradiated solution was stirred continuously by a small magnet. Photochemical reactions were performed at 0 f 1". The photosensitizers were added to the enzyme solution in the dark; the sample was then divided into equal parts, one was kept in the dark as a control, and the other was exposed to light.

    CD-spectroscopy CD-spectra were moritored with a Cary 61 spectropolarimeter (Varian) equipped with a Wang 2000 for signal averaging. The elhpticities between 200 and 240nm were calculated as mean residue ellipticities (MRW: 116), for other wavelength ranges as molar ellipticities (MW: 6.8 x lo4).

    The CD spectra of the enzyme were corrected for the contribution of the nucleotide at each concentration used in the titration; it was assumed that the spectrum of the nucleotide did not alter upon interaction with the enzyme.

    The content of secondary structures was estimated according to Greenfield & Fasman (1969).


    Characterization of the NADPH-cytochrome c reductase from pig liver microsomes The amino acid analysis of the unmodified NADPH-cytochrome c reductase purified from pig liver microsomes differs distinctly from the data reported by Baggott & Langdon (1970) but is in good agreement with the results published for the homologous enzyme from rat liver (Gum & Strobel, 1979)(Table 1). Spectro- photometric titration of the tryptophan residues by N-bromosuccinimide indicated that porcine NADPHcytochrome c reductase contains 7.5 f 0.5 residues of Trp/mol. This value is corrected


    TABLk 1 Amino acid composition of the pig liver NADPH-

    cyrochrome c reductase

    Mol '7c No. of residues/mol

    Lysine Histidine Arginine Aspartate Threonine Serine Glutarnate Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Pheny lalanine Tryptophana Cysteineb

    6.2 3.8 8.7 9.5 4.1 4.0

    14.3 4.6 3.4 5.2 5.9 2.8 3.8 9.3 1.2 5.5 1.9 0.9

    33 19 31 56 21 31 75 3 2 38 50 43 14 23 56 30 25

    7-8 6

    a Determined by titration of the NADPH-cytochrome c reductase with N-brornosuccinimide.

    Determined by the reaction of 1 rnM Nbs, with NADPH-cytochrorne c reductase in the presence of 2% SDS.

    for the N-bromosuccinimide consumption by thiol group oxidation. The COOH-terminal sequence cf pig liver NADPHcytochrome c reductase could be determined to {Val, Leu, Asp)-Ser COOH.

    The NH2-terininal residue is identified as glutamic acid by the dansyl chloride technique.

    Number of NADPH binding sites in the NADPH-cytochrome c reductase The identification of a single cysteinyl residue/ mol reductase essential for NADPH binding by the enzyme leads to the suggestion that only one NADPH binding site exists in the NADPH- cytochrome c reductase (Lazar et al., 1977). Further evidence for this assumption is obtained by changes of the CD spectrum of the NADPH- cytochrome c reductase induced by NADP' (Ki = 2.3 PM) (Fig. 1). Titration of the enzyme with NADP' flattens the CD band with negative ellipticity at 262nm. The transition reaches a maximum at 1 : l molar ratio of enzyme to



    1 240 260 280 300 nm

    , ,q A, '+

    B /

    FIGURE 1 CD spectrum of NADPH-cyto- chrome c reductase (1OpM) in buffer A (pH = 7.5) alone (1) and after titration with 6.45 (2), 14.5 (3) and 38.7 (4) p M NADPH. In- set, the change in ellipticity at 255nm as...


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