the functional role of thiol groups in protease-solubilized nadph—cytochrome c reductase from...

Eur. J. Biochem. 76, 365-371 (1977)

The Functional Role of Thiol Groups in Protease-Solubilized NADPH - Cytochrome c Reductase from Pork-Liver Microsomes Tibor LAZAR, Helga EHRIG, and Ludwig LUMPER

Zentrum fur Biochemie der Justus-Liebig-Universitat GieIjen

(Received October 21, 1976/April 1, 1977)

The total - SH content of protease-solubilized NADPH -cytochrome c reductase (EC 1.6.2.4) from pork liver microsomes was determined to be 5.9 k 0.3 mol thiol groups per mol of protein. Only three - SH groups of the protease-solubilized NADPH - cytochrome c reductase could be modified by 0.34 or 1.0 mM 5,5'-dithio-bis(2-nitrobenzoate) at pH 7.5 and + 4 "C. More than 95% of the original enzymatic activity was lost during this treatment. In the presence of 1 mM NADP+ only two -SH groups of the NADPH-cytochrome c reductase reacted with 5,5'-dithio-bis(2-nitrobenzoate). After removal of the competitive inhibitor NADP' , an enzyme derivative with the specific activity of the unmodified enzyme was obtained, containing two cysteinyl residues as mixed disulfides with 2-nitro-5-thiobenzoate. Renewed treatment of the modified reductase with 0.34 or 1 .O mM 5,5'-dithio-bis(2-nitrobenzoate) resulted in the modification of one additional thiol group under complete inactivation ( k for modification = 0.027 min-', k for inactivation = 0.039 min-' at 0.34 mM Nbsz, pH 7.5, + 4 "C). Kinetic analysis confirmed the suggestion that a single thiol group of the NADPH-cytochrome c reductase was protected by NADP+ against the reaction with 5,5'-dithio-bis(2-nitrobenzoate). Cytochrome c (58 pM) apparently enhanced the effect of NADP'. The modification of the accessible thiol groups by 1 mM 5,5'-dithio-bis(2-nitrobenzoate) did not affect the half-reduced state of the NADPH - cytochrome c reductase.

NADPH - cytochrome c reductase is part of the microsomal electron transport system. It could be solubilized as a protease-resistant core by trypsin [l] or bromelain [2]. The catalytic activity of the protease- solubilized reductase has been thoroughly investigated with physiological and artificial electron acceptors [3]. The enzyme contains per mol of protein, one FMN and one FAD [4]. This is the molecular basis for the exceptional redox properties of the enzyme which has been clarified by Masters et al. [3].

Dedicated to Prof. H. Zahn on occasion of his 60th birthday.

Abbreviations. E(SsbN)z, modified NADPH -cytochrome c reductase obtained by the reaction of native enzyme with 1 mM Nbsz in the presence of 1 mM NADP'; Nbsz, 5,5'-dithio-bis(2- nitrobenzoate) (Ellman's reagent); Nbs-, 2-nitro-5-thiobenzoate anion.

Enzymes. NADPH-cytochrome c reductase (EC 1.6.2.4): trypsin (EC 3.4.21.4); bromelain (EC 3.4.22.4).

This paper forms part of doctoral theses Mrs Helga Ehrig (Fachbereich Chemie Justus-Liebig-Universitat GieRen 1974) and Tibor Lazar (in preparation).

The correlation between the protein structure and the enzymatic properties of the reductase has not yet been studied. Neither the flavin binding sites nor the NADPH binding sequence has been characterized. Experiments with thiol reagents (e.g. p-chloromercuribenzoate, iodoacetate, mercuric chloride) showed that the enzymatic activity of NADPH - cytochrome c reductase depends on its cysteinyl residues [5]. These more global studies were not intended to elucidate the functional role of -SH groups as parts of the catalytic or binding centres in this enzyme. The study of the reaction between the enzyme and 5,5'-dithio- bis(2-nitrobenzoate) promised to give detailed an- swers to these unsolved questions of its structure.

MATERIALS AND METHODS

Materials

NADPH, NADP', cytochrome c, trypsin (2 x recrystallized), bromelain, FAD and FMN were

366 Functional Role of - SH Groups in NADPH ~ Cytochrome c Reductase

obtained from Boehringer Mannheim GmbH, 53'- dithio-bis(2-nitrobenzoate) (Nbs2) from EGA Chemie K.G. (Heidenheim, F.R.G.), Sepharose from Deutsche Pharmacia GmbH (Frankfurt, F.R.G.), DEAE-cellu- lose (DE-32, microgranular) from Whatman Bio- chemicals Ltd (Springfield Mill, England). Acryl- amide, N,N '-methylenebisacrylamide were products of Serva (Heidelberg, F.R.G.). Ampholine was pur- chased from LKB Producter (Uppsala, Sweden). Bovine serum albumin was obtained from Behring- Werke AG (Marburg, F.R.G.).

Purification of NADPH- Cytochrome c Reductase

Solubilization of the enzyme has been achieved by proteolysis according to Omura and Takesue [l] or Pederson et al. [2]. Both methods of purification led to essentially the same preparation of reductase with respect to homogeneity as proved by disc electrophoresis and specific activity.

Preparative Isotachophoresis of Purified NADPH- Cytochrome c Reductase

Preparative isotachophoresis was performed with the Uniphor system (LKB producter, Uppsala, Swe- den) according to the LKB application note number 146. Both buffers had to be renewed every 3 h during an electrophoresis run. Conditions of experiment were as follows. The gel was 2.5 cm in diameter, 9.5 cm in height, with total monomer concentration, acrylamide + N,N '-methylenebisacrylamide, of 7.5 % (w/v) and concentration of bisacrylamide expressed as a percentage of total monomer concentration 2.9 %. The terminating electrolyte contained 30 g glycine, 6 g Tris base filled up to 2 1 with bidistilled water and adjusted to pH 8.4 with 5 M HC1; anode and elution buffer contained 121 ml 1 M sulfuric acid, 32 g Tris base filled up to 4 1 with quartz-distilled water and adjusted to pH 7.3 with 5 M HC1. During electrophoresis a constant power of 10 W was maintained.

Assay of NADPH- Cytochrome c Reductase

The activity of NADPH - cytochrome c reductase was determined according to Phillips and Langdon [7] from the linear increase of the absorbance at 550 nm during the first 30 s after addition of 120 nmol NADPH to a test system containing 30 nmol cytochrome c, 5 - 50 p1 enzyme solution in a total volume of 0.5 ml. All substances except the enzyme were dissolved in 0.05 M potassium phosphate buffer pH 7.5. The activity of reductase was measured at 25 "C. The millimolar absorption coefficient of 21 mM-l cm-I for the difference in absorption between reduced and oxidized cytochrome c at 550 nm was used in calculating enzyme activity. One unit (U)

of enzyme activity was defined as reduction of 1 pmol of cytochrome c per min under the assay conditions.

Spectrophotornetric Determination of NADPH- Cytochrome c Reductase

The concentration of NADPH - cytochrome c reductase was determined spectroscopically using the molar absorption coefficients d &273 = 1.25 x lo5 M-l cm-' and A6455 = 18.5 x lo3 Mpl cm-l. With purified preparations of the reductase a ratio A&273/ A&+55 = 6.8 f 0.4wasdetermined.

Determinution of Protein Content

Protein was estimated according to Lowry et al. [8] with bovine serum albumin (dried over P205 in vacuo) as reference protein. For purified reductase, protein determinations with the Lowry method and from d A273 using d &273 = 1.25 x lo5 M-l cm-I gave identical values within the limits f 5.6 %.

Disc Electrophoresis

amide gel; total monomer concentration = 7.5% (w/v), degree of cross linkage = 2.6%; pH of separa- tion = 9.5. Staining of the protein zones was achieved with Amidoschwarz 10B (1 %, v/v) in 7 % acetic acid and 30% (v/v) methanol. The gels were destained in 7 % (v/v) acetic acid/30 % (v/v) methanol with charcoal.

This was performed in a medium-pore polyacryl-.

Determination of - S H Content in Reductase Preparations

The number of (reactive) -SH groups per mol NADPH - cytochrome c reductase was estimated with Ellman's reagent (Nbs2) using a molar absorption coefficient of 14 140 M-l cm-' for 2-nitro-5-thiobenzoate anion [9] and a molecular weight of 68000 for the protease-solubilized NADPH - cytochrome c reductase [4]. The degree of - SH group modification was calculated from the amount of Nbs- liberated assuming 1 : 1 stoichiometry between Nbs- ions and mol thiol groups reacted.

The total number of - SH groups per mol reductase was determined with Nbsz after denaturation of the enzyme by 2 % (w/v) sodium dodecyl sulfate.

Estimation of Fluvin

according to the method of Faeder and Siege1 [lo]. Estimation of FMN and FAD was performed

Reaction of the 'Half-Reduced' State of NADPH- Cytochrome c Reductase with Nbs2

NADPH - cytochrome c reductase was aerobically reduced to the 02-stable 'semiquinone' form by

T. Lazar, H. Ehrig, and L. Lumper 367

addition of NADPH (final concentration 0.31 mM) at pH 7.5. The solution was saturated with oxygen by bubbling air through the medium till a constant value of AA5so was reached. The cosubstrate was removed by gel filtration on Sephadex G-25 and the enzyme solution concentrated by ultrafiltration through an Amicon PM-30 membrane. A ratio A A455 / A A580 = 2.74 was determined for the pre- treated reductase. This value indicated that more than 90% of the enzyme was transformed into the 0 2 -

stable half-reduced state ( A A455/4 A585 = 2.73 - 2.92 [4]). Nbs2 (final concentration 1 mM) was added to the half-reduced reductase and the absorbances at 412 nm and 580 nm monitored continuously for 5 h at +4 "C.

RESULTS

Total Content of - SH Groups in NADPH- Cytochrome c Reductase

The study of the functional role of thiol groups in NADPH - cytochrome c reductase was performed with enzyme preparations migrating as a single protein zone in 7.5% (w/v) polyacrylamide gel during electrophoresis and sedimenting as one symmetric peak through a 8 - 32 % (w/w) sucrose gradient. With the procedures of purification used [l, 21, specific activities of NADPH - cytochrome c reductase between 25 and 30 U/mg protein (ionic strength : 0.13 M) were achieved. By isotachophoresis of these preparations a fast-migrating component (specific activity <= 10 U/mg protein) could be separated off from the main fraction. The specific activity of the purified NADPH - cytochrome c reductase could be increased to 35-40 U/mg protein. A total content of 5.9 f 0.3 mol thiol groups/mol reductase was determined with Ellman's reagent (Nbs2) after denaturation of the protein with 2% (w/v) sodium dodecyl sulfate. The number of cysteinyl residues estimated per mol reductase was independent of the procedure of purification, if enzyme preparations with specific activities between 25 and 40 U/mg protein were used.

Reaction of Thiol Reagents with NADPH- Cytochrome c Reductase

The reactivity of thiol reagents with the cysteinyl residues of NADPH - cytochrome c reductase differed significantly. Masters et al. succeeded in inhibiting the reductase with 6 mol p-chloromercuribenzoate/ mol enzyme-bound flavin [3]. Our results confirmed the observations that 1 mM iodoacetamide fails to inhibit the NADPH - cytochrome c reductase (1 O3 mol reagent/mol reductase) even after incubation periods up to 24h at 4°C. On the other hand we could demonstrate that 0.3- 1.0 mM Nbsz modifies the

sulfhydryl groups of the reductase and inhibits the activity of this enzyme.

Reaction between Nbs2 and the NADPH- Cytochrome c Reductase

The kinetics of thiol group modification by Nbs2 were studied at +4 "C, pH 7.5 and ionic strength 0.13 M, if not mentioned otherwise. The progress curve for the liberation of 2-nitro-5-thiobenzoate anion (Nbs-) as a function of time during the reaction of the reductase (8 - 20 pM) with Nbsz reached a final value asymptotically within 24 h (Fig. 1). Using an Nbsz concentration between 0.34 mM and 1.0 mM, 90% of the accessible -SH groups were modified within 8 -9 h (Fig. 1). The number of reactive -SH groups in the reductase was calculated from the mol Nbs- liberated per mol enzyme within 24 h. At +4 "C 2.3 0.2 mol -SH groups/mol reductase were accessible to 0.34 mM Nbsz. This value increased to 2.9 k 0.1 using 1.0 mM Nbsz under identical conditions of reaction.

The molar ratio Nbs2 to reductase was kept in the range 50-100. This relation was sufficient to provide pseudo-first-order conditions with respect to Nbs2 during the modification of the reductase. Higher molar ratios Nbsz/enzyme could not be used, since even with 3.4 mM Nbsz intense precipitation of protein occurred.

The reactivity of the - SH groups in NADPH - cytochrome c reductase is strongly dependent on the ionic strength I of the reaction mixture, which was varied by the addition of KC1. With 0.34mM Nbs2 one -SH group was modified within 60 min at I = 0.07 M, but 2.1 mol thiol group/mol enzyme at I = 1.67 M (+4.8 "C). A distinctly higher accessibility of thiol groups was observed at 25 "C: 2.3 mol thiol groups/mol enzyme reacted within the same time interval at I = 0.07 M and 4.5 mol thiol groups/mol enzyme at I = 1.67 M.

The effects of the disulfide exchange between Nbsz and the reductase considered were (a) the modification of thiol groups essential for flavin binding and (b) conformational changes, which destabilize the binding forces between the flavocoenzymes and the protein component of the reductase. After complete reaction of reductase with 0.34- 1.0 mM Nbsz only a negligible amount of free flavin could be detected by fluorescence measurements in the reaction mixture itself or its low-molecular-weight fraction, which was isolated by gel filtration on Sephadex G-25. The loss of flavin caused by the modification of the NADPH - cytochrome c reductase was therefore at most 2 - 3 % of its total content.

2-Nitro-5-thiobenzoate anion could be liberated from the Nbs2-modified reductase by 1 mM dithioerythritol at pH 8.4 (+4 "C) after removal of the

368 Functional Role of -SH Groups in NADPH-Cytochrome c Reductase

Time ( m i n )

Fig. 1. Time course of the inactivation and modification of NADPH-eytochrome c reductase. Reaction conditions: (e0) and ( x -x) 10 pM reductase, 0.34mM Nbsz in 50mM sodium phosphate buffer containing 1 mM NaZEDTA pH 7.5 (+4"C); (*---O) and (x- - - -x) 17 pM reductase, 1 mM Nbsz in 50 mM sodium phosphate buffer containing 1 mM NazEDTA pH 7.5 (+4 "C). ( x ) Time course of reductase inactivation; (0) time course of thiol group modification

excess reagent. The yield of 2-nitro-5-thiobenzoate anion liberated/mol -SH group modified was 70- 75 % for preparations of NADPH - cytochrome c reductase, which were incubated with 0.34 or 1 mM Nbs2 under liberation of 2 or 3 mol Nbs- respectively.

Inactivation and Reactivation ofNADPH- Cytochrome c Reductase

During the reaction of NADPH - cytmchrome c reductase with 0.34- 1.0 mM Nbsz the enzyme is inactivated to more than 95% within 24 h. The semilogarithmic plot of log [E]/[El0 vs. time for the inactivation of native reductase by 0.34- 1.0 mM Nbs2 is nonlinear. During the first 40 min of reaction a fast phase is observed.

The activity can be completely recovered by incubation of the modified enzyme with 1 mM dithioerythritol (pH 8.4, I = 0.13 M ; molar ratio dithioerythritol/reductase = 100) for 10- 20 min.

Reaction ofNbs2 with NADPH- Cytochrome c Reductase in the Presence 0fNADP'

NADP' and 2'-AMP are potent inhibitors of the NADPH-cytochrome c reductase [l l] . The inhibition constants Ki are 2.0 pM and 21 pM respectively at pH 7.5 and ionic strength 0.09 M [7].

In the presence of 1.0 mM NADP' we observed a diminished number of thiol groups in reductase accessible to Nbs2. Only 2.0 f 0.1 mol -SH groups/

Table 1. Inhibition of the NADPH- cytochrome c reductase and decrease of - S H group modification by Nbsz in the presence of

Final reaction conditions (other than the competitive inhibitor varied): 8-20 pM reductase and 1 mM Nbsz in 50 mM sodium phosphate buffer pH 7.5 ( I = 0.13 M) at 4 "C. The reaction was terminated after 23 h by removing excess Nbsz and NADP' by gel filtration on Sephadex G-25 (20 x 2 cm, elution buffer 50 mM sodium phosphate pH 7.5). The activity gives the percentage of the original activity of the unmodified cytochrome c reductase. The apparent Ki of NADP' was taken as 2 pM [11]

N A D P +

[NADP'] [NADP+]/K, Nbsz Accessible Activity - SH groups

mM mol/mol enzyme p:

0 0 100 3.0 0 0.01 5 100 3.0 0 0.1 50 74 2.3 30 1 .o 500 54 2.0 100

mol protein reacted at an Nbs2 concentration of 1.0 mM within 23 h. This means that only two - SH groups can be modified in the presence of saturating concentrations of NADP' , whereas one thiol group is protected against modification. The protective effect of NADP' against the conversion of this single sulfhydryl group to a mixed disulfide showed a strong dependence on the ratio between the concentrations of the complex reductase . NADP' and the unliganded enzyme, which is identical with the value of [NADP']/ Ki (Table 1).


- - ~ 2.0 @ ,-" 100.0

> E . - -

% - - .- " n m

m aJ n

\ 4 \ .- c

- L

. 1.0 ;; Y) n z

1 I 0 150 200

Time ( m i n )

Fig.2. Reaction between E(SsbNi2 and Nbs2 at + 4 "C. ( 0 4 ) and ( x ~

phosphate buffer pH 7.5; (*---a) and ( x ~

of Nbs- liberation; ( x ) kinetics of enzyme inactivation

x ) 15.8 pM reductase and 1 mM Nbsz in 50 mM sodium ~ x ) 17 pM reductase, 0.34 mM Nbsz in 50 mM sodium phosphate buffer pH 7.5. (0) Kinetics

Reductase modified in the presence of 1 mM NADP' exhibited the same specific activity after removal of the competitive inhibitor and excess Nbsz as the unmodified enzyme (Table 1). The reductase derivative E(SsbN)z was stable at pH 7.5. No liberation of Nbs- could be observed during incubation periods up to 24 h at +4 "C.

The progress curve for the liberation of Nbs- in the presence of 1 mM NADP' gave a non-linear semilogarithmic plot of log (fraction of - SH groups remaining) vs. time. According to Ray and Koshland [12] two pseudo-first-order processes with the rate constants kl = 0.016 min-' and kZ = 0.003 min-l (reductase concentration = 18 pM) could be separated. The Ztraight line for the fast part of the progress curve intersected the ordinate at 0.56.

Kinetics of - SH Group Mod6cation and Inactivation of the Modified Reductase E(SsbN)z

The modified reductase E(SsbN)z lost more than 95% of i t s original activity during the reaction with 0.34- 1.0 mM Nbs2 at + 4 "C under liberation of 1 mol2-nitro-5-thiobenzoate/mol enzyme (Fig. 2). The kinetics of the modification of E(SsbN)z and the simultaneous inactivation gave linear semilogarithmic plots. This was consistent with the reaction of one single thiol group in the modified enzyme E(SsbN)z. The rate constants of activity destruction and modification were (practically) identical (Table 2) and were linear functions of [Nbsz] at constant concentration of enzyme (15.8 pM).

-

Table 2. Pseudo-first-order rate constunts for the - SH group modification and inactivation of the NADPH- cytochrome c reductase derivative E(SsbN)z by Nbsz Conditions of experiment: 15.8 pM reductase, 50 mM sodium phosphate buffer pH 7.5 ( 4 T , I = 0.13 M)

Nbsz k s H kinactivation

mM min-'

0.34 0.027 0.039 1.0 0.069 0.11

The action of irreversible inhibitors at ligand binding sites of enzyme could be overcome by protection with substances, e.g. (co)substrates or inhibitors, which bind reversibly at the same site of the enzyme. Kinetic evidence for the reaction of an irreversible inhibitor at a ligand binding site could be drawn by analysis of the concurrence reaction between inhibitor and ligand at the enzyme using the approach applied by Vinogradov et al. [13], if the functional groups in the ligand-protected site are completely masked against the attack by the irreversible inhibitor. This condition is valid for the masking of the 'essential' -SH group in the reductase by NADP+ (Table 2). The reaction of an enzyme at an functionally necessary site with an irreversible inhibitor follows the rate law

370 Functional Role of -SH Groups in NADPH- Cytochrome c Reductase

: i i

10 11 d,* I , ,a

0.01 0.05 0.10 [NADP'] ( m M )

Fig.3. Inactivation plot for the reaction of N A D P H - cytochrome c reductase with Nbsz in the presence and in the ahsence of cytochrome c. Reaction conditions for (A): 8.8 pM reductase, 1.0 mM .Nbsz, 10- 100 pM NADP+, 58 pM cytochrome c in 50 mM sodium phosphate buffer pH 7 . 5 ; (B): identical reaction mixture without cytochrome c. Reaction conditions : temperature + 4 "C; reaction time t = 3 h ; after this time aliquots of the reaction mixture were removed and diluted 500 times with 50 mM phosphate buffer pH 7 . 5 for the determination of enzymatic activity. The data are presented as t/ln [E]o/[E], vs. [NADP+]. [E]0/[Elt is the ratio enzymatic activity at t = O/enzymatic activity at t. Intercept on the negative abscissa is interpreted as KNAop

in the presence of the reversible bound ligand NADP' (KNAD~ = dissociation constant for the complex between NADP' and the reductase, [NADP'] = concentration of NADP+, [I] = concentration of the irreversibly reacting inhibitor Nbsz). The integrated equation could be rearranged into the following form :

The graph t/(ln [E]o/[E],) vs. [NADP'] gave a straight line with Nbs2 as irreversible inhibitor (Fig. 3). This result was to be expected, if the irreversible inhibition was caused by a reaction with only one functional group at the ligand binding site of the enzyme. The numerical value of the intercept on the abscissa was not identical with that of Ki for NADP' (2 pM at ionic strength 0.09 M [7]) obtained under the conditions of the assay system for the estimation of the reductase activity. In contrast KNADP (about 3 pM) calculated from the plot t/(ln [E]o/[E],) vs. [NADP'] for the concurrence reaction between NADP' and Nbs2 in the presence of cytochrome c (58 pM) agreed very closely with the value of Ki determined by Phillips and Langdon [7] (Fig. 3).

Influence of the Modijication by Nbs2 on the Stability of the 'Half-Reduced' Form ofthe NADPH- Cytochrome c Reductase

The semiquinoid state of the reductase was not destroyed by modification of 2.2 mol thiol groups/ mol semiquinoid reductase by Nbs2 (+4 "C, pH 7.5, see Materials and Methods). During the action of Nbs2 99 % of the original enzymatic activity were lost.

DISCUSSION

One - SH group in NADPH - cytochrome c reductase from pork liver microsomes could be protected by NADP' against inactivation by Nbsz. NADP' is a competitive inhibitor of the reductase with respect to the cosubstrate NADPH. It could be demonstrated by kinetic methods that this thiol group is part of the cosubstrate binding site of the reductase. The basis of this suggestion was the following results : (a) the modification constant for the reaction between Nbsz and the modified reductase E(SsbN)z at the single -SH group, which was unaccessible in the presence of saturating concentrations of NADP' , were identical with the activity destruction constant; (b) the complete loss of activity during the reaction of the single thiol group in E(SsbN)2 with Nbs2 ; (c) the kinetic evidence that NADP' and Nbs2 interact at the same site of the reductase.

The inactivation of the NADPH - cytochrome c reductase by the formation of a mixed disulfide between the essential thiol group and the Nbs moiety does not imply that this functional group is directly involved into the binding of NADP' by the enzyme. The introduction of such a bulky group as Nbs into the NADPH binding region or its vicinity can inhibit the enzymatic activity of the reductase not only by modifying the binding groups for the cosubstrate itself but also in a more indirect way by steric hindrance or electrostatic effects on these groups. By kinetic analysis of the concurrence between the irreversible inhibitor Nbsz and the competitive inhibitor NADP' at the enzyme using the method of Vinogradov et al. [13] we obtained a value for the apparent dissociation constant KNADP for the complex reductase . NADP' , which differed by a factor of about 7 from Ki values determined by measuring the initial rates of the reductase-catalysed cytochrome c reduction in the presence of varying concentrations of NADP' [l l] . The numerical values of both constants agreed very well, if the concurrence experiments were performed in the presence of cytochrome c (58 pM). Direct estimates of the dissociation constant for the complex reductase . NADP' are desirable to establish the significance of Ki.

The kinetic evidence that Nbsz and NADP+ attack the same site of the reductase was based on the


linearity of the function t/(ln [E]o/[E],) vs. [NADP'] and should not be doubted considering the differences between KNADP found in the absence of cytochrome c and Ki.

Equilibrium binding studies demonstrated that the kinetically determined K, values of cytochrome c (2- 6 pM) are true dissociation constants of the reductase . cytochrome c complex [14]. Varying the NADPH concentration at a series of constant cytochrome c concentrations resulted in a family of parallel lines in reciprocal plots [14]. These results indicate that K, for NADPH is apparently dependent on cytochrome c concentration and a random addition of the two substrates [14]. The competition experiments between the dead-end inhibitor NADP' and Nbs2 demonstrated a decrease of apparent Ki for NADP' in the presence of cytochrome c (Fig. 3). This observation was consistent with the formation of a ternary complex during the electron transfer from NADPH to cytochrome c mediated by the reductase and did not favor an ordered addition with NADPH as the first substrate ('binary' ping-pong mechanism [4]).

The three accessible - SH groups of the NADPH- cytochrome c reductase showed small second-order- rate constants compared to sulfhydryl groups specified as essential in other enzymes. Wengenmayer et al. [15] discussed the idea that the slow liberation of Nbs- anion indicates an intramolecular disulfide exchange of the protein. This possibility could very probably be excluded in the case of NADPH-cytochrome c reductase by the following results: (a) the amounts of Nbs- anion liberated during the formation of E(SsbN)s by the reaction of reductase with Nbsz and during the reduction of E(SsbN)s with dithioerythritol were identical within the limits of experimental accuracy and (b) no liberation of Nbs- anion could be observed during the incubation of E(SsbN)2 up to

24 h at 4 "C (pH 7.5). Both observations seemed to be sufficient evidence that even partial intramolecular disulfide exchange plays no role during the modification of the NADPH - cytochrome c reductase with Nbs2.

This work was supported by grants of the Deutsche Forschungs- gemeinschaft (Bonn-Bad Godesberg, F.R.G.). The skilful technical assistance of Mr F. Busch and Mrs Becker is gratefully acknowl- edged.

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T. Lazar, H. Ehrig, and L. Lumper, Zentrum fur Biochemie der Justus-Liebig-Universitat GieBen, FriedrichstraBe 24, D-6300 GieBen, Federal Republic of Germany

the functional role of thiol groups in protease-solubilized nadph—cytochrome c reductase from...

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