- Email: [email protected]
Electron transfer in the membranes of endoplasmic reticulum
ARCHIVES
OF BIOCHEMISTRY
Electron
AND
Transfer
The Interaction
166, 308-312 (1975)
BIOPHYSICS
in the Membranes
of NADPH-
of Endoplasmic
and NADH-Specific
Reticulum
Electron-Transfer
Chains
in
Microsomesl A. I. ARCHAKOV,
V. M. DEVICHENSKY,
I. I. KARUZINA,
AND
A. V. KARJAKIN
Central Research Laboratory, N. I. Pirogov Second Medical Institute, Moscow 119435, USSR Received March 4, 1974 Data on the stimulating effect of NADH on the rate of NADPH-dependent hydroxylation reactions of dimethylaniline and ethylmorphine favor the possibility of electron transfer from the NADH-oxidation chain to the NADPH-oxidation chain. The presence of the stimulating effect of NADH at a low concentration of NADPH may explain the existence of the low electron-transfer rate from NADH-redox chain to the NADPH-redox chain to the NADPH-redox chain. The exchange of reducing equivalents between the chains occurs behind the flavoprotein sites of the chains. 100 UE/p 1 mg of protein). Other reagents were USSR-made chemical grade preparations. White male rats weighing 200-250 g were used. The methods for preparing microsomal fractions and measuring the rate of the redox reactions of cytochromes b, and P-450 using aerobic conditions as well as the rate of the reduction reactions of these hemoproteins under anaerobic conditions have been described in previous papers (l-3). The activity of the hydroxylation system of microsomes was determined using 1.0 ml incubation mixture containing 40 mM Tris-HCl buffer, pH 7.5, 16 mM MgCl,, 6 mM dimethylaniline, or 1 mM ethylmorphine; 2.5 mg of microsomal protein. The samples were incubated for 20 min at 37°C with constant shaking. The reactions were interrupted by adding 1.0 ml of mixture of equal volumes of 25% ZnSO, and a saturated solution of Ba(OH),. The samples were centrifuged at 7OOOgfor 10 min. One hundred twenty microliters of Nash solution (5) were added to 240 ~1 of the supernatant fraction. Measurements were taken in an SF 4A spectrophotometer at 412 nm in a microcuvette.
The possibility of cytochrome b, participation in NADPH-oxidation reactions has been demonstrated previously (l-4). The whole pool of cytochrome b, in the membranes of the endoplasmic reticulum was postulated to be heterogeneous: one of its subfractions is included in the NADPHspecific chain of electron transfer and the other is involved in the NADH-oxidation system. The data obtained gave grounds to suggest a scheme of electron transfer in microsomes, according to which an exchange of reducing equivalents should exist between the two chains (I). It is only with such an assumption that the experimental evidence obtained can be rationalized. The aim of this investigation is to obtain additional support to the idea of electron transfer from one chain to the other and to find out where the sites of this exchange are localized. EXPERIMENTAL
PROCEDURES
RESULTS
NADPH and NADH were obtained from C. F. Boehringer and Pronase (from Streptomyces griseus) from Koch-Lvht Laboratories Ltd, England. Propylgallate was purchased from Sigma Chemical Co., USA, glucose-6-phosphate dehydrogenase from Fluka ‘This
paper is the second in a series.
Electron transfer from the NADHdependent respiratory chain to the NADPH oxidation chain is readily demonstrated in the experiments on the stimulation of the NADPH-specific hydroxylation of DMA and ethylmorphine by NADH.
308 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
ELECTRON
TRANSFER
As is seen from Table I, with low concentrations of NADPH in the incubation medium, addition of NADH enhances demethylation of DMA and ethylmorphine. This effect is increased by using an NADPH-generating system instead of NADPH (Table II). Thus, it is not the consequence of a saving effect for NADPH but is a result of the increase in the hydroxylation rate caused by NADH. Analyzing the results obtained we may suppose that the NADH reducing equivalents, in the process of its oxidation, may be brought to the NADPH-oxidation chain and if this chain is not fully saturated with its substrate, they produce a stimulating effect on the hydroxylation reaction. This effect was revealed by Cohen and Estabrook (6). The fact that such an effect is absent with high-saturating NADPH concentrations points to the fact that the rate of electron transfer from the NADH-oxidation chain to the NADPH-oxidation chain is much lower than that in the hydroxylating chain. This conclusion is in good correlation with the data obtained by us earlier (1). The direct measurement of the rate of the cytochrome P-450 reduction in anaerobic condition by NADH permitted us to calculate the apparent first-order rate constant of this reaction. It was 0.2 min’ at 12”C, much lower that those in the NADPHredox chain (7 min- ‘) . Further, an attempt was made to find out at which sites reducing equivalents are transferred from one enzyme complex to another. Scheme I presents all formally possible pathways of exchange for reducing equivalents between microsomal electron-transfer chains. NADPH I
---
Fp 1-
cyt. b, -
cyt. P-450
IN MICROSOMES.
309
II
TABLE I THE EFFECT OF NADH ON THE RATE OF N-DEMETHYLATION OF DIMETHYLANILINE AND ETHYLMORPHINE~ Experimental conditions
Y-Demethylatior of dimethylaniline nmoles IrIg-‘mm’
NADPH 3 mM NADPH 3 mM + NADH 1 mM NADPH 0.5 mM NADPH 0.5 mM + NADH 1 mM NADH 1 mM
9%
11u’-Demethylation of T ethylmorphine 90
nmoles mg-’ min-’
8.0 8.1
100 100
13.3 13.2
100 100
4.8 6.6
60 80
9.5 12.6
70 95
0.46
5
0.69
5
a One milliliter of incubation mixture contained 40 mM Tris-buffer, pH 7.5; 16 mM MgCl,, 3 mM NADPH, 1.5 mg of microsomal protein, and 6 mM of dimethylaniline in the case of N-demethylation of dimethylaniline and in the case of N-demethylation of ethylmorphine the incubation mixture contained 0.75 mg of microsomal protein and 1 mM of ethylmorphine. The results are average of five experiments. TABLE II THE EFFECT OF NADH ON THE RATE OF NDEMETHYLATION OF DIMETHYLANILINE IN THE PRESENCEOF AN NADPHGENERATING SYSTEM” N-Demethylation dimethylaniline nmoles. rng-‘.min’ NADPH 3 mM NADH 1 mM NADP+ 0.15 mM NADP+ 0.15 mM $ NADH 1 mM NADP+ 0.6 mM NADP+ 0.6 mM + NADH 1 mM
of %
12.3 1.0 5.1 9.1
100 8 40 75
9.0 10.3
75 85
NADH
a One milliliter of incubation mixture for N-demethylation of dimethylaniline in the presence of an NADPH-generating system contained 40 mM Tris buffer, pH 7.5; 16 mM MgCl,; 10 mM nicotinamide, 10 mM glucose&phosphate (sodium salt), 1 UE glucose6.phosphate dehydrogenase, 1.5 mg of the microsomal protein. The reaction was initiated by addition of 6 mM dimethylaniline.
Many authors (1, 8, 9) do not recognize the existence of an NADH-NADP+-trans-
hydrogenase reaction in the microsomal fraction, but others have reported that
1
310
ARCHAKOV
purified Fp, can interact with NADH (10). We have undertaken a special investigation to show that the electron exchange between the chains occurs not at the initial flavoprotein site of the chains, but somewhere at the cytochrome level. With this aim we studied the action of pronase, the enzyme which at low concentrations selectively solubilizes the NADPH-specific flavoprotein (ll), on the reduction rate of cytochromes b, and P-450 in the presence of NADPH and NADH (Fig. 1). Addition of pronase to the incubation mixture causes a considerable inhibition of cytochrome b, reduction by NADPH and does not affect its reduction by NADH. So, if cytochrome P-450 were reduced by NADH, i.e., the electron-transfer reaction from the NADH-oxidation chain to the NADPH-oxidation chain occurred at the level of the NADPH-specific flavoprotein, in pronase-treated microsomes it would be inhibited in the same manner as in the other reactions of NADPH-specific electron transfer. However, the curves given in Fig. 1 show that the treatment of microsomes by pronase does not affect the rate of the NADH-cytochrome P-450 reNADPH
ET AL
ductase reaction. This means that the NADPH-specific flavoprotein is not involved in electron transfer from the NADH-oxidation chain to the NADPHoxidation chain, i.e., reactions 1 and 2 in Scheme II should be ignored. This conclusion was supported by studying the inhibiting effect of propylgallate on the rate of demethylation of DMA when NADPH and NADH are used as substrates. If the electron transfer of the NADH-oxidation chain to the NADPH-oxidation chain occurred at the initial flavoprotein site, then addition of propylgallate, an inhibitor interacting with the NADPH-specific flavoprotein (la), to the incubation mixture should remove the stimulating effect of NADH on the rate of demethylation in the presence of low concentrations of NADPH. As is seen in Table III, no such effect is observed. Propylgallate produced a decreasing effect on the level of reduction of cytochromes b, and P-450 only when NADPH was used as substrate (Fig. 2). This effect is explained by the interaction of propylgallate with the NADPH-specific flavoprotein and results in the interruption of electron flow from NADPH. The absence of this NADH
s’
0.07 Q, 2 0.08p” ok 0.05 2
0.04-
4 Q
0.03-
0.02 -
Time (sec.) FIG. 1. The effect of pronase on the reduction rate of cytochrome 6, and P-450 by NADPH and NADH using anaerobic conditions. Incubation mixture contained 50 mM Tris-HCl buffer, pH 7.5: 1 mg.ml-’ of microsomal protein, 1 rng’rnl-’ of protein of submitochondrial particles, 2 FM rotenone, 15 mM succinate. The reaction was initiated by adding 30 pM NADPH or 8 FM NADH. The moment of addition of the substrate is indicated by the arrow. Pronase content, 7 pg.rng-’ microsomal protein. The time of incubation of microsomes with pronase 0, 5, 10, 15 min. The record was made using a Hitachi 356 spectrophotometer and the wavelength pairs for cyt. b, AA,08.,2,, solid line, and for cyt. P-450 AA,50.,75, broken line.
ELECTRON TABLE
TRANSFER
IN MICROSOMES.
311
II
III
It is also possible to demonstrate experimentally that there is no electron transfer from the NADPH-oxidation chain via cytochrome P-450 to cytochrome b, of the AND NADH NADH-oxidation chain (reaction 9, scheme 2). If this were the case, addition of J-Demethylation Experimental” N-Demethylation conditions of dimethylf ethylmorphinec carbon monoxide to the incubation mix(nmoles~mg-‘~ anilineb ture would result in a lower level of reducmin- ‘) (nmoles .mg-’ tion of cytochrome b, when NADPH is min-‘1 used as substrate. On the contrary, it 15.3 NADPH 3 mM 13.0 shows a considerable increase in the level NADPH 3 mM + 16.1 13.1 of the hemoprotein reduction to take place NADHlmM (1, 2). NADPH 3 mM + 9.6 5.1 Electron transfer from the NADH-cytoPropylgallate chrome b, reductase complex to the 0.2 rnM NADPH-oxidation chain cannot proceed NADPH 3 mM + 10.9 6.3 via the pathway indicated by arrow 4 in NADH 1 mM + Propylgallate scheme 2, since in this case the NADPH0.2 mM specific flavoprotein should reduce cytoNADH 1 mM 1.0 0.8 chain chrome b, in the NADPH-oxidation at a very high rate. The affinity of this DThe experimental conditions are those described flavoprotein to cytochrome b, is known to for Table I. be very high. With such a pathway, the *The average of four experiments. rate of electron transfer from the NADHc The average of six experiments. dependent respiratory chain to the cytoeffect on the reduction of cytochrome P-450 chrome P-450 oxygenase system should be by NADH indicates the existence of an very high, and this is not so. electron-transfer pathway from NADH to Thus, out of all the possible ways of cytochrome P-450 which does not include electron exchange between the two microthe NADPH-specific flavoprotein. The results of these experiments lend NADH NADPH support to our assumption that electron 0.08 A transfer from the NADH-oxidation chain to the NADPH-oxidation chain is accomplished at the site localized behind the NADPH-specific flavoprotein. Consequently, reactions 4, 5, 6 and 8 are indicated in scheme 2 and illustrate possible pathways of electron transfer. THE EFFECT OF PROPYLCALLATE ON THE RATE OF IV-DEMETHYLATION OF DIMETHYLANILINE AND ETHYLMORPHINE IN THE PRESENCE OF NADPH
009
DISCUSSION
The results on the inhibiting action of pronase and propylgallate on the rate of reduction of cytochromes b, and P-450 in the presence of NADPH, as well as the absence of the inhibiting effect of pronase and propylgallate on the NADH-stimulating effect in the hydroxylation reaction, permit the conclusion that at the initial site (reactions 1 and 2, scheme 2) there is no electron transfer from the NADHspecific chain to the NADPH-specific chain.
123
?
4
2
3
4
llme(mln)
FIG. 2. The effect of propylgallate on the redox behavior of cytochromes b, and P-450 reduced by NADPH and NADH using aerobic conditions. Control incubation mixture contained 50 mM Tris-HCI buffer, pH 7.5; 1 mg.ml-’ microsomal protein (1). Experimental incubation mixture was the same as described in Ref. 1 with 200 pM of propylgallate (2). The reaction was initiated by adding of 8 FM NADPH or NADH. Solid line, cytochrome b,. Broken line, cytochrome P-450.
312
ARCHAKOV
somal respiratory chains, the reactions indicated by arrows 3, 5, 6, 7, and 8 in scheme 2 are realistic. It is most probable that electron exchange between t,he chains proceeds at the cytochrome b, level via reactions 6 and 7. However, pathway 5 is not to be ruled out as the potent NADH-Fp,-cytochrome b, reductase system in the cell is responsible for cytochrome b, in the NADH-oxidation chain being always in the fully reduced state (13). In these conditions an excess of NADH may initiate reaction 5: NADH 4 Fp, + cyt. P-450. Ichikawa and Loehr (14) obtained experimental data about a similar enzyme complex existing in microsomes. Thus, the transfer of reducing equivalents from the NADPH-oxidation chain to the NADH-oxidation chain may take place either due to cytochrome b, reduction in the NADH-oxidation chain directly by the NADPH-specific flavoprotein as a result of electrons coming to this cytochrome from cytochrome b, of the NADPH-oxidation chain. The rate of this reaction is equal to that of cytochrome b, reduction in the NADPH-oxidation chain. Three possibilities should be pointed out with regard to electron transfer from the NADH-specific chain to the NADPH-oxidation chain: the first pathway is directly from the NADH-specific flavoprotein to cytochrome P-450, the second, from cytochrome b, of the NADH-oxidation chain to cytochrome b, of the NADPH-oxidation chain, and the third, directly from cytochrome b, of the NADH-oxidation chain to cytochrome P-450. Thus, we may offer a more detailed scheme of the reactions of electron ex-
ET AL.
change between tory chains:
the microsomal
NADPH -
Fp, -
NADH .
Fp, dcyt.
cyt. b, d
respiracyt. P-450
b, ’
REFERENCES 1. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND SEVERINA, v. A. (1969) Biochimia 34, 782-790. 2. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARJAKIN, A. V. (0000) Arch. Biochem. Biophys. 3. ARCHAKOV, A. I., KARUZINA, I. I., KOKAREVA, I. S. AND BACHMANOVA, G. I. (1973) Biochem. J. 136, 371-379. 4. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARUZINA, I. I. (1973) IX Int. Biochem. Cong. 7 B, 22, p. 336. Stockholm. 5. NASH, T. (1953) Biochem. J. 55, 416-421. 6. COHEN, B. S., AND ESTABROOK, R. W. (1971) Arch. Biochem. Biophys. 143, 37-53. 7. ARCHAKOV, A. I., AND DEVICHENSKY, V. M. (0000) Arch. Biochem. Biophys. 8. ORRENIUS, S., BERG, A., AND ERNSTEH, L. (1969) Eur. J. Biochem. 11, 193-200. 9. KRISH, K., AND STAUDINGER, H. (1961) Biochem. 2. 334,312-327. 10. ICHIKAWA, Y., YAMANO, T., AND FUJISHIMA, H. (1969) B&him. Biophys. Acta 171, 32-46. 11. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARUZINA, I. I. (1973) Dokl. Akad. Nauk. SSSR 209,973-976. 12. SLATER, T. F., AND TORIELL, M. V. (1971) Biothem. J. 121, 40 P. 13. ARCHAKOV, A. I., KARJAKIN, A. V., AND SKULACHEV, V. P. (1973) Dokl. Akad. Nauk SSSR 209, 221-223. 14. ICHIKAWA, Y., AND LOEHR, J. S. (1972) Biochem. Biophys. Res. Commun. 46, 1187-1193.
OF BIOCHEMISTRY
Electron
AND
Transfer
The Interaction
166, 308-312 (1975)
BIOPHYSICS
in the Membranes
of NADPH-
of Endoplasmic
and NADH-Specific
Reticulum
Electron-Transfer
Chains
in
Microsomesl A. I. ARCHAKOV,
V. M. DEVICHENSKY,
I. I. KARUZINA,
AND
A. V. KARJAKIN
Central Research Laboratory, N. I. Pirogov Second Medical Institute, Moscow 119435, USSR Received March 4, 1974 Data on the stimulating effect of NADH on the rate of NADPH-dependent hydroxylation reactions of dimethylaniline and ethylmorphine favor the possibility of electron transfer from the NADH-oxidation chain to the NADPH-oxidation chain. The presence of the stimulating effect of NADH at a low concentration of NADPH may explain the existence of the low electron-transfer rate from NADH-redox chain to the NADPH-redox chain to the NADPH-redox chain. The exchange of reducing equivalents between the chains occurs behind the flavoprotein sites of the chains. 100 UE/p 1 mg of protein). Other reagents were USSR-made chemical grade preparations. White male rats weighing 200-250 g were used. The methods for preparing microsomal fractions and measuring the rate of the redox reactions of cytochromes b, and P-450 using aerobic conditions as well as the rate of the reduction reactions of these hemoproteins under anaerobic conditions have been described in previous papers (l-3). The activity of the hydroxylation system of microsomes was determined using 1.0 ml incubation mixture containing 40 mM Tris-HCl buffer, pH 7.5, 16 mM MgCl,, 6 mM dimethylaniline, or 1 mM ethylmorphine; 2.5 mg of microsomal protein. The samples were incubated for 20 min at 37°C with constant shaking. The reactions were interrupted by adding 1.0 ml of mixture of equal volumes of 25% ZnSO, and a saturated solution of Ba(OH),. The samples were centrifuged at 7OOOgfor 10 min. One hundred twenty microliters of Nash solution (5) were added to 240 ~1 of the supernatant fraction. Measurements were taken in an SF 4A spectrophotometer at 412 nm in a microcuvette.
The possibility of cytochrome b, participation in NADPH-oxidation reactions has been demonstrated previously (l-4). The whole pool of cytochrome b, in the membranes of the endoplasmic reticulum was postulated to be heterogeneous: one of its subfractions is included in the NADPHspecific chain of electron transfer and the other is involved in the NADH-oxidation system. The data obtained gave grounds to suggest a scheme of electron transfer in microsomes, according to which an exchange of reducing equivalents should exist between the two chains (I). It is only with such an assumption that the experimental evidence obtained can be rationalized. The aim of this investigation is to obtain additional support to the idea of electron transfer from one chain to the other and to find out where the sites of this exchange are localized. EXPERIMENTAL
PROCEDURES
RESULTS
NADPH and NADH were obtained from C. F. Boehringer and Pronase (from Streptomyces griseus) from Koch-Lvht Laboratories Ltd, England. Propylgallate was purchased from Sigma Chemical Co., USA, glucose-6-phosphate dehydrogenase from Fluka ‘This
paper is the second in a series.
Electron transfer from the NADHdependent respiratory chain to the NADPH oxidation chain is readily demonstrated in the experiments on the stimulation of the NADPH-specific hydroxylation of DMA and ethylmorphine by NADH.
308 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
ELECTRON
TRANSFER
As is seen from Table I, with low concentrations of NADPH in the incubation medium, addition of NADH enhances demethylation of DMA and ethylmorphine. This effect is increased by using an NADPH-generating system instead of NADPH (Table II). Thus, it is not the consequence of a saving effect for NADPH but is a result of the increase in the hydroxylation rate caused by NADH. Analyzing the results obtained we may suppose that the NADH reducing equivalents, in the process of its oxidation, may be brought to the NADPH-oxidation chain and if this chain is not fully saturated with its substrate, they produce a stimulating effect on the hydroxylation reaction. This effect was revealed by Cohen and Estabrook (6). The fact that such an effect is absent with high-saturating NADPH concentrations points to the fact that the rate of electron transfer from the NADH-oxidation chain to the NADPH-oxidation chain is much lower than that in the hydroxylating chain. This conclusion is in good correlation with the data obtained by us earlier (1). The direct measurement of the rate of the cytochrome P-450 reduction in anaerobic condition by NADH permitted us to calculate the apparent first-order rate constant of this reaction. It was 0.2 min’ at 12”C, much lower that those in the NADPHredox chain (7 min- ‘) . Further, an attempt was made to find out at which sites reducing equivalents are transferred from one enzyme complex to another. Scheme I presents all formally possible pathways of exchange for reducing equivalents between microsomal electron-transfer chains. NADPH I
---
Fp 1-
cyt. b, -
cyt. P-450
IN MICROSOMES.
309
II
TABLE I THE EFFECT OF NADH ON THE RATE OF N-DEMETHYLATION OF DIMETHYLANILINE AND ETHYLMORPHINE~ Experimental conditions
Y-Demethylatior of dimethylaniline nmoles IrIg-‘mm’
NADPH 3 mM NADPH 3 mM + NADH 1 mM NADPH 0.5 mM NADPH 0.5 mM + NADH 1 mM NADH 1 mM
9%
11u’-Demethylation of T ethylmorphine 90
nmoles mg-’ min-’
8.0 8.1
100 100
13.3 13.2
100 100
4.8 6.6
60 80
9.5 12.6
70 95
0.46
5
0.69
5
a One milliliter of incubation mixture contained 40 mM Tris-buffer, pH 7.5; 16 mM MgCl,, 3 mM NADPH, 1.5 mg of microsomal protein, and 6 mM of dimethylaniline in the case of N-demethylation of dimethylaniline and in the case of N-demethylation of ethylmorphine the incubation mixture contained 0.75 mg of microsomal protein and 1 mM of ethylmorphine. The results are average of five experiments. TABLE II THE EFFECT OF NADH ON THE RATE OF NDEMETHYLATION OF DIMETHYLANILINE IN THE PRESENCEOF AN NADPHGENERATING SYSTEM” N-Demethylation dimethylaniline nmoles. rng-‘.min’ NADPH 3 mM NADH 1 mM NADP+ 0.15 mM NADP+ 0.15 mM $ NADH 1 mM NADP+ 0.6 mM NADP+ 0.6 mM + NADH 1 mM
of %
12.3 1.0 5.1 9.1
100 8 40 75
9.0 10.3
75 85
NADH
a One milliliter of incubation mixture for N-demethylation of dimethylaniline in the presence of an NADPH-generating system contained 40 mM Tris buffer, pH 7.5; 16 mM MgCl,; 10 mM nicotinamide, 10 mM glucose&phosphate (sodium salt), 1 UE glucose6.phosphate dehydrogenase, 1.5 mg of the microsomal protein. The reaction was initiated by addition of 6 mM dimethylaniline.
Many authors (1, 8, 9) do not recognize the existence of an NADH-NADP+-trans-
hydrogenase reaction in the microsomal fraction, but others have reported that
1
310
ARCHAKOV
purified Fp, can interact with NADH (10). We have undertaken a special investigation to show that the electron exchange between the chains occurs not at the initial flavoprotein site of the chains, but somewhere at the cytochrome level. With this aim we studied the action of pronase, the enzyme which at low concentrations selectively solubilizes the NADPH-specific flavoprotein (ll), on the reduction rate of cytochromes b, and P-450 in the presence of NADPH and NADH (Fig. 1). Addition of pronase to the incubation mixture causes a considerable inhibition of cytochrome b, reduction by NADPH and does not affect its reduction by NADH. So, if cytochrome P-450 were reduced by NADH, i.e., the electron-transfer reaction from the NADH-oxidation chain to the NADPH-oxidation chain occurred at the level of the NADPH-specific flavoprotein, in pronase-treated microsomes it would be inhibited in the same manner as in the other reactions of NADPH-specific electron transfer. However, the curves given in Fig. 1 show that the treatment of microsomes by pronase does not affect the rate of the NADH-cytochrome P-450 reNADPH
ET AL
ductase reaction. This means that the NADPH-specific flavoprotein is not involved in electron transfer from the NADH-oxidation chain to the NADPHoxidation chain, i.e., reactions 1 and 2 in Scheme II should be ignored. This conclusion was supported by studying the inhibiting effect of propylgallate on the rate of demethylation of DMA when NADPH and NADH are used as substrates. If the electron transfer of the NADH-oxidation chain to the NADPH-oxidation chain occurred at the initial flavoprotein site, then addition of propylgallate, an inhibitor interacting with the NADPH-specific flavoprotein (la), to the incubation mixture should remove the stimulating effect of NADH on the rate of demethylation in the presence of low concentrations of NADPH. As is seen in Table III, no such effect is observed. Propylgallate produced a decreasing effect on the level of reduction of cytochromes b, and P-450 only when NADPH was used as substrate (Fig. 2). This effect is explained by the interaction of propylgallate with the NADPH-specific flavoprotein and results in the interruption of electron flow from NADPH. The absence of this NADH
s’
0.07 Q, 2 0.08p” ok 0.05 2
0.04-
4 Q
0.03-
0.02 -
Time (sec.) FIG. 1. The effect of pronase on the reduction rate of cytochrome 6, and P-450 by NADPH and NADH using anaerobic conditions. Incubation mixture contained 50 mM Tris-HCl buffer, pH 7.5: 1 mg.ml-’ of microsomal protein, 1 rng’rnl-’ of protein of submitochondrial particles, 2 FM rotenone, 15 mM succinate. The reaction was initiated by adding 30 pM NADPH or 8 FM NADH. The moment of addition of the substrate is indicated by the arrow. Pronase content, 7 pg.rng-’ microsomal protein. The time of incubation of microsomes with pronase 0, 5, 10, 15 min. The record was made using a Hitachi 356 spectrophotometer and the wavelength pairs for cyt. b, AA,08.,2,, solid line, and for cyt. P-450 AA,50.,75, broken line.
ELECTRON TABLE
TRANSFER
IN MICROSOMES.
311
II
III
It is also possible to demonstrate experimentally that there is no electron transfer from the NADPH-oxidation chain via cytochrome P-450 to cytochrome b, of the AND NADH NADH-oxidation chain (reaction 9, scheme 2). If this were the case, addition of J-Demethylation Experimental” N-Demethylation conditions of dimethylf ethylmorphinec carbon monoxide to the incubation mix(nmoles~mg-‘~ anilineb ture would result in a lower level of reducmin- ‘) (nmoles .mg-’ tion of cytochrome b, when NADPH is min-‘1 used as substrate. On the contrary, it 15.3 NADPH 3 mM 13.0 shows a considerable increase in the level NADPH 3 mM + 16.1 13.1 of the hemoprotein reduction to take place NADHlmM (1, 2). NADPH 3 mM + 9.6 5.1 Electron transfer from the NADH-cytoPropylgallate chrome b, reductase complex to the 0.2 rnM NADPH-oxidation chain cannot proceed NADPH 3 mM + 10.9 6.3 via the pathway indicated by arrow 4 in NADH 1 mM + Propylgallate scheme 2, since in this case the NADPH0.2 mM specific flavoprotein should reduce cytoNADH 1 mM 1.0 0.8 chain chrome b, in the NADPH-oxidation at a very high rate. The affinity of this DThe experimental conditions are those described flavoprotein to cytochrome b, is known to for Table I. be very high. With such a pathway, the *The average of four experiments. rate of electron transfer from the NADHc The average of six experiments. dependent respiratory chain to the cytoeffect on the reduction of cytochrome P-450 chrome P-450 oxygenase system should be by NADH indicates the existence of an very high, and this is not so. electron-transfer pathway from NADH to Thus, out of all the possible ways of cytochrome P-450 which does not include electron exchange between the two microthe NADPH-specific flavoprotein. The results of these experiments lend NADH NADPH support to our assumption that electron 0.08 A transfer from the NADH-oxidation chain to the NADPH-oxidation chain is accomplished at the site localized behind the NADPH-specific flavoprotein. Consequently, reactions 4, 5, 6 and 8 are indicated in scheme 2 and illustrate possible pathways of electron transfer. THE EFFECT OF PROPYLCALLATE ON THE RATE OF IV-DEMETHYLATION OF DIMETHYLANILINE AND ETHYLMORPHINE IN THE PRESENCE OF NADPH
009
DISCUSSION
The results on the inhibiting action of pronase and propylgallate on the rate of reduction of cytochromes b, and P-450 in the presence of NADPH, as well as the absence of the inhibiting effect of pronase and propylgallate on the NADH-stimulating effect in the hydroxylation reaction, permit the conclusion that at the initial site (reactions 1 and 2, scheme 2) there is no electron transfer from the NADHspecific chain to the NADPH-specific chain.
123
?
4
2
3
4
llme(mln)
FIG. 2. The effect of propylgallate on the redox behavior of cytochromes b, and P-450 reduced by NADPH and NADH using aerobic conditions. Control incubation mixture contained 50 mM Tris-HCI buffer, pH 7.5; 1 mg.ml-’ microsomal protein (1). Experimental incubation mixture was the same as described in Ref. 1 with 200 pM of propylgallate (2). The reaction was initiated by adding of 8 FM NADPH or NADH. Solid line, cytochrome b,. Broken line, cytochrome P-450.
312
ARCHAKOV
somal respiratory chains, the reactions indicated by arrows 3, 5, 6, 7, and 8 in scheme 2 are realistic. It is most probable that electron exchange between t,he chains proceeds at the cytochrome b, level via reactions 6 and 7. However, pathway 5 is not to be ruled out as the potent NADH-Fp,-cytochrome b, reductase system in the cell is responsible for cytochrome b, in the NADH-oxidation chain being always in the fully reduced state (13). In these conditions an excess of NADH may initiate reaction 5: NADH 4 Fp, + cyt. P-450. Ichikawa and Loehr (14) obtained experimental data about a similar enzyme complex existing in microsomes. Thus, the transfer of reducing equivalents from the NADPH-oxidation chain to the NADH-oxidation chain may take place either due to cytochrome b, reduction in the NADH-oxidation chain directly by the NADPH-specific flavoprotein as a result of electrons coming to this cytochrome from cytochrome b, of the NADPH-oxidation chain. The rate of this reaction is equal to that of cytochrome b, reduction in the NADPH-oxidation chain. Three possibilities should be pointed out with regard to electron transfer from the NADH-specific chain to the NADPH-oxidation chain: the first pathway is directly from the NADH-specific flavoprotein to cytochrome P-450, the second, from cytochrome b, of the NADH-oxidation chain to cytochrome b, of the NADPH-oxidation chain, and the third, directly from cytochrome b, of the NADH-oxidation chain to cytochrome P-450. Thus, we may offer a more detailed scheme of the reactions of electron ex-
ET AL.
change between tory chains:
the microsomal
NADPH -
Fp, -
NADH .
Fp, dcyt.
cyt. b, d
respiracyt. P-450
b, ’
REFERENCES 1. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND SEVERINA, v. A. (1969) Biochimia 34, 782-790. 2. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARJAKIN, A. V. (0000) Arch. Biochem. Biophys. 3. ARCHAKOV, A. I., KARUZINA, I. I., KOKAREVA, I. S. AND BACHMANOVA, G. I. (1973) Biochem. J. 136, 371-379. 4. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARUZINA, I. I. (1973) IX Int. Biochem. Cong. 7 B, 22, p. 336. Stockholm. 5. NASH, T. (1953) Biochem. J. 55, 416-421. 6. COHEN, B. S., AND ESTABROOK, R. W. (1971) Arch. Biochem. Biophys. 143, 37-53. 7. ARCHAKOV, A. I., AND DEVICHENSKY, V. M. (0000) Arch. Biochem. Biophys. 8. ORRENIUS, S., BERG, A., AND ERNSTEH, L. (1969) Eur. J. Biochem. 11, 193-200. 9. KRISH, K., AND STAUDINGER, H. (1961) Biochem. 2. 334,312-327. 10. ICHIKAWA, Y., YAMANO, T., AND FUJISHIMA, H. (1969) B&him. Biophys. Acta 171, 32-46. 11. ARCHAKOV, A. I., DEVICHENSKY, V. M., AND KARUZINA, I. I. (1973) Dokl. Akad. Nauk. SSSR 209,973-976. 12. SLATER, T. F., AND TORIELL, M. V. (1971) Biothem. J. 121, 40 P. 13. ARCHAKOV, A. I., KARJAKIN, A. V., AND SKULACHEV, V. P. (1973) Dokl. Akad. Nauk SSSR 209, 221-223. 14. ICHIKAWA, Y., AND LOEHR, J. S. (1972) Biochem. Biophys. Res. Commun. 46, 1187-1193.