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Stereochemistry of hydrogen-transfer in the energy-linked pyridine nucleotide transhydrogenase and related reactions
BIOCHIMICA ET BIOPHYSICA ACTA
397
STEREOCHEMISTRY OF HYDROGEN-TRANSFER IN THE ENERGYLINKED PYRIDINE NUCLEOTIDE TRANSHYDROGENASE AND RELATED REACTIONS* CHUAN-PU LEE, N. SIMARD-DUQUESNE"" AND L. ERNSTER Wenner-Gren Institute, Un iversity of Stockholm, Stockholm (Sweden) AND H. D . HOBERMAN Department of B iochemistry, Albert Einstein College of Medicine , New York, N .Y. (U.S.A .) (Received March 4th, 1965)
SUMMARY
The stereochemistry of hydrogen-transfer in the energy-linked and non-energylinked pyridine nucleotide transhydrogenase, the respiratory chain-linked NADH dehydrogenase, and the DT diaphorase-catalyzed reactions have been investigated with tritiated pyridine nucleotides. Both the energy-linked and the non-energy-linked pyridine nucleotide transhydrogenase reactions, catalyzed by submitochondrial particles from beef heart, involve the 4A hydrogen atom of NADH, and the 413 hydrogen atom of NADPH. The reactions involve no intrinsic exchange of hydrogen atoms between the reduced pyridine nucleotides and water. The aerobic oxidation of NADH, and the energy-linked reduction of NAD+ by succinate, catalyzed by the same particles, involve the 4B hydrogen atom of NADH, and are connected with a rapid exchange of hydrogen atoms between the latter and water. The reaction catalyzed by purified rat-liver DT diaphorase involves the 4A hydrogen atom of either NADH or NADPH, and involves no exchange of hydrogen atoms between the reduced pyridine nucleotides and water. Studies with IdC-labelled NADH indicate that the energy-linked pyridine nucleotide transhydrogenase reaction proceeds without an exchange of the nicotinamide moieties of the two pyridine nuc1eotides. These results are discussed in relation to the mechanism of the energy-linked pyridine nucleotide transhydrogenase reaction.
INTRODUCTION Studies by DANIELSON AND ERNSTER 2- 4 have revealed that submitochondrial particles catalyze an energy-dependent conversion ofNADH and NADP+into NAD+ " Essential parts of this work have already been briefly communicated at An International Symposium on Oxidases and Related Oxidation Reduction Systems, Amherst, Mass., July 15-19, 1964 (ref. I) , and in a verbal report at the Symposium on Bioenergetics, 6th International Congress of Biochemistry, New York, July 26--August I, 1964. Similar results concerning the stereospecificity of the pyrid ine nucleotide transhydrogenase reactions were simultaneously reported by D . E. GRIFF1TFlS, Oxford. "" Present address: Institut de Cardiologic de Montreal, Montreal, P.Q. (Canada).
Biochim. Bjoph)'s. Acta,
T05
(r965) 397-409
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et at.
and NADPH, coupled to the energy-transfer system of the respiratory chain. This reaction, which appears to explain earlier observations reported in the literature'
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATION
399
cuvettes of r-em light-path. The temperature was 30°. Pyridine nucleotide transhydrogenation was carried out 20 by incubating NADH + NADP+ or NAD+ NADPH, suitably labelled, with particles. The reaction was usually terminated by heat-inactivation, and the concentrations of NADH and NADPH were determined by enzymic methods. In the case of the energy-linked transhydrogenase reaction, the energy was supplied either by the simultaneous aerobic oxidation of succinate or by added ATP. Succinate-linked NAD+ reduction was assayed in the presence of ATP as the source of energy and KCN to block NADH oxidase. Details of the single experiments are described in the figure- and table-legends. [14C]NAD+ (specific activity 0.3 fJ-C/fJ-mole) was isolated from extracts of the liver of rats given [7-14C]nicotinamide. [4-3H]NAD+ and [4-3H]NADP+, with specific activities of 0.7 and o.oS ttC/ttmole, respectively, were prepared as described by KRAKOW et al. 21• [r- 3H]Ethanol (specific activity 2S ftC/fJ-mole) was purchased from New England Nuclear Company. [14C]NADH was generated by reduction of [14CJNAD+ with ethanol in the presence of yeast alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.r) and semicarbazide at pH 7.8. The reduction was essentially complete as revealed by the change in absorbancy at 340 mfJ-. The same method was used for the generation of [4B-3H]NADH from [4-3H]NAD+ and unlabelled ethanol (yeast alcohol dehydrogenase being, by convention, A-specific 22). [4A-3H]NADH was generated either by the converse system, using unlabelled NAD+ and [I- 3H]ethanol, or by incubating [4-3H]NAD+ with unlabelled UDPG and the B-specific UDPG dehydrogenase's (UDPglucose :NAD+ oxidoreductase, EC 1.1.1.22). After having established that the succinate-linked NAD+ reduction catalyzed by the submitochondrial particles involves the 4B H-atom of NADH, [4A- 3H ]NADH was also generated by incubating [4-8H]NAD+ and unlabelled succinate with particles in the presence of ATP and KCN. [¢-3H]NADPH and C4B-3H]NADPH were prepared by reduction of [4-3H]NADP+ with unlabelled glucose 6-phosphate + the B-specific glucose-6-phosphate dehydrogenases" (n-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) and with unlabelled isocitrate + the A-specific isocitrate dehydrogenase 24 (Ls-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1-42), respectively. In all cases, the NADH- or NADPH-generating systems were inactivated by heat-denaturation prior to further use of the reduced pyridine nucleotides. In the case of the particle-catalyzed generation of C¢-3H]NADH, addition of rotenone was sufficient, without heat-denaturation of the particles, to terminate the succinatelinked NAD+ reduction, after which the system could be supplemented with NADP+ and used for the study of the transhydrogenase reactions. Radioactivity was measured with a Packard Model 314 EX Tri-Carb liquid scintillation counter, using IS ml of scintillation fluid (containing 4 g 2,s-diphenyloxazole, 40 mg I,4-bis-2-(4-methyl-s-phenyloxazolyl)-benzene, 770 ml toluene and 230 ml ethanol/l) in each bottle. The samples were counted in aliquots of 0.2 ml, Water was isolated from the reaction mixtures in the following way: 0.3 m1 of the mixture was transferred to the side-bulb of a Thunberg tube and frozen by immersion of the bulb in a dry ice-acetone mixture. The tube was evacuated, sealed off from the atmosphere by rotating the side-bulb to the closed position, and the body of the tube then immersed in dry ice-acetone mixture until all of the water from the sidebulb had condensed in the immersed portion of the tube. Localization of 3H in [4-3H]NADH was done by oxidation of the compound
+
Biochim. Biophys. Acta, 105 (1965) 397-409
C-P. LEE et
40 0
at.
with pyruvate and the A-specific lactate dehydrogenasev (L-Iactate:NAD+ oxidoreductase, EC I.I.I.27) and subsequent separation of the NAD+ and lactate formed on a Dowex-r formate column with a three-chamber gradient system of formic acid as the eluent. The same chromatographic system was used for the separation of NAD+ and NADP+ in the experiments with 14C-labelled pyridine nucleotides. [4-3HJNADPH was treated with oxidized glutathione and the B-specific glutathione reductase 26 (NADPH:glutathione oxidoreductase, EC r.6.4.z). The resulting [S-3H]glutathione undergoes instantaneous detritiation. The amount of 3H in water appearing after treatment of the sample with glutathione reductase is thus a measure of the quantity of tritium in [4B- 3H ]NAD PH . All enzymes used for the generation and degradation of reduced pyridine nucleotides were commercial products, except glutathione reductase, which was prepared from Escherichia coli, and kindly supplied by Professor P. REICHARD, Stockholm. RESULTS
Respiratory chain-linked NADH dehydrogenase When submitochondrial particles were incubated aerobically with [¢-3HJNADH or [4B-3H]NADH, the former underwent little or no detritiation, while the latter was rapidly detritiated (Fig. r). When the detritiation of the [4B-3HJNADH was complete, all NADH was oxidized as measured spectrophotometrically. These
'-'
.",
I
30
E
;:-
$2 ~ 20
E
"til C o :;:;
E'
10
tJl
2c
iii
::g I
'"
~"""===l~=r===::!l!.==*==1~ 2
4
6
a
10
16
Time (min)
Fig. I. Detritiation of [4A-3H]NADH (x-x). [4B-3H]NADH (0-0), and of [4-3H]NAD+ reduced by succinate (e-e) by submitochondrial particles from beef heart. [4A.3H]NADH and [4B-3H]NADH were generated from [4·3H]NAD+ by incubation with DDPG + UDPG dehydrogenase and ethanol + alcohol dehydrogenase, respectively (ef. METHODS). After heatinactivation of the NADH-generating systems, submitochondrial particles (0.43 mg protein) were added to a reaction mixture containing 0.18 mM labelled NADH, 200 mM sucrose, 50 mM Tris-acetate buffer, (pH 7.8) in a final volume of 3 ml, Succinate-linked NAD+ reduction was carried out in a reaction mixture containing submitochondrial particles (0.43 mg protein), 0.2 mM labelled NAD+, 5 mM succinate, 2 mM ATP, 10 mM MgSO,j, 1.6 mM KeN, 200 mM sucrose, and 50 mM tris-acetate buffer, (pH 7.8) in a final volume of 3 ml, The reaction was started by the addition of the particles. All incubations were performed at 30°. Aliquots of 0.3 ml were removed at the time intervals indicated, and handled for determination of 3R in H 2 0 as described in METHODS.
Biochim, Biophys. Acta, 105 (1955) 397-409
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATION
4°1
findings are consistent with the conclusion that the NADH dehydrogenase reaction of the respiratory chain involves the 4B H-atom of NADH. The detritiation was not inhibited when the aerobic oxidation of NADH was blocked by cyanide, antimycin A, or rotenone, indicating that the reduced NADH dehydrogenase flavoprotein catalyzes a rapid exchange of H-atoms between NADH and water. As also shown in Fig. I, there occurred no appreciable detritiation of [4-3H} NADH when this was generated from [4-3H]NAD+ through energy-linked reduction with unlabelled succinate. The ATP-supported reduction of [4-3H]NAD+ by succinate was monitored spectrophotometrically. The reduction of the [4-3H]NAD+was virtually complete after 10 min. After heat-denaturation of the enzyme system, and subsequent incubation with pyruvate + lactate dehydrogenase (cf. METHODS), the 3H wasquantitatively recovered in lactate, indicating that the NADH resulting from the succinatelinked NAD+ reduction was labelled in A-position. Thus, the succinate-linked NAD+ reduction involves the 4B hydrogen atom of NADH, a conclusion consistent with the concepts that the reaction proceeds by way of the respiratory chain-linked NADH dehydrogenase, and that the latter enzyme is 4B-specific with respect to NADH. Pyridine nucleotide transhydrogenase reactions
The possibility has been considered-" that the energy-linked reduction of NADP+byNADH might involve a group-transfer (rather than a transfer of hydrogen) , such as a transfer of the z-phosphate group of NADP+, or an exchange of the aclenylate moieties of the two pyridine nucleotides. In either case, the reaction should result in an exchange of the nicotinamide moieties of NADH and NADP+. In order to
U
50 25
4
8
12
16
20
E .g :z: o, o «z
24
Time (min)
Fig. 2. Energy-linked reduction of NADP+ by [4A-SH1NADH. [4A-3H]NADH was generated from [4-3H1NAD+ by reduction with succinate as described in Fig. I, except that 10.5 mg particle protein and a final volume of 2I ml were used. After virtual completion of the reduction of NAD+, the reaction mixture was heated in boiling water-bath for I min, and denatured protein was removed by centrifugation. The supernatant fluid, containing 0.I9 mM [4A-3H]NADH, was supplemented with submitochondrial particles (4·5 mg protein), 3.3,uM rotenone, 2 mM ATP, and 0.3 mM NADP+. The final volume was I8 ml. The reaction was started by the addition of the particles. Temperature was 30°. Aliquots of 3 ml were removed at the time intervals indicated and assayed, as described in METHODS, for: (a) NADPH by the glutathione reductase reaction (dotted line); and (b) for "H in H 2 0 before the glutathione reductase assay (solid line I), and the increment of 3H in H 2 0 after the glutathione reductase assay (solid line II).
Biochim, Biophys, Acta, I05 (1965) 397-409
4°2
C-P. LEE
et ai.
investigate this possibility, the energy-linked pyridine nucleotide interaction was carried out in the presence ofl 4C-labelled NADH generated from [nicotinamide-7-14C} NAD by alcohol dehydrogenase and unlabelled ethanol. When virtually all NADP+ was reduced, an excess of a-ketoglutarate NH 4 + glutamate dehydrogenase (t-glutamate :NAD(P)+ oxidoreductase (deaminating), EC I,4.I.3) was added, whereby both NADH and NADPH were oxidized instantaneously. After chromatographic separation, no radioactivity was found in the fraction containing NADP+, and all radioactivity was recovered in the fraction containing NAD+. It may thus be concluded that the energy-linked pyridine nucleotide interaction involves no grouptransfer of the types considered above, and hence, that it involves a transfer of hydrogen. Data relating to the mechanism of hydrogen transfer in the energy-linked reduction of NADP+ by NADH are shown in Fig. 2. In this experiment [¢_8H]_ NADH was generated by incubating submitochondrial particles with [4-8H]NAD+ and unlabelled succinate in the presence of ATP and KCN. When virtually all NAD+ was reduced, rotenone and more ATP were added (the former in order to block further hydrogen transfer from succinate, and the latter in order to ensure an ample supply of energy for the subsequent NADH-Hnked NADP+ reduction), followed by NADP+. Aliquots were removed at various time intervals and assayed, on one hand, for NADPH by the glutathione reductase reaction (dotted line), and, on the other hand, for 3H in H 20 before (solid line I) and after the glutathione reductase assay; the increment of 3H content in H 20 after the glutathione reductase assay (solid line II) is indicative of 8H in the 4B-Position of NADPH (ef. METHODS). It may be seen that, in the initial phase ofthe energy-dependent transhydrogenase reaction, the formation
+
9. 14
+
....!?..
..SL
I
(
EO 12 ;r..
b ';; 10 c
~B
c
250
/
200 (5
E
::!.
E 150 :;;
Q
....0
100
L
m
I1J
+'
E
-E L .2 I
4
0..
C
iii
50
;§ 2
0
« z
I
'" B
16
32 Time (min)
32
Fig. 3. Energy-linked (a) and non-energy-linked (b) reduction of NADP+ by [4A-SH]NADH. For the energy-linked system, the conditions were as in Fig. 2, except that the heat-treatment following the generation of [4A-SH]N ADH was omitted and no second addition of particles was made. The amount of particles was 7.2 mg protein, and the final volume 18.3 ml. The non-energylinked system contained the same components as the energy-linked one, except that the second addition of ATP was omitted and replaced by the addition of 30 pg oligomycin. The lines in the figure indicate: amount of NADPH formed (dotted line); 3H in H 20 before the glutathione reductase assay (solid line I); and the increment of sH in H 20 after the glutathione reductase assay (solid line II).
Biochim, Biopbys. Acta, 105 (1965) 397-409
STEREOCHEMISTRY OF
NAD-NADP
Tl{A~SHYDROGEI\ATIO~
4°3
of NADPH was quantitatively paralleled by a transfer of 3H from the 4A-position of NADH to the 4B-Position of NADPH. After a short lag-phase, 3H began to appear in H 20, and simultaneously the specific activity of KADPH decreased. Similar results were obtained when [4A-3H]NADH was generated from unlabelled NAD+ and labelled ethanol, or when the energy for the transhydrogenase reaction was supplied by the aerobic oxidation of succinate rather than by added ATP. Fig. 3 compares the stereospecificity of hydrogen-transfer in the energy-linked and non-energy-linked transhydrogenase reactions. The conditions for the energylinked reaction were similar to those in Fig. 2, except that a larger amount of enzyme was used. The non-energy-linked reaction was measured under conditions analogous to those employed for the energy-linked reaction, but the second addition of ATP was omitted, and oligomycin was added in order to inhibit energy supply from ATP possibly present from the first addition. As could be anticipatedw, the rate of formation of NADPH (dotted line) was much slower in the non-energy-linked system than in the energy-linked one, and leveled off when the expected equilibrium was reached. The fate of 3H was the same as in the energy-linked reaction: 3H appeared in the 4Bposition of NADPH (solid line II) parallel to the formation of the latter; and it appeared in water at a slow initial but gradually increasing rate (solid line I). The initial rate of appearance of 3H in water was slower in the non-energy-linked than in the energy-linked system, and it seemed to be proportional to the concentration of NADPH present at any given time. The possible implications ofthese findings will be discussed later (cf. DISCUSSION). The stereospecificity of the non-energy-linked transhydrogenase reaction was also tested in the reverse direction. This was done by incubating submitochondrial particles with [4-SH]NAD+ and unlabelled NADPH. On the basis of the foregoing results, the transhydrogenase reaction in this system should give rise to [4B-3H]NADI-I, which in turn should undergo rapid detritiation via the respiratory chain(3 I
'"
E:
120
24
~
E
.......
o'"
~
•
E
~ 16
80
.........
t '"0
'" c o
E
".;:;
o'-
o
40 .....
5, 8
I
II>
~ Z
+-'
c
~ J:
ro
10
20
30
Time (min)
Fig. 4. Reduction of [4-8HJNAD+ by NADPH. The reaction mixture contained submitochondrial particles (4.2 mg protein), 0.2 mM [4-'HJNAD+, 0.4 m M NADPH, 3.3 f.lM rotenone, 200 mM sucrose, and 50 mM Tris-acetate buffer, (pH 7.8) in a final volume of 21 ml. Temperature, 30°. Aliquots were removed at the time intervals indicated, and heated in boiling water-bath for I min. NADH formed (dotted line) and BI-! in 1-1.0 (solid line) were determined as described in METHODS.
Biochim. Biopbys. Acta.
TO;;
(1965) 397-409
Cop. LEE
et al.
linked NADH dehydrogenase. In Fig. 4 it is shown that this indeed was the case. From the results presented in this section it may thus be concluded that both the energy-linked and the non-energy-linked transhydrogenase reactions involve the 4A H-atom of NADH and the 4B El-atom of NADPH. DT diaphorase
Since it has been suggested'" that the energy-linked transhydrogenase reaction might be related to the pyridine nucleotide-non-specific, highly dicoumarol-sensitive f1avoenzyme, DT diaphorase-", it was of interest to determine the stereospecificity of the hydrogen-transfer catalyzed by this enzyme. Although the submitochondrial particles used in the present studies did contain some DT diaphorasew, its activity was too low to allow accurate determination of the stereospecificity. A purified preparation of DT diaphorase from rat liver was therefore used for this purpose. The enzyme was incubated in the presence of bovine serum albumin as activator-s, and with [4A-3H]NADH or [4B-3H]NADH, or with [4A- 3H ]NADPH or [4B-3HJNADPH as substrate (Table I). No detritiation of any of the labelled pyridine nucleotides occurred in the absence of hydrogen acceptor. When 2,6-dichlorophenol indophenol was added, NADH and NADPH labelled in the ¢-position underwent substantial detritiation, whereas when labelled in the 4B-Position the nucleotides were not significantly detritiated in the course of their oxidation. In complementary experiments TABLE! STEREOSPECtFICITY OF
DT
DIAPHORASE
[4A-3H]NADH and [4B·3H]NADH were generated from [4-3H]NAD+ by UDPG + UDPG dehydrogenase and by ethanol + alcohol dehydrogenase, respectively. [4A-3H]NADPH and [4B·3H]NADPH were generated from [4-3H]NADP+ by glucose 6·phosphate + glucose-6-phosphate dehydrogenase and by isocitrate + isocitrate dehydrogenase, respectively (ej. METHODS). A sample of purified DT diaphorase from rat liver (cf. ref. IS) with an activity of 14,umoles NAD(P)H oxidizedjminjml by 2,6·dichlorophenolindophenol (DCPIP) in the presence of 0.07% bovine serum albumin was used. The reaction mixtures for the assay of DT diaphorase contained, in the case of NADH, 0.094 mM [4A-3H]NADH or 0.13 mM [4B-3H]NADH, 0.07% bovine serum albumin, 20 mM phosphate buffer, (pH 8.S) and 2!tl DT diaphorase, in a final volume of 2.7 ml; and in the case of NADPH, 0.97 mM [4A-3H]NADPH or LI5 mM [4B-3H]NADPH, 0.07% bovine serum albumin, 20 mM phosphate buffer, (pH 7.5) and 10 fJ,1 DT diaphorase, in a final volume of 0.7 ml. The samples were incubated for 5 min, after which aliquots of 0.3 ml were removed for determination of 3H in H 2 0 . To the remainders dichlorophenolindophenolwas added to the NADH system in a final concentration of 0.194 mM, and to the NADPH system in a final concentration of 0.65 mM;' the latter addition was made in 0.13 mM portions in order to minimize inhibition of the enzyme by the oxidized dye (ef. ref. IS). After a further 5 to 7 min of incubation, the samples were frozen and 3H in H 20 was determined as described in METHODS.
Substrate
8H (disintegrations/min
Added as NAD(P)H
[4A-3H]NADH S.OI [4B.8H]NADH II.07 [4A•3H]NADPH 4· I 7 [4B• 3H]NADPH ·4·95
X
Io- 4/ml sample)
Found in H 20 before addition ofDT diaphorase
0·3 I
0.440.5 2 0.67
Biochim. Biophys. Acta, 105 (1965) 397-409
after incubation after incubation with DT with DT diaphorase diaphorase + DCPIP 0.24 0.4 6 0·52 0·7 I
8.7 6 0.7 6
3.5 0 0.69
STEREOCHEMISTRY OF NAD-NADP TRANSHYDROGENATION it was also found that the detritiation of the 4A-labelled reduced pyridine nucleotides paralleled stoichiometrically the reduction of dichlorophenolindophenol, and that it was inhibited by dicoumarol and other inhibitors of DT diaphorase. These results clearly show that the DT diaphorase-catalyzed reaction involves the
105
(I96S) 397-4 09
406
C-P. LEE
et at.
The detritiation observed in connection with the transhydrogenase reactions appears from its kinetics to be secondary and not an intrinsic part of the reaction. The following sequence of reactions seems to provide a reasonable explanation for this detritiatiori:
+ NADP+~NAD+ + [4B·BH}NADPH [4A-BH}NADH + Fp + H+~ [4-BHJNAD+ + FpH B NADPH + [4-3H]NAD+ ~ NADP+ + [4B-3H]NADH [4B-3H]NADH + Fp + H+ ~ NAD+ + [3H]FpH. [BHJFpH. + Hp ~ FpH. + [3H]H.O [4A-BH]NADH
When [4A-8H]NADH is incubated with submitochondrial particles in the presence of NADP+ and a respiratory inhibitor (i.e. under the conditions used here to assay the transhydrogenase reactions), it enters two types of reaction: it reacts with NADP+ giving rise to unlabelled NAD+ and [4B-3H]NADPH (Reaction I); and it equilibrates with NADH-dehydrogenase (Fp), with the formation of a catalytic amount of [4-3H]NAD+ (Reaction 2). The latter reacts with NADPH (formed in the first reaction *J, through the reversed transhydrogenases reaction (cf. Fig. 4), giving rise to [4B-3H]NADH (Reaction 3). This, in turn, will undergo detritiation via NADH dehydrogenase (Reactions 4 and 5). This explanation is consistent with the finding that the rate of detritiation was increasing with time and proportional to the concentration ofNADPH. The difference in stereospecificity between succinate-linked NAD+ reduction and NADH-linked NADP+ reduction with respect to NADH eliminates the possibility that the two reactions might involve a common hydrogen-transfer step at the energized pyridine nucleotide level ("symmetric" and "asymmetric" transhydrogenase) as postulated by HOMMES 38. The latter conclusion has already been criticized on several groundsw, e.g. on the basis of observations with octylguanidine, which inhibits the ATP-supported reduction of NAD+ by succinate but not that of NADP+ by NADH. The identical stereospecificities of the energy-linked and non-energy-linked transhydrogenase reactions favour the view that the two types of reaction involve a common transhydrogenase, This is also supported by the recent findings of KAWASAIG et al.3 9 that antibodies produced against the purified non-energy-linked transhydrogenase suppress the energy-linked transhydrogenase activity of submitochondrial particles. Identity of the enzymes involved in the two types of transhydrogenase reaction has earlier been concluded by ESTABROOK and associates40,41 on the basis of similar activation energies (of the order of 20 kcal/mole) and inhibitions by triiodothyronine. However, as has been pointed out elsewhere30, these features apply to a large number of enzyme reactions, and cannot therefore be accepted as sufficient criteria for establishing the identity of the two enzymes. Assuming that the two types of transhydrogenase reaction involve the same * It is understood that both labelled and unlabelled NADPI-I are formed in Reaction T. In the following only unlabelled NADPH will be considered. since the concentration of the labelled form is, of course, exceedingly small as compared to the unlabelled form. l4B·3H1NADPH might likewise react with [4-BHJNAD-r via Reaction 3, giving rise to [4AB-3H}NADH, which again will undergo detritiation according to Reactions 4 and 5· Biocbim. Biophys. Acta,
105
(19 6 5) 397-409
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATIO~
4°7
transhydrogenase, the question arises as to the difference in the reaction mechanisms. That one enzyme can catalyze two types of reaction is not an uncommon phenomenon. The terminal enzyme in the oxidative phosphorylation sequence, for example, is often considered (cf. ref. 30 for review) to be able to act in two ways: in a coupled reaction, transferring energy from a high-energy intermediate to phosphorylate ADP (or vice versa); and in an uncoupled reaction, as ATPase. The two reactions differ not only in reactants and products but also in thermodynamic equilibrium. In an analogous manner, it may be visualized that the enzyme, NAD-I-·NADP+ transhydrogenase, may act in a coupled anel an uncoupled fashion. The coupled reaction may involve an energized form of either NADH or NADP+ as a reactant, which, when entering hydrogen transfer with the counter-part pyridine nucleotide, simultaneously loses its high-energy character. In equations, the reaction sequence may be written as follows (where INX is a high-energy intermediate of the respiratory chain-linked energytransfer system) : NADH (or NADP+)
+I
'"
NADH", I (or NADP ...... I)
NADH '" I (or NADP '" I) + X + NADP+ (or NADH) -+ NAD+ + NADPH + I
X~
(6) (7)
This reaction thus would differ from the conventional, non-coupled, transhydrogenase reaction: NADPH
+
NAD+ ~ NADP+
+
NADH
(8)
with respect both to reactants and products and to thermodynamic equilibrium. Energized forms ofNADH (refs. 7, 42-44) and NADP+ (ref. 45) have been considered to occur in mitochondria on the basis of various experimental findings. Prompted by recent findings of GRIFFITHS 46 regarding a compound with the probable structure of phosphoryl-NADH, it has also been speculated- that NADH phosphorylated by the 2-phosphate group of NADP+ might be an intermediate in the energy-linked transhydrogenase reaction. This possibility might be investigated in the future by studying the incorporation of 18 0 from water into the 2-phosphate group of NADPH in the course of the energy-linked transhydrogenase reaction", Finally, the previous findings that Mg2+ (refs. 20,40,41) and adenine nucleotidess? are competitive inhibitors of the non-energy-linked, but not of the energy-linked, transhydrogenase reaction, may be explained on the basis of the mechanism discussed here, if one assumes that these compounds compete with NADH or NADP+ but not with NADHNI or NADPNI. Alternatively to the mechanism just considered, the energy-linked transhydrogenase reaction might be explained on the basis of an active transport of pyridine nucleotides as suggested by KLINGENBERG12. It is conceivable, for example, that NAD+ is removed from the site of the transhydrogenase in the particle structure by an active ion transport mechanism: NADtn
+ I",
X -+ NAD 6ut + I
+X
(9)
which thus would maintain the NAD+ concentration at a low level in the vicinity of the enzyme, thereby promoting the rate and extent of NADPH formation by Reaction 8. GREENSPAN AND PURVIS48 have recently reported evidence for an energy• \Ve are indebted to Professor P. D. (el. ref. r].
BOYER
for directing our attention to this possibility
Biochim, BiopJzys. Acta, 105 (1965) 397-409
408
C·P. LEE
et al,
linked exchange between extra- and intra-mitochondrial NAD+ (but not NADP+) which would seem to be in line with the present possibility. Further information on this interesting reaction, and, in general, on the mechanism of active transport in mitochondria and submitochondrial particles (cj. refs. 15 and 30) will be required to settle this question.
ACKNOWLEDGEMENTS
This work has been supported by grants from the Swedish Cancer Society, the Swedish Natural Science Research Council, and the Jane Coffin Childs Memorial Fund for Medical Research. Chuan-Pu Lee is Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, 1963-1965. N. Simard-Duquesne is Recipient of a Schering Travelling Fellowship. H. D. Hoberman was visiting Professor at the Wenner-Gren Institute, University of Stockholm, May-June, 196+ His research was supported in part by Public Health Service Grant CA-03641-07 from the National Cancer Institute, Grant GB-764 from the National Science Foundation, and by a grant from the Commonwealth Fund. REFERENCES I L. ERNSTER, in T. E. KING, H. S. MASON AND M. MORRISON, An Intern. Symp, on Oxidases and Related Oxidation Reduction Systems, Amherst, Mnss., I964, 'Wiley & Sons, New York, in the press. 2 L. DANIELSON AND L. ERNSTER, Biochem, Biophys. Res. Commtm., 10 (1963) 9I. 3 L. DANIELSON AND L. ERNSTER, in B. CHANCE, Johnson Foundation Colloquium on EnergyLinked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 157. 4 L. DANIELSON AND L. ERNSTER, Biochem: Z., 338 (1963) 188. 5 M. KLINGENBERG AND W. SLENCZKA, Biochem: Z., 331 (1959) 486. 6 M. KLINGENBERG AND P. SCHOLLMEYER, in E. C. SLATER. Symp . on Intracellular Respiration. 5th Intern. Congr, of Biochemistry, Moscow, I96I, Vol. 5, Pergamon Press, Oxford, Ig63, p. 46. 7 R. W. ESTABROOK AND S. P. NISSLEY, in P. KARLSON, Symp. iibe» die fttnktionelle und morphologische Orgamisation der Zelle, Rottach-Egern, I962, Springer-Verlag, Heidelberg, 1963, p, IIg. 8 G. E. GLOCK AND P. McLEAN, Biochem. ]., 61 (Ig55) 381, 388. 9 H. A. KREBS AND H. L. KORNBERG, Ergeb. Physiol., 49 (Ig58) 212. 10 T. BUCHER AND M. KLINGENBERG, Angew. Chem., 70 (1958) 552. I f F. DICKENS, G. E. GLOCK AND P. McLEAN, in G. E. W. WOLSTENHOLME AND C. M. O'CONNOR, Ciba Foundation Sym]», on the Regulation of Cell Metabolism, London, I9S8, Churchill, London, 1959, p. IS0. 12 M. KLINGENBERG, in B. CHANCE, Johnson Foundation Colloquium on Energy-Linked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 121. 13 E. C. ST_ATER AND J. M. TAGER, in B. CHANCE, Johnson Foundation Colloquium on EnergyLinked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 97. r4 C. P. LEE, G. F. AZZONE, AND L. ERNSTER, Nature, 201 (1964) 152. IS C. P. LEE AND L. ERNSTER, Biochem, Biophys. Res. Commun., 18 (1965) 523. 16 L. ERNSTER AND C. P. LEE, in Symp. on Bioenergetics, 6th Intern. Congr, of Biochemistry, New York, I964, p. 768. 17 N. O. KAPLAN, S. P. COLOWICK AND E. F. NEUFELD, J. Bioi. Chem., 205 (1953) I. 18 L. ERNSTER, L. DANIELSON AND M. LJUNGGREN, Biochim. Biophys. Acta, 58 (1962) 171. 19 H. Low AND 1. VALLlN, Biochim. Biophys. Acta, 69 (Ig63) 36I. 20 C. P. LEE AND L. ERNSTER, Biochim, Biopbys. Acta, 81 (Ig64) 187. 21 G. KRAKOW, J. LUDOWIEG, J. H. MATHER, W. M. NORMORE, L. TOSl, S. UDAKA AND B. VENNESLAND, Biochemistry, 2 (1963) 1009. 22 B. VENNESLAND, in E. C. SLATER, Symp. on Intracellular Respiration, 5th Intern. Congr. of Biochemistry, Moscow, I96I, Vol. 5, Pergamon Press, Oxford, 1963, p. 38. 23 B. K. STERN AND B. VENNESLAND. ]. Biol. Chem., 235 (1960) 205.
Biochim; Biophys, Acta, l0S (Ig65) 397-409
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NAD-NADP
TRANSHYDROGENATION
24 T. NAKAMOTO AND B. VENNESLAND, j. Bioi. Chem., 235 (1960) 202. 25 F. A. LOEWUS, P.OFNER, H. F. FISHER, F. H. VVESTHEIMER AND B. VENNESLAND, J. Bioi. Chem., 202 (1953) 699. 26 B. K. STERN AND B. VENNESLAND, J. Bioi. cu«, 235 (1960) 209. 27 G. MCGUIRE, L. PESCH AND H. FANNING, Nature, 200 (1963) 71. 28 W. P. CUNNINGHAM, F. L. CRANE AND G. L. SOTTOCASA, personal communication. 29 H. D. HOBERMAN, L. PROSKY, P. G. HEMPSTEAD AND H. W. AFRIN, Biochem; Biophys, Res. Comrnun., 17 (1964) 490. 30 L. ERNSTER AND C. P. LEE, Ann. Rev. Biochem., 33 (1964) 729. 31 O. GAWRON, A. G. GLAID, S. NOBEL AND M. GAN, Biochem. Biopnys. Res. Commun., 16 (1964) 43 2 • 32 G. R. DRYSDALE AND M. COHN, Biochim. Biophys. Acta, 21 (1956) 397. 33 V. MASSEY AND C. VEEGER, Ann. Rev. Biochem., 32 (1963) 579. 34 G. R. DRYSDALE, M. G. SPIEGEL AND P. STRITTMATTER, J. Bioi. cse«, 236 (1961) 2323. 35 P. SRTITTMATTER, j. Bioi. Chem., 237 (1962) 3250. 36 P. STRITTMATTER, J. Bioi. cs-«, 238 (1963) 2213. 37 G. L. SOTTOCASA AND L. ERNSTER, Abstr, and Meeting of the Federation of European Biochemical Societies, Vienna, I96S. 38 F. A. HOMMES, in B. CHANCE, Johnson Foundation Colloquium on Energy-Linked Functions of Mitochondria, Philadelpbia, I963, Academic Press, New York, 19 6 3 , p. 39· 39 T. KAWASAKI, K. SATOH AND N. O. KAPLAN, Biochem, Biophys. Res. Commun., 17 (1964) 64 8 . 40 F. A. HOMMES AND R. VV. ESTABROOK, Biochem, Biopbys. Res. Commun., II (1963) I. 41 R. W. ESTABROOK, F. A. HOMMES AND J. GONZE, in B. CHANCE, Johnson Foundation Colloquium on Energy-Linked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. I43, p. 112. 42 B. CHANCE AND R. G. WILLIAMS, Advan. Eneymol., 17 (1956) 65. 43 B. CHANCE AND H. BALTSCHEFFSKY, ]. Bioi. Chem., 233 (1958) 73 6. 44 R. VV. ESTABROOK, ]. GONZE AND S. P. NISSLEY, Federation Proc., 22 (1963) 1071. 45 J. L. PURVIS, Biochim. Biophys, Acta, 38 (1960) 435· 46 D. E. GRIFFITHS, Federation Proc., 22 (1963) 1064. 47 C. P. LEE AND L. ERNSTER, Abstr. 6th Intern. Congr. of Biochemistry, New York, I9 64, p. 783. 4 8 M. D. GREENSPAN AND J. L. PURVIS, Biochim. Biophys. Acta, 99 (1965) 19 1.
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397
STEREOCHEMISTRY OF HYDROGEN-TRANSFER IN THE ENERGYLINKED PYRIDINE NUCLEOTIDE TRANSHYDROGENASE AND RELATED REACTIONS* CHUAN-PU LEE, N. SIMARD-DUQUESNE"" AND L. ERNSTER Wenner-Gren Institute, Un iversity of Stockholm, Stockholm (Sweden) AND H. D . HOBERMAN Department of B iochemistry, Albert Einstein College of Medicine , New York, N .Y. (U.S.A .) (Received March 4th, 1965)
SUMMARY
The stereochemistry of hydrogen-transfer in the energy-linked and non-energylinked pyridine nucleotide transhydrogenase, the respiratory chain-linked NADH dehydrogenase, and the DT diaphorase-catalyzed reactions have been investigated with tritiated pyridine nucleotides. Both the energy-linked and the non-energy-linked pyridine nucleotide transhydrogenase reactions, catalyzed by submitochondrial particles from beef heart, involve the 4A hydrogen atom of NADH, and the 413 hydrogen atom of NADPH. The reactions involve no intrinsic exchange of hydrogen atoms between the reduced pyridine nucleotides and water. The aerobic oxidation of NADH, and the energy-linked reduction of NAD+ by succinate, catalyzed by the same particles, involve the 4B hydrogen atom of NADH, and are connected with a rapid exchange of hydrogen atoms between the latter and water. The reaction catalyzed by purified rat-liver DT diaphorase involves the 4A hydrogen atom of either NADH or NADPH, and involves no exchange of hydrogen atoms between the reduced pyridine nucleotides and water. Studies with IdC-labelled NADH indicate that the energy-linked pyridine nucleotide transhydrogenase reaction proceeds without an exchange of the nicotinamide moieties of the two pyridine nuc1eotides. These results are discussed in relation to the mechanism of the energy-linked pyridine nucleotide transhydrogenase reaction.
INTRODUCTION Studies by DANIELSON AND ERNSTER 2- 4 have revealed that submitochondrial particles catalyze an energy-dependent conversion ofNADH and NADP+into NAD+ " Essential parts of this work have already been briefly communicated at An International Symposium on Oxidases and Related Oxidation Reduction Systems, Amherst, Mass., July 15-19, 1964 (ref. I) , and in a verbal report at the Symposium on Bioenergetics, 6th International Congress of Biochemistry, New York, July 26--August I, 1964. Similar results concerning the stereospecificity of the pyrid ine nucleotide transhydrogenase reactions were simultaneously reported by D . E. GRIFF1TFlS, Oxford. "" Present address: Institut de Cardiologic de Montreal, Montreal, P.Q. (Canada).
Biochim. Bjoph)'s. Acta,
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(r965) 397-409
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et at.
and NADPH, coupled to the energy-transfer system of the respiratory chain. This reaction, which appears to explain earlier observations reported in the literature'
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATION
399
cuvettes of r-em light-path. The temperature was 30°. Pyridine nucleotide transhydrogenation was carried out 20 by incubating NADH + NADP+ or NAD+ NADPH, suitably labelled, with particles. The reaction was usually terminated by heat-inactivation, and the concentrations of NADH and NADPH were determined by enzymic methods. In the case of the energy-linked transhydrogenase reaction, the energy was supplied either by the simultaneous aerobic oxidation of succinate or by added ATP. Succinate-linked NAD+ reduction was assayed in the presence of ATP as the source of energy and KCN to block NADH oxidase. Details of the single experiments are described in the figure- and table-legends. [14C]NAD+ (specific activity 0.3 fJ-C/fJ-mole) was isolated from extracts of the liver of rats given [7-14C]nicotinamide. [4-3H]NAD+ and [4-3H]NADP+, with specific activities of 0.7 and o.oS ttC/ttmole, respectively, were prepared as described by KRAKOW et al. 21• [r- 3H]Ethanol (specific activity 2S ftC/fJ-mole) was purchased from New England Nuclear Company. [14C]NADH was generated by reduction of [14CJNAD+ with ethanol in the presence of yeast alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.r) and semicarbazide at pH 7.8. The reduction was essentially complete as revealed by the change in absorbancy at 340 mfJ-. The same method was used for the generation of [4B-3H]NADH from [4-3H]NAD+ and unlabelled ethanol (yeast alcohol dehydrogenase being, by convention, A-specific 22). [4A-3H]NADH was generated either by the converse system, using unlabelled NAD+ and [I- 3H]ethanol, or by incubating [4-3H]NAD+ with unlabelled UDPG and the B-specific UDPG dehydrogenase's (UDPglucose :NAD+ oxidoreductase, EC 1.1.1.22). After having established that the succinate-linked NAD+ reduction catalyzed by the submitochondrial particles involves the 4B H-atom of NADH, [4A- 3H ]NADH was also generated by incubating [4-8H]NAD+ and unlabelled succinate with particles in the presence of ATP and KCN. [¢-3H]NADPH and C4B-3H]NADPH were prepared by reduction of [4-3H]NADP+ with unlabelled glucose 6-phosphate + the B-specific glucose-6-phosphate dehydrogenases" (n-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) and with unlabelled isocitrate + the A-specific isocitrate dehydrogenase 24 (Ls-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1-42), respectively. In all cases, the NADH- or NADPH-generating systems were inactivated by heat-denaturation prior to further use of the reduced pyridine nucleotides. In the case of the particle-catalyzed generation of C¢-3H]NADH, addition of rotenone was sufficient, without heat-denaturation of the particles, to terminate the succinatelinked NAD+ reduction, after which the system could be supplemented with NADP+ and used for the study of the transhydrogenase reactions. Radioactivity was measured with a Packard Model 314 EX Tri-Carb liquid scintillation counter, using IS ml of scintillation fluid (containing 4 g 2,s-diphenyloxazole, 40 mg I,4-bis-2-(4-methyl-s-phenyloxazolyl)-benzene, 770 ml toluene and 230 ml ethanol/l) in each bottle. The samples were counted in aliquots of 0.2 ml, Water was isolated from the reaction mixtures in the following way: 0.3 m1 of the mixture was transferred to the side-bulb of a Thunberg tube and frozen by immersion of the bulb in a dry ice-acetone mixture. The tube was evacuated, sealed off from the atmosphere by rotating the side-bulb to the closed position, and the body of the tube then immersed in dry ice-acetone mixture until all of the water from the sidebulb had condensed in the immersed portion of the tube. Localization of 3H in [4-3H]NADH was done by oxidation of the compound
+
Biochim. Biophys. Acta, 105 (1965) 397-409
C-P. LEE et
40 0
at.
with pyruvate and the A-specific lactate dehydrogenasev (L-Iactate:NAD+ oxidoreductase, EC I.I.I.27) and subsequent separation of the NAD+ and lactate formed on a Dowex-r formate column with a three-chamber gradient system of formic acid as the eluent. The same chromatographic system was used for the separation of NAD+ and NADP+ in the experiments with 14C-labelled pyridine nucleotides. [4-3HJNADPH was treated with oxidized glutathione and the B-specific glutathione reductase 26 (NADPH:glutathione oxidoreductase, EC r.6.4.z). The resulting [S-3H]glutathione undergoes instantaneous detritiation. The amount of 3H in water appearing after treatment of the sample with glutathione reductase is thus a measure of the quantity of tritium in [4B- 3H ]NAD PH . All enzymes used for the generation and degradation of reduced pyridine nucleotides were commercial products, except glutathione reductase, which was prepared from Escherichia coli, and kindly supplied by Professor P. REICHARD, Stockholm. RESULTS
Respiratory chain-linked NADH dehydrogenase When submitochondrial particles were incubated aerobically with [¢-3HJNADH or [4B-3H]NADH, the former underwent little or no detritiation, while the latter was rapidly detritiated (Fig. r). When the detritiation of the [4B-3HJNADH was complete, all NADH was oxidized as measured spectrophotometrically. These
'-'
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I
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4
6
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16
Time (min)
Fig. I. Detritiation of [4A-3H]NADH (x-x). [4B-3H]NADH (0-0), and of [4-3H]NAD+ reduced by succinate (e-e) by submitochondrial particles from beef heart. [4A.3H]NADH and [4B-3H]NADH were generated from [4·3H]NAD+ by incubation with DDPG + UDPG dehydrogenase and ethanol + alcohol dehydrogenase, respectively (ef. METHODS). After heatinactivation of the NADH-generating systems, submitochondrial particles (0.43 mg protein) were added to a reaction mixture containing 0.18 mM labelled NADH, 200 mM sucrose, 50 mM Tris-acetate buffer, (pH 7.8) in a final volume of 3 ml, Succinate-linked NAD+ reduction was carried out in a reaction mixture containing submitochondrial particles (0.43 mg protein), 0.2 mM labelled NAD+, 5 mM succinate, 2 mM ATP, 10 mM MgSO,j, 1.6 mM KeN, 200 mM sucrose, and 50 mM tris-acetate buffer, (pH 7.8) in a final volume of 3 ml, The reaction was started by the addition of the particles. All incubations were performed at 30°. Aliquots of 0.3 ml were removed at the time intervals indicated, and handled for determination of 3R in H 2 0 as described in METHODS.
Biochim, Biophys. Acta, 105 (1955) 397-409
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATION
4°1
findings are consistent with the conclusion that the NADH dehydrogenase reaction of the respiratory chain involves the 4B H-atom of NADH. The detritiation was not inhibited when the aerobic oxidation of NADH was blocked by cyanide, antimycin A, or rotenone, indicating that the reduced NADH dehydrogenase flavoprotein catalyzes a rapid exchange of H-atoms between NADH and water. As also shown in Fig. I, there occurred no appreciable detritiation of [4-3H} NADH when this was generated from [4-3H]NAD+ through energy-linked reduction with unlabelled succinate. The ATP-supported reduction of [4-3H]NAD+ by succinate was monitored spectrophotometrically. The reduction of the [4-3H]NAD+was virtually complete after 10 min. After heat-denaturation of the enzyme system, and subsequent incubation with pyruvate + lactate dehydrogenase (cf. METHODS), the 3H wasquantitatively recovered in lactate, indicating that the NADH resulting from the succinatelinked NAD+ reduction was labelled in A-position. Thus, the succinate-linked NAD+ reduction involves the 4B hydrogen atom of NADH, a conclusion consistent with the concepts that the reaction proceeds by way of the respiratory chain-linked NADH dehydrogenase, and that the latter enzyme is 4B-specific with respect to NADH. Pyridine nucleotide transhydrogenase reactions
The possibility has been considered-" that the energy-linked reduction of NADP+byNADH might involve a group-transfer (rather than a transfer of hydrogen) , such as a transfer of the z-phosphate group of NADP+, or an exchange of the aclenylate moieties of the two pyridine nucleotides. In either case, the reaction should result in an exchange of the nicotinamide moieties of NADH and NADP+. In order to
U
50 25
4
8
12
16
20
E .g :z: o, o «z
24
Time (min)
Fig. 2. Energy-linked reduction of NADP+ by [4A-SH1NADH. [4A-3H]NADH was generated from [4-3H1NAD+ by reduction with succinate as described in Fig. I, except that 10.5 mg particle protein and a final volume of 2I ml were used. After virtual completion of the reduction of NAD+, the reaction mixture was heated in boiling water-bath for I min, and denatured protein was removed by centrifugation. The supernatant fluid, containing 0.I9 mM [4A-3H]NADH, was supplemented with submitochondrial particles (4·5 mg protein), 3.3,uM rotenone, 2 mM ATP, and 0.3 mM NADP+. The final volume was I8 ml. The reaction was started by the addition of the particles. Temperature was 30°. Aliquots of 3 ml were removed at the time intervals indicated and assayed, as described in METHODS, for: (a) NADPH by the glutathione reductase reaction (dotted line); and (b) for "H in H 2 0 before the glutathione reductase assay (solid line I), and the increment of 3H in H 2 0 after the glutathione reductase assay (solid line II).
Biochim, Biophys, Acta, I05 (1965) 397-409
4°2
C-P. LEE
et ai.
investigate this possibility, the energy-linked pyridine nucleotide interaction was carried out in the presence ofl 4C-labelled NADH generated from [nicotinamide-7-14C} NAD by alcohol dehydrogenase and unlabelled ethanol. When virtually all NADP+ was reduced, an excess of a-ketoglutarate NH 4 + glutamate dehydrogenase (t-glutamate :NAD(P)+ oxidoreductase (deaminating), EC I,4.I.3) was added, whereby both NADH and NADPH were oxidized instantaneously. After chromatographic separation, no radioactivity was found in the fraction containing NADP+, and all radioactivity was recovered in the fraction containing NAD+. It may thus be concluded that the energy-linked pyridine nucleotide interaction involves no grouptransfer of the types considered above, and hence, that it involves a transfer of hydrogen. Data relating to the mechanism of hydrogen transfer in the energy-linked reduction of NADP+ by NADH are shown in Fig. 2. In this experiment [¢_8H]_ NADH was generated by incubating submitochondrial particles with [4-8H]NAD+ and unlabelled succinate in the presence of ATP and KCN. When virtually all NAD+ was reduced, rotenone and more ATP were added (the former in order to block further hydrogen transfer from succinate, and the latter in order to ensure an ample supply of energy for the subsequent NADH-Hnked NADP+ reduction), followed by NADP+. Aliquots were removed at various time intervals and assayed, on one hand, for NADPH by the glutathione reductase reaction (dotted line), and, on the other hand, for 3H in H 20 before (solid line I) and after the glutathione reductase assay; the increment of 3H content in H 20 after the glutathione reductase assay (solid line II) is indicative of 8H in the 4B-Position of NADPH (ef. METHODS). It may be seen that, in the initial phase ofthe energy-dependent transhydrogenase reaction, the formation
+
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32
Fig. 3. Energy-linked (a) and non-energy-linked (b) reduction of NADP+ by [4A-SH]NADH. For the energy-linked system, the conditions were as in Fig. 2, except that the heat-treatment following the generation of [4A-SH]N ADH was omitted and no second addition of particles was made. The amount of particles was 7.2 mg protein, and the final volume 18.3 ml. The non-energylinked system contained the same components as the energy-linked one, except that the second addition of ATP was omitted and replaced by the addition of 30 pg oligomycin. The lines in the figure indicate: amount of NADPH formed (dotted line); 3H in H 20 before the glutathione reductase assay (solid line I); and the increment of sH in H 20 after the glutathione reductase assay (solid line II).
Biochim, Biopbys. Acta, 105 (1965) 397-409
STEREOCHEMISTRY OF
NAD-NADP
Tl{A~SHYDROGEI\ATIO~
4°3
of NADPH was quantitatively paralleled by a transfer of 3H from the 4A-position of NADH to the 4B-Position of NADPH. After a short lag-phase, 3H began to appear in H 20, and simultaneously the specific activity of KADPH decreased. Similar results were obtained when [4A-3H]NADH was generated from unlabelled NAD+ and labelled ethanol, or when the energy for the transhydrogenase reaction was supplied by the aerobic oxidation of succinate rather than by added ATP. Fig. 3 compares the stereospecificity of hydrogen-transfer in the energy-linked and non-energy-linked transhydrogenase reactions. The conditions for the energylinked reaction were similar to those in Fig. 2, except that a larger amount of enzyme was used. The non-energy-linked reaction was measured under conditions analogous to those employed for the energy-linked reaction, but the second addition of ATP was omitted, and oligomycin was added in order to inhibit energy supply from ATP possibly present from the first addition. As could be anticipatedw, the rate of formation of NADPH (dotted line) was much slower in the non-energy-linked system than in the energy-linked one, and leveled off when the expected equilibrium was reached. The fate of 3H was the same as in the energy-linked reaction: 3H appeared in the 4Bposition of NADPH (solid line II) parallel to the formation of the latter; and it appeared in water at a slow initial but gradually increasing rate (solid line I). The initial rate of appearance of 3H in water was slower in the non-energy-linked than in the energy-linked system, and it seemed to be proportional to the concentration of NADPH present at any given time. The possible implications ofthese findings will be discussed later (cf. DISCUSSION). The stereospecificity of the non-energy-linked transhydrogenase reaction was also tested in the reverse direction. This was done by incubating submitochondrial particles with [4-SH]NAD+ and unlabelled NADPH. On the basis of the foregoing results, the transhydrogenase reaction in this system should give rise to [4B-3H]NADI-I, which in turn should undergo rapid detritiation via the respiratory chain(3 I
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Fig. 4. Reduction of [4-8HJNAD+ by NADPH. The reaction mixture contained submitochondrial particles (4.2 mg protein), 0.2 mM [4-'HJNAD+, 0.4 m M NADPH, 3.3 f.lM rotenone, 200 mM sucrose, and 50 mM Tris-acetate buffer, (pH 7.8) in a final volume of 21 ml. Temperature, 30°. Aliquots were removed at the time intervals indicated, and heated in boiling water-bath for I min. NADH formed (dotted line) and BI-! in 1-1.0 (solid line) were determined as described in METHODS.
Biochim. Biopbys. Acta.
TO;;
(1965) 397-409
Cop. LEE
et al.
linked NADH dehydrogenase. In Fig. 4 it is shown that this indeed was the case. From the results presented in this section it may thus be concluded that both the energy-linked and the non-energy-linked transhydrogenase reactions involve the 4A H-atom of NADH and the 4B El-atom of NADPH. DT diaphorase
Since it has been suggested'" that the energy-linked transhydrogenase reaction might be related to the pyridine nucleotide-non-specific, highly dicoumarol-sensitive f1avoenzyme, DT diaphorase-", it was of interest to determine the stereospecificity of the hydrogen-transfer catalyzed by this enzyme. Although the submitochondrial particles used in the present studies did contain some DT diaphorasew, its activity was too low to allow accurate determination of the stereospecificity. A purified preparation of DT diaphorase from rat liver was therefore used for this purpose. The enzyme was incubated in the presence of bovine serum albumin as activator-s, and with [4A-3H]NADH or [4B-3H]NADH, or with [4A- 3H ]NADPH or [4B-3HJNADPH as substrate (Table I). No detritiation of any of the labelled pyridine nucleotides occurred in the absence of hydrogen acceptor. When 2,6-dichlorophenol indophenol was added, NADH and NADPH labelled in the ¢-position underwent substantial detritiation, whereas when labelled in the 4B-Position the nucleotides were not significantly detritiated in the course of their oxidation. In complementary experiments TABLE! STEREOSPECtFICITY OF
DT
DIAPHORASE
[4A-3H]NADH and [4B·3H]NADH were generated from [4-3H]NAD+ by UDPG + UDPG dehydrogenase and by ethanol + alcohol dehydrogenase, respectively. [4A-3H]NADPH and [4B·3H]NADPH were generated from [4-3H]NADP+ by glucose 6·phosphate + glucose-6-phosphate dehydrogenase and by isocitrate + isocitrate dehydrogenase, respectively (ej. METHODS). A sample of purified DT diaphorase from rat liver (cf. ref. IS) with an activity of 14,umoles NAD(P)H oxidizedjminjml by 2,6·dichlorophenolindophenol (DCPIP) in the presence of 0.07% bovine serum albumin was used. The reaction mixtures for the assay of DT diaphorase contained, in the case of NADH, 0.094 mM [4A-3H]NADH or 0.13 mM [4B-3H]NADH, 0.07% bovine serum albumin, 20 mM phosphate buffer, (pH 8.S) and 2!tl DT diaphorase, in a final volume of 2.7 ml; and in the case of NADPH, 0.97 mM [4A-3H]NADPH or LI5 mM [4B-3H]NADPH, 0.07% bovine serum albumin, 20 mM phosphate buffer, (pH 7.5) and 10 fJ,1 DT diaphorase, in a final volume of 0.7 ml. The samples were incubated for 5 min, after which aliquots of 0.3 ml were removed for determination of 3H in H 2 0 . To the remainders dichlorophenolindophenolwas added to the NADH system in a final concentration of 0.194 mM, and to the NADPH system in a final concentration of 0.65 mM;' the latter addition was made in 0.13 mM portions in order to minimize inhibition of the enzyme by the oxidized dye (ef. ref. IS). After a further 5 to 7 min of incubation, the samples were frozen and 3H in H 20 was determined as described in METHODS.
Substrate
8H (disintegrations/min
Added as NAD(P)H
[4A-3H]NADH S.OI [4B.8H]NADH II.07 [4A•3H]NADPH 4· I 7 [4B• 3H]NADPH ·4·95
X
Io- 4/ml sample)
Found in H 20 before addition ofDT diaphorase
0·3 I
0.440.5 2 0.67
Biochim. Biophys. Acta, 105 (1965) 397-409
after incubation after incubation with DT with DT diaphorase diaphorase + DCPIP 0.24 0.4 6 0·52 0·7 I
8.7 6 0.7 6
3.5 0 0.69
STEREOCHEMISTRY OF NAD-NADP TRANSHYDROGENATION it was also found that the detritiation of the 4A-labelled reduced pyridine nucleotides paralleled stoichiometrically the reduction of dichlorophenolindophenol, and that it was inhibited by dicoumarol and other inhibitors of DT diaphorase. These results clearly show that the DT diaphorase-catalyzed reaction involves the
105
(I96S) 397-4 09
406
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et at.
The detritiation observed in connection with the transhydrogenase reactions appears from its kinetics to be secondary and not an intrinsic part of the reaction. The following sequence of reactions seems to provide a reasonable explanation for this detritiatiori:
+ NADP+~NAD+ + [4B·BH}NADPH [4A-BH}NADH + Fp + H+~ [4-BHJNAD+ + FpH B NADPH + [4-3H]NAD+ ~ NADP+ + [4B-3H]NADH [4B-3H]NADH + Fp + H+ ~ NAD+ + [3H]FpH. [BHJFpH. + Hp ~ FpH. + [3H]H.O [4A-BH]NADH
When [4A-8H]NADH is incubated with submitochondrial particles in the presence of NADP+ and a respiratory inhibitor (i.e. under the conditions used here to assay the transhydrogenase reactions), it enters two types of reaction: it reacts with NADP+ giving rise to unlabelled NAD+ and [4B-3H]NADPH (Reaction I); and it equilibrates with NADH-dehydrogenase (Fp), with the formation of a catalytic amount of [4-3H]NAD+ (Reaction 2). The latter reacts with NADPH (formed in the first reaction *J, through the reversed transhydrogenases reaction (cf. Fig. 4), giving rise to [4B-3H]NADH (Reaction 3). This, in turn, will undergo detritiation via NADH dehydrogenase (Reactions 4 and 5). This explanation is consistent with the finding that the rate of detritiation was increasing with time and proportional to the concentration ofNADPH. The difference in stereospecificity between succinate-linked NAD+ reduction and NADH-linked NADP+ reduction with respect to NADH eliminates the possibility that the two reactions might involve a common hydrogen-transfer step at the energized pyridine nucleotide level ("symmetric" and "asymmetric" transhydrogenase) as postulated by HOMMES 38. The latter conclusion has already been criticized on several groundsw, e.g. on the basis of observations with octylguanidine, which inhibits the ATP-supported reduction of NAD+ by succinate but not that of NADP+ by NADH. The identical stereospecificities of the energy-linked and non-energy-linked transhydrogenase reactions favour the view that the two types of reaction involve a common transhydrogenase, This is also supported by the recent findings of KAWASAIG et al.3 9 that antibodies produced against the purified non-energy-linked transhydrogenase suppress the energy-linked transhydrogenase activity of submitochondrial particles. Identity of the enzymes involved in the two types of transhydrogenase reaction has earlier been concluded by ESTABROOK and associates40,41 on the basis of similar activation energies (of the order of 20 kcal/mole) and inhibitions by triiodothyronine. However, as has been pointed out elsewhere30, these features apply to a large number of enzyme reactions, and cannot therefore be accepted as sufficient criteria for establishing the identity of the two enzymes. Assuming that the two types of transhydrogenase reaction involve the same * It is understood that both labelled and unlabelled NADPI-I are formed in Reaction T. In the following only unlabelled NADPH will be considered. since the concentration of the labelled form is, of course, exceedingly small as compared to the unlabelled form. l4B·3H1NADPH might likewise react with [4-BHJNAD-r via Reaction 3, giving rise to [4AB-3H}NADH, which again will undergo detritiation according to Reactions 4 and 5· Biocbim. Biophys. Acta,
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(19 6 5) 397-409
STEREOCHEMISTRY OF
NAD-NADP
TRANSHYDROGENATIO~
4°7
transhydrogenase, the question arises as to the difference in the reaction mechanisms. That one enzyme can catalyze two types of reaction is not an uncommon phenomenon. The terminal enzyme in the oxidative phosphorylation sequence, for example, is often considered (cf. ref. 30 for review) to be able to act in two ways: in a coupled reaction, transferring energy from a high-energy intermediate to phosphorylate ADP (or vice versa); and in an uncoupled reaction, as ATPase. The two reactions differ not only in reactants and products but also in thermodynamic equilibrium. In an analogous manner, it may be visualized that the enzyme, NAD-I-·NADP+ transhydrogenase, may act in a coupled anel an uncoupled fashion. The coupled reaction may involve an energized form of either NADH or NADP+ as a reactant, which, when entering hydrogen transfer with the counter-part pyridine nucleotide, simultaneously loses its high-energy character. In equations, the reaction sequence may be written as follows (where INX is a high-energy intermediate of the respiratory chain-linked energytransfer system) : NADH (or NADP+)
+I
'"
NADH", I (or NADP ...... I)
NADH '" I (or NADP '" I) + X + NADP+ (or NADH) -+ NAD+ + NADPH + I
X~
(6) (7)
This reaction thus would differ from the conventional, non-coupled, transhydrogenase reaction: NADPH
+
NAD+ ~ NADP+
+
NADH
(8)
with respect both to reactants and products and to thermodynamic equilibrium. Energized forms ofNADH (refs. 7, 42-44) and NADP+ (ref. 45) have been considered to occur in mitochondria on the basis of various experimental findings. Prompted by recent findings of GRIFFITHS 46 regarding a compound with the probable structure of phosphoryl-NADH, it has also been speculated- that NADH phosphorylated by the 2-phosphate group of NADP+ might be an intermediate in the energy-linked transhydrogenase reaction. This possibility might be investigated in the future by studying the incorporation of 18 0 from water into the 2-phosphate group of NADPH in the course of the energy-linked transhydrogenase reaction", Finally, the previous findings that Mg2+ (refs. 20,40,41) and adenine nucleotidess? are competitive inhibitors of the non-energy-linked, but not of the energy-linked, transhydrogenase reaction, may be explained on the basis of the mechanism discussed here, if one assumes that these compounds compete with NADH or NADP+ but not with NADHNI or NADPNI. Alternatively to the mechanism just considered, the energy-linked transhydrogenase reaction might be explained on the basis of an active transport of pyridine nucleotides as suggested by KLINGENBERG12. It is conceivable, for example, that NAD+ is removed from the site of the transhydrogenase in the particle structure by an active ion transport mechanism: NADtn
+ I",
X -+ NAD 6ut + I
+X
(9)
which thus would maintain the NAD+ concentration at a low level in the vicinity of the enzyme, thereby promoting the rate and extent of NADPH formation by Reaction 8. GREENSPAN AND PURVIS48 have recently reported evidence for an energy• \Ve are indebted to Professor P. D. (el. ref. r].
BOYER
for directing our attention to this possibility
Biochim, BiopJzys. Acta, 105 (1965) 397-409
408
C·P. LEE
et al,
linked exchange between extra- and intra-mitochondrial NAD+ (but not NADP+) which would seem to be in line with the present possibility. Further information on this interesting reaction, and, in general, on the mechanism of active transport in mitochondria and submitochondrial particles (cj. refs. 15 and 30) will be required to settle this question.
ACKNOWLEDGEMENTS
This work has been supported by grants from the Swedish Cancer Society, the Swedish Natural Science Research Council, and the Jane Coffin Childs Memorial Fund for Medical Research. Chuan-Pu Lee is Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, 1963-1965. N. Simard-Duquesne is Recipient of a Schering Travelling Fellowship. H. D. Hoberman was visiting Professor at the Wenner-Gren Institute, University of Stockholm, May-June, 196+ His research was supported in part by Public Health Service Grant CA-03641-07 from the National Cancer Institute, Grant GB-764 from the National Science Foundation, and by a grant from the Commonwealth Fund. REFERENCES I L. ERNSTER, in T. E. KING, H. S. MASON AND M. MORRISON, An Intern. Symp, on Oxidases and Related Oxidation Reduction Systems, Amherst, Mnss., I964, 'Wiley & Sons, New York, in the press. 2 L. DANIELSON AND L. ERNSTER, Biochem, Biophys. Res. Commtm., 10 (1963) 9I. 3 L. DANIELSON AND L. ERNSTER, in B. CHANCE, Johnson Foundation Colloquium on EnergyLinked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 157. 4 L. DANIELSON AND L. ERNSTER, Biochem: Z., 338 (1963) 188. 5 M. KLINGENBERG AND W. SLENCZKA, Biochem: Z., 331 (1959) 486. 6 M. KLINGENBERG AND P. SCHOLLMEYER, in E. C. SLATER. Symp . on Intracellular Respiration. 5th Intern. Congr, of Biochemistry, Moscow, I96I, Vol. 5, Pergamon Press, Oxford, Ig63, p. 46. 7 R. W. ESTABROOK AND S. P. NISSLEY, in P. KARLSON, Symp. iibe» die fttnktionelle und morphologische Orgamisation der Zelle, Rottach-Egern, I962, Springer-Verlag, Heidelberg, 1963, p, IIg. 8 G. E. GLOCK AND P. McLEAN, Biochem. ]., 61 (Ig55) 381, 388. 9 H. A. KREBS AND H. L. KORNBERG, Ergeb. Physiol., 49 (Ig58) 212. 10 T. BUCHER AND M. KLINGENBERG, Angew. Chem., 70 (1958) 552. I f F. DICKENS, G. E. GLOCK AND P. McLEAN, in G. E. W. WOLSTENHOLME AND C. M. O'CONNOR, Ciba Foundation Sym]», on the Regulation of Cell Metabolism, London, I9S8, Churchill, London, 1959, p. IS0. 12 M. KLINGENBERG, in B. CHANCE, Johnson Foundation Colloquium on Energy-Linked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 121. 13 E. C. ST_ATER AND J. M. TAGER, in B. CHANCE, Johnson Foundation Colloquium on EnergyLinked Functions of Mitochondria, Philadelphia, I963, Academic Press, New York, 1963, p. 97. r4 C. P. LEE, G. F. AZZONE, AND L. ERNSTER, Nature, 201 (1964) 152. IS C. P. LEE AND L. ERNSTER, Biochem, Biophys. Res. Commun., 18 (1965) 523. 16 L. ERNSTER AND C. P. LEE, in Symp. on Bioenergetics, 6th Intern. Congr, of Biochemistry, New York, I964, p. 768. 17 N. O. KAPLAN, S. P. COLOWICK AND E. F. NEUFELD, J. Bioi. Chem., 205 (1953) I. 18 L. ERNSTER, L. DANIELSON AND M. LJUNGGREN, Biochim. Biophys. Acta, 58 (1962) 171. 19 H. Low AND 1. VALLlN, Biochim. Biophys. Acta, 69 (Ig63) 36I. 20 C. P. LEE AND L. ERNSTER, Biochim, Biopbys. Acta, 81 (Ig64) 187. 21 G. KRAKOW, J. LUDOWIEG, J. H. MATHER, W. M. NORMORE, L. TOSl, S. UDAKA AND B. VENNESLAND, Biochemistry, 2 (1963) 1009. 22 B. VENNESLAND, in E. C. SLATER, Symp. on Intracellular Respiration, 5th Intern. Congr. of Biochemistry, Moscow, I96I, Vol. 5, Pergamon Press, Oxford, 1963, p. 38. 23 B. K. STERN AND B. VENNESLAND. ]. Biol. Chem., 235 (1960) 205.
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TRANSHYDROGENATION
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