BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 846-851
Vol. 176, No. 2, 1991 April 30, 1991
Steady-State Kinetics of Autoxidation of NAD(P)H Initiated by Hydroperoxyl Radical, the Acid Form of Superoxide Anion Radical Ken Fujimori* and Hiromitsu Nakajima Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, 305, Japan Received March 14, 1991
Summary: Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 -I- 0.13 x 104 M-is -1 and 9.26 4-_0.58 x 104 M-is -1 at 25°C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated. ~ 1991Ao~demioP..... ~,~o.
Numerous studies have been made on chemical reactivities of superoxide during the last two decades, 1~ because of the presence of much chances to produce superoxide in a living organism, e.g. NADPH oxidase dependent respiratory burst at phagocytes, 2) leak of electron from flavoenzymes to molecular oxygen, 3~ oxidation of xanthine by xanthine oxidase (XO) 4) etc.
On the other hand, superoxide initiated
autoxidation of NADH was postulated in the HRP clock reaction by Yamazaki et al.,~ while Land and Swallow found that superoxide anion radical is practically inert to NADH (second-order rate constant of the reaction between 02- and NADH < 27 M-is -1 ).6) Later, Dunford et al. demonstrated that hydroperoxyl radical, the acid form of O2-, * To whom correspondence should be addressed.
Abbreviations NAD(P)H: NADPH and NADH, NAD(P)+: NADP+ and NAD +, NAD(P)': NADP" and NAD °, NAD(P)O2°: NADPO2 ° and NADO2 °, NAD(P)O2H: NADPO2H and NADO2H, G-6-P: glucose-6-phosphate, G-6-P DH: glucose-6-phosphate dehydrogenase, SOD: superoxide dismutase, XO: xanthine oxidase, Gill: ferricytochrome C, Oil: ferrocytochrome C, Ri: rate of superoxide production by xanthine/XO system. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
846
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
reacts with NADH (k2 = 1.8 x 105 M-is -1 at 20°C). 7~ Although NADPH and NADH (NAD(P)H) are often electron donors to molecular oxygen generating superoxide in vivo,2. ~) no steady-state kinetics has been reported for superoxide initiated autoxidation of NAD(P)H. promoted
by hydroperoxyl
Thus, we investigated the autoxidation of NAD(P)H radical which was generated continuously with
xanthine/XO system. Materials and Methods
NADPH, NADP +, NAD +, D-glucose-6-phosphate (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-P DH; EC 1.1.1.49, from leuconostoc mesenteroides) were purchased from Oriental Yeast. Superoxide dismutase (SOD; Cu, Zn type, EC 1.15.1.1) and NADH were from Wako Pure Chemical Ind. Xanthine sodium salt, butter milk xanthine oxidase (XO; EC 1.1.3.22, grade III, Lot 69F-3779), horse heart cytochrome C (type III) and deferoxamine were obtained from Sigma. And the other chemicals used were of special grade. UV-visible spectra were recorded on JASCO Ubest-50 spectrophotometer equipped with thermostatic control. Kinetics of oxidation of NAD(P)H were carried out at 25°C under aerobic conditions. XO assay: Buffer solution (0.1 M potassium phosphate, 200 gM xanthine, pH 7.4, 500 Ixl) containing various concentrations of ferricytochrome C (C m) was placed in a UV-cuvette. The reaction was started by adding XO, and recorded increasing absorbance at 550 nm due to ferrocytochrome C (Cll). Under the condition XO converted 114 nM xanthine to uric acid per sec. The fate of O2- generated in the system may be expressed by Scheme 1, in which superoxide disappears via three paths, reaction with C m, dismutation, and other bimolecular reactions with substances such as xanthine etc. involved in the reaction solution. The rate of superoxide generation (Ri) could be obtained as the rate of reduction of C "i to C II with the infinite concentration of C m. From the Hanes-Woolf's treatment of experimentally observed initial rates of C Ill reduction as the variable [Cm]o (Fig. 1), the xanthine/XO system was found to generate 63.3 nM O2-/sec. This commercially obtained XO utilized 28% of total consumption of xanthine for superoxide production. The calculation was based on EM(CII-C III)= 19.6 mM-lcm -1 reported by Yonetani for the horse heart cytochrome 0. 8)
Autoxidation of NAD(P)H initiated by Superoxide: Potassium phosphate buffer (0.1 M, pH 7.4, 500 lal) containing 200 I.tM xanthine was placed in a UV-cuvette. Aqueous solution of deferoxamine (10 mM, 5 Ixl) and NAD(P)H solution were added. Then the
xanthine ~
02 ~ k r e d [CIII]]..............~ CII
uric acid"" ~
"~ O~" /
kdis[°OOH], [H+]
-~ 02 .-[- H202
inactive species [Can] [Cm] ka~ [.OOH] + kothers d[Cm]/dt = Ri + kred Ri Scheme 1 847
(Eq. 1)
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS 6O 5O
,0r f -
3°V
-/
~ 20,
10~
o
0~
fl
:oo ,oo
;'
;
I
1'o 1'5 20
1'0 [O"]I'~M) [ Cca ] (I.tM)
20
Fig. I, Plot of the initial rate of cytochrome C reduction with xanthine/XO system as a function of [Cm]0. The incubation contained XO which convened 114 nM of xanthine to uric acid per sec. Interset is the Hanes-Woolf plot.
reaction was initiated by adding XO (final volume 560 p.I), and the decrease of absorbance at 340 nm due to NAD(P)H was recorded. Kinetics of irreversible degradation of NAD(P)H: Buffer solution (0.1 M potassium phosphate, pH 7.4, 500 #t) containing 200 I~M xanthine, 4 mM G-6-P, 2 mM magnesium chloride, and 219 I~M NADP+ (or 223 #M NAD +) was placed in a UVcuvette. Deferoxamine (10 mM, 5 l~l) and G-6-P DH (200 units/mi, 10 t~1)were added into the cuvette. After all NAD(P) + were reduced to NAD(P)H, XO (Ri= 63.3 nM O2/sec) was added and recorded decreasing absorbance at 340 nm.
Results and Discussion When the initial concentration of NAD(P)H was fixed, the initial rates of NAD(P)H oxidation with the xanthine/XO system under aerobic conditions were found to correlate linearly with square roots of [XO], i.e. Ri1/2 (Fig. 2). Meanwhile, under the conditions of constant [XO], the initial rates of NAD(P)H oxidations increased linearly with the increase of [NAD(P)H]o (Fig. 3).
Control experiments in the absence of
xanthine showed that NAD(P)H were practically not to be the substrates for XO 9~. 50
~
4O
=O 40
'~o 30
-~, 30
50
0
~ 20 10 (
o
5 10 I5 Ri lt2 (rffd 0 2-/sec) lf2
t'o
20
20
Ri 1/2(riM 0 2"/sec) 1/2
Fig. 2, Effect of Ri on the initial rate of the oxidation of NAD(P)H with xanthine/XO system. The incubate contained t75 ~tM NADPH or 172 gM NADH. 848
v{5°
VOI. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~ 50!
=o 40
40
=
-=~
O
30
~ 3c
2o
2c < 10 z
O" '
0
'
'
'__ "
'
100 200 300 [ NADPH ]o (~VI)
"
100
400
200 300 [ NADH ]0 (gNl)
400
Fig. 3. Effect of NAD(P)H concentrations on the initial rate of the oxidation of NAD(P)H with xanthine/XO system ( R i = 63.3 nM O2-/sec).
These kinetic observations satisfy well the steady-state kinetic equation 2, which is derived from the autoxidation mechanism of Scheme 2 assuming the autoxidation of NAD(P)H is initiated by hydrogen atom abstraction at 4-position of nicotinamide moiety of NAD(P)H with hydroperoxyl radical. There are three possible reaction paths for NAD(P)', the reactions with 0 2 (k= 1.9 x 109 M-is -1 lo); propagation reaction) and superoxide (termination), and dimerization (termination). If the rate of the reaction with molecular oxygen predominates over those of the above two termination reactions, the autoxidation rates of NAD(P)H under air ([02] = 278 gM) should be identical to those under pure molecular oxygen atmosphere ([02] = 1.38 mM). In fact, this was the case; hence both the reaction of NAD(P) ° with superoxide and the dimerization of NAD(P)" may be ignored. Under the aerobic condition with [NADPH]o = 174 gM and Ri = 63.3 nM O2-/sec, one nmol of superoxide produced by XO oxidized 0.37 nmol of NADPH, while with [NADPH]o= 317 gM 0.77 nmol of NADPH was consumed under the condition. Initiation Xan~ine
+
02 •02H
Propagation "02H NAD(P)-
_ K
= ~,
0202.
+ i_i+
+
NAD(P)H
kp
~-
H202
+
02
k
=
NAD(P)+ +
O2-
02-
kt W
=
I-I202
02
+
Termination •02H
Ri XO
d [NAD(P)H] dt
+
+
[I-1+] ~1/2 R1/2 Scheme 2 849
NAD(P).
(Eq. 2)
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
From Ri=63.3 nM O2-/sec, kt= 9.7x 107 M-is -1, ~) K= 1.58x 10-5 M, 1) and [H+] = 3.98 x 10 .8 M, the steady-state concentration of the total superoxide([O2-]t), i.e. the sum of the acidic and the basic forms of superoxide, was calculated to be 3.44 #M. The kp values were calculated to be 9.69 x 104 M-is -1 for NADPH and 8.68 x 104 M-is -1 for NADH from the [O2-]t and the slope of Fig. 2, while kp = 9.94 x 104 M-is -1 for NADPH and 9.84x 104 M-is -1 for NADH were obtained from [O2-]t and the slope of Fig. 3. This good agreement of the two kp values obtained from slopes of Figs. 2 and 3 supports the autoxidation mechanism shown in Scheme 2. The kp value of 9.26 + 0.58 x 104 Mls-1 for NADH at 25°C determined in this work is smaller than the kp value of 1.8 x 105 M-is -1 at 20°C reported by Nadezhdin and Dunford who measured the rate of disappearing NADH in the reaction with superoxide generated by a flash photolysis of hydrogen peroxide under air. 7> In the above discussion, we assumed that hydroperoxyl radical attacks exclusively the hydrogen atom at 4-position of dihydronicotinamide moiety of. NAD(P)H affording NAD(P) °. In order to confirm this assumption NAD(P)H were exposed to superoxide generated by the xanthine/XO system in the presence of G-6-P and G-6-P DH, where NAD(P) + formed during the oxidation of NAD(P)H with superoxide were immediately reduced back to NAD(P)H.
Experimental results shown in Fig• 4 reveal that
NADPH
NADH
Control
>. Control
o
O
~
15
~
~ tx~
0~
_
_
L
_
j
I
15
Time (mia)
Time (rain)
I . ~''L
0 kst
Fig. 4, Time courses of absorbance at 340 nm of NAD(P)H in the presence of G-6-P/G-6-P DH and xanthine/XO. Identical results were obtained when two times concentrated G-6-P DH was used. Control: minus XO. Conditions: see experimental section. 850
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
irreversible degradation of NAD(P)H by superoxide did take place but much smaller than that of the oxidation of NAD(P)H (rate ratio = 0.10:1 for NADPH, 0.14:1 for NADH). The irreversible oxidation of NAD(P)H would be attributed to the degradation of NAD(P) ° through coupling of NAD(P) ° and molecular oxygen at 4 or 6-position of the nicotinamide moiety to afford NAD(P)O2" which still regenerates NAD(P)" by the reaction with NAD(P)H and eventually changes into degraded products other than NAD(P) + (Eqs 3-5). Decrease of the steady-state concentration of superoxide by such side reactions is, however, negligibly small. NAD(P)" + 0 2
~ NAD(P)O2"
NAD(P)O2 ° + NAD(P)H NAD(P)O2H
~- NAD(P)O2H + NAD(P)"
= degraded products
(3) (4) (5)
These observations suggest that one must take account of the superoxide initiated autoxidation of NAD(P)H in the investigation of NAD(P)H supported monooxygenase reactions in which superoxide leaks out from the enzyme active sites during the activation of dioxygen, for example cytochrome P-450. ~1~
Acknowledgment: We gratefully acknowledge support by the Ministry of Education of Japan.
References 1. Bielski,B.H.J.; CabelI,D.E.; Arudi,R.L.; Ross,A.B., (1985), J.Phys.Chem.Ref Data, 14, 1041-1100. 2. a) Babior, B.M.; Kipnes,R.S.; Curnutte,J.T., (1973), J.Clin~lnvest., 52, 741-744. b) Makino,R.; Tanaka,T.; lizuka,T.;Ishimura,Y.;Kanegasaki,S.,(1986), J.Biol.Chem., 261, 11444-11447. 3. Yamazaki,l.; Piette,L.H.; Grover,T.A., (1990), J.Biol.Chem., 265, 652-659. 4. a) Bray,R.C., (1975), The Enzymes, Ed. Boyer, P.D., 303-387, Academic Press, N.Y. b) Roy, R.S.; McCord,J.M., (1983), Oxy Radicals and Their Scavenger Systems, Ed. Cohen,G.; Greennald,M.D., vol. 2, 143-153, Elsevier Publ., N.Y.. 5. a) Yokota, K.; Yamazaki,l., (1965), Biochim. Biophys.Acta, 105, 301-312. b) Yokota, K.; Yamazaki,l., (1977), Biochemistry, 16, 1913-1920. 6. Land,E.J.; Swallow,A.J., (1971), Biochim.Biophys.Acta, 234, 34-42. 7. Nazezhdin,A; Dunford,H.B., (1979), J.Phys.Chem., 83, 1957-1961. 8. Yonetani,T., (1965), J.Biol.Chem., 240,.4509-4514. 9. The pseudo first-order rate constants for the oxidation of NADPH: 1.15 x 10-4 s q with xanthine/XO system (Ri = 63.3 nM O2-/sec); 2.35 x 10 -6 s-1 with XO in the absence of xanthine. 10. Willson,R.L., (1970), J.Chem.Soc.Chem.Commun., 1005. 11. Kuthan,H.; Tsuji,H.; Graf, H.; UIIrich,U.; Werringloer,J.; Estabrook, R.W., (1978), FEBS Lett., 91,343-346.
851
Vol. 176, No. 2, 1991 April 30, 1991
Steady-State Kinetics of Autoxidation of NAD(P)H Initiated by Hydroperoxyl Radical, the Acid Form of Superoxide Anion Radical Ken Fujimori* and Hiromitsu Nakajima Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, 305, Japan Received March 14, 1991
Summary: Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 -I- 0.13 x 104 M-is -1 and 9.26 4-_0.58 x 104 M-is -1 at 25°C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated. ~ 1991Ao~demioP..... ~,~o.
Numerous studies have been made on chemical reactivities of superoxide during the last two decades, 1~ because of the presence of much chances to produce superoxide in a living organism, e.g. NADPH oxidase dependent respiratory burst at phagocytes, 2) leak of electron from flavoenzymes to molecular oxygen, 3~ oxidation of xanthine by xanthine oxidase (XO) 4) etc.
On the other hand, superoxide initiated
autoxidation of NADH was postulated in the HRP clock reaction by Yamazaki et al.,~ while Land and Swallow found that superoxide anion radical is practically inert to NADH (second-order rate constant of the reaction between 02- and NADH < 27 M-is -1 ).6) Later, Dunford et al. demonstrated that hydroperoxyl radical, the acid form of O2-, * To whom correspondence should be addressed.
Abbreviations NAD(P)H: NADPH and NADH, NAD(P)+: NADP+ and NAD +, NAD(P)': NADP" and NAD °, NAD(P)O2°: NADPO2 ° and NADO2 °, NAD(P)O2H: NADPO2H and NADO2H, G-6-P: glucose-6-phosphate, G-6-P DH: glucose-6-phosphate dehydrogenase, SOD: superoxide dismutase, XO: xanthine oxidase, Gill: ferricytochrome C, Oil: ferrocytochrome C, Ri: rate of superoxide production by xanthine/XO system. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
846
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
reacts with NADH (k2 = 1.8 x 105 M-is -1 at 20°C). 7~ Although NADPH and NADH (NAD(P)H) are often electron donors to molecular oxygen generating superoxide in vivo,2. ~) no steady-state kinetics has been reported for superoxide initiated autoxidation of NAD(P)H. promoted
by hydroperoxyl
Thus, we investigated the autoxidation of NAD(P)H radical which was generated continuously with
xanthine/XO system. Materials and Methods
NADPH, NADP +, NAD +, D-glucose-6-phosphate (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-P DH; EC 1.1.1.49, from leuconostoc mesenteroides) were purchased from Oriental Yeast. Superoxide dismutase (SOD; Cu, Zn type, EC 1.15.1.1) and NADH were from Wako Pure Chemical Ind. Xanthine sodium salt, butter milk xanthine oxidase (XO; EC 1.1.3.22, grade III, Lot 69F-3779), horse heart cytochrome C (type III) and deferoxamine were obtained from Sigma. And the other chemicals used were of special grade. UV-visible spectra were recorded on JASCO Ubest-50 spectrophotometer equipped with thermostatic control. Kinetics of oxidation of NAD(P)H were carried out at 25°C under aerobic conditions. XO assay: Buffer solution (0.1 M potassium phosphate, 200 gM xanthine, pH 7.4, 500 Ixl) containing various concentrations of ferricytochrome C (C m) was placed in a UV-cuvette. The reaction was started by adding XO, and recorded increasing absorbance at 550 nm due to ferrocytochrome C (Cll). Under the condition XO converted 114 nM xanthine to uric acid per sec. The fate of O2- generated in the system may be expressed by Scheme 1, in which superoxide disappears via three paths, reaction with C m, dismutation, and other bimolecular reactions with substances such as xanthine etc. involved in the reaction solution. The rate of superoxide generation (Ri) could be obtained as the rate of reduction of C "i to C II with the infinite concentration of C m. From the Hanes-Woolf's treatment of experimentally observed initial rates of C Ill reduction as the variable [Cm]o (Fig. 1), the xanthine/XO system was found to generate 63.3 nM O2-/sec. This commercially obtained XO utilized 28% of total consumption of xanthine for superoxide production. The calculation was based on EM(CII-C III)= 19.6 mM-lcm -1 reported by Yonetani for the horse heart cytochrome 0. 8)
Autoxidation of NAD(P)H initiated by Superoxide: Potassium phosphate buffer (0.1 M, pH 7.4, 500 lal) containing 200 I.tM xanthine was placed in a UV-cuvette. Aqueous solution of deferoxamine (10 mM, 5 Ixl) and NAD(P)H solution were added. Then the
xanthine ~
02 ~ k r e d [CIII]]..............~ CII
uric acid"" ~
"~ O~" /
kdis[°OOH], [H+]
-~ 02 .-[- H202
inactive species [Can] [Cm] ka~ [.OOH] + kothers d[Cm]/dt = Ri + kred Ri Scheme 1 847
(Eq. 1)
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS 6O 5O
,0r f -
3°V
-/
~ 20,
10~
o
0~
fl
:oo ,oo
;'
;
I
1'o 1'5 20
1'0 [O"]I'~M) [ Cca ] (I.tM)
20
Fig. I, Plot of the initial rate of cytochrome C reduction with xanthine/XO system as a function of [Cm]0. The incubation contained XO which convened 114 nM of xanthine to uric acid per sec. Interset is the Hanes-Woolf plot.
reaction was initiated by adding XO (final volume 560 p.I), and the decrease of absorbance at 340 nm due to NAD(P)H was recorded. Kinetics of irreversible degradation of NAD(P)H: Buffer solution (0.1 M potassium phosphate, pH 7.4, 500 #t) containing 200 I~M xanthine, 4 mM G-6-P, 2 mM magnesium chloride, and 219 I~M NADP+ (or 223 #M NAD +) was placed in a UVcuvette. Deferoxamine (10 mM, 5 l~l) and G-6-P DH (200 units/mi, 10 t~1)were added into the cuvette. After all NAD(P) + were reduced to NAD(P)H, XO (Ri= 63.3 nM O2/sec) was added and recorded decreasing absorbance at 340 nm.
Results and Discussion When the initial concentration of NAD(P)H was fixed, the initial rates of NAD(P)H oxidation with the xanthine/XO system under aerobic conditions were found to correlate linearly with square roots of [XO], i.e. Ri1/2 (Fig. 2). Meanwhile, under the conditions of constant [XO], the initial rates of NAD(P)H oxidations increased linearly with the increase of [NAD(P)H]o (Fig. 3).
Control experiments in the absence of
xanthine showed that NAD(P)H were practically not to be the substrates for XO 9~. 50
~
4O
=O 40
'~o 30
-~, 30
50
0
~ 20 10 (
o
5 10 I5 Ri lt2 (rffd 0 2-/sec) lf2
t'o
20
20
Ri 1/2(riM 0 2"/sec) 1/2
Fig. 2, Effect of Ri on the initial rate of the oxidation of NAD(P)H with xanthine/XO system. The incubate contained t75 ~tM NADPH or 172 gM NADH. 848
v{5°
VOI. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~ 50!
=o 40
40
=
-=~
O
30
~ 3c
2o
2c < 10 z
O" '
0
'
'
'__ "
'
100 200 300 [ NADPH ]o (~VI)
"
100
400
200 300 [ NADH ]0 (gNl)
400
Fig. 3. Effect of NAD(P)H concentrations on the initial rate of the oxidation of NAD(P)H with xanthine/XO system ( R i = 63.3 nM O2-/sec).
These kinetic observations satisfy well the steady-state kinetic equation 2, which is derived from the autoxidation mechanism of Scheme 2 assuming the autoxidation of NAD(P)H is initiated by hydrogen atom abstraction at 4-position of nicotinamide moiety of NAD(P)H with hydroperoxyl radical. There are three possible reaction paths for NAD(P)', the reactions with 0 2 (k= 1.9 x 109 M-is -1 lo); propagation reaction) and superoxide (termination), and dimerization (termination). If the rate of the reaction with molecular oxygen predominates over those of the above two termination reactions, the autoxidation rates of NAD(P)H under air ([02] = 278 gM) should be identical to those under pure molecular oxygen atmosphere ([02] = 1.38 mM). In fact, this was the case; hence both the reaction of NAD(P) ° with superoxide and the dimerization of NAD(P)" may be ignored. Under the aerobic condition with [NADPH]o = 174 gM and Ri = 63.3 nM O2-/sec, one nmol of superoxide produced by XO oxidized 0.37 nmol of NADPH, while with [NADPH]o= 317 gM 0.77 nmol of NADPH was consumed under the condition. Initiation Xan~ine
+
02 •02H
Propagation "02H NAD(P)-
_ K
= ~,
0202.
+ i_i+
+
NAD(P)H
kp
~-
H202
+
02
k
=
NAD(P)+ +
O2-
02-
kt W
=
I-I202
02
+
Termination •02H
Ri XO
d [NAD(P)H] dt
+
+
[I-1+] ~1/2 R1/2 Scheme 2 849
NAD(P).
(Eq. 2)
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
From Ri=63.3 nM O2-/sec, kt= 9.7x 107 M-is -1, ~) K= 1.58x 10-5 M, 1) and [H+] = 3.98 x 10 .8 M, the steady-state concentration of the total superoxide([O2-]t), i.e. the sum of the acidic and the basic forms of superoxide, was calculated to be 3.44 #M. The kp values were calculated to be 9.69 x 104 M-is -1 for NADPH and 8.68 x 104 M-is -1 for NADH from the [O2-]t and the slope of Fig. 2, while kp = 9.94 x 104 M-is -1 for NADPH and 9.84x 104 M-is -1 for NADH were obtained from [O2-]t and the slope of Fig. 3. This good agreement of the two kp values obtained from slopes of Figs. 2 and 3 supports the autoxidation mechanism shown in Scheme 2. The kp value of 9.26 + 0.58 x 104 Mls-1 for NADH at 25°C determined in this work is smaller than the kp value of 1.8 x 105 M-is -1 at 20°C reported by Nadezhdin and Dunford who measured the rate of disappearing NADH in the reaction with superoxide generated by a flash photolysis of hydrogen peroxide under air. 7> In the above discussion, we assumed that hydroperoxyl radical attacks exclusively the hydrogen atom at 4-position of dihydronicotinamide moiety of. NAD(P)H affording NAD(P) °. In order to confirm this assumption NAD(P)H were exposed to superoxide generated by the xanthine/XO system in the presence of G-6-P and G-6-P DH, where NAD(P) + formed during the oxidation of NAD(P)H with superoxide were immediately reduced back to NAD(P)H.
Experimental results shown in Fig• 4 reveal that
NADPH
NADH
Control
>. Control
o
O
~
15
~
~ tx~
0~
_
_
L
_
j
I
15
Time (mia)
Time (rain)
I . ~''L
0 kst
Fig. 4, Time courses of absorbance at 340 nm of NAD(P)H in the presence of G-6-P/G-6-P DH and xanthine/XO. Identical results were obtained when two times concentrated G-6-P DH was used. Control: minus XO. Conditions: see experimental section. 850
Vol. 176, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
irreversible degradation of NAD(P)H by superoxide did take place but much smaller than that of the oxidation of NAD(P)H (rate ratio = 0.10:1 for NADPH, 0.14:1 for NADH). The irreversible oxidation of NAD(P)H would be attributed to the degradation of NAD(P) ° through coupling of NAD(P) ° and molecular oxygen at 4 or 6-position of the nicotinamide moiety to afford NAD(P)O2" which still regenerates NAD(P)" by the reaction with NAD(P)H and eventually changes into degraded products other than NAD(P) + (Eqs 3-5). Decrease of the steady-state concentration of superoxide by such side reactions is, however, negligibly small. NAD(P)" + 0 2
~ NAD(P)O2"
NAD(P)O2 ° + NAD(P)H NAD(P)O2H
~- NAD(P)O2H + NAD(P)"
= degraded products
(3) (4) (5)
These observations suggest that one must take account of the superoxide initiated autoxidation of NAD(P)H in the investigation of NAD(P)H supported monooxygenase reactions in which superoxide leaks out from the enzyme active sites during the activation of dioxygen, for example cytochrome P-450. ~1~
Acknowledgment: We gratefully acknowledge support by the Ministry of Education of Japan.
References 1. Bielski,B.H.J.; CabelI,D.E.; Arudi,R.L.; Ross,A.B., (1985), J.Phys.Chem.Ref Data, 14, 1041-1100. 2. a) Babior, B.M.; Kipnes,R.S.; Curnutte,J.T., (1973), J.Clin~lnvest., 52, 741-744. b) Makino,R.; Tanaka,T.; lizuka,T.;Ishimura,Y.;Kanegasaki,S.,(1986), J.Biol.Chem., 261, 11444-11447. 3. Yamazaki,l.; Piette,L.H.; Grover,T.A., (1990), J.Biol.Chem., 265, 652-659. 4. a) Bray,R.C., (1975), The Enzymes, Ed. Boyer, P.D., 303-387, Academic Press, N.Y. b) Roy, R.S.; McCord,J.M., (1983), Oxy Radicals and Their Scavenger Systems, Ed. Cohen,G.; Greennald,M.D., vol. 2, 143-153, Elsevier Publ., N.Y.. 5. a) Yokota, K.; Yamazaki,l., (1965), Biochim. Biophys.Acta, 105, 301-312. b) Yokota, K.; Yamazaki,l., (1977), Biochemistry, 16, 1913-1920. 6. Land,E.J.; Swallow,A.J., (1971), Biochim.Biophys.Acta, 234, 34-42. 7. Nazezhdin,A; Dunford,H.B., (1979), J.Phys.Chem., 83, 1957-1961. 8. Yonetani,T., (1965), J.Biol.Chem., 240,.4509-4514. 9. The pseudo first-order rate constants for the oxidation of NADPH: 1.15 x 10-4 s q with xanthine/XO system (Ri = 63.3 nM O2-/sec); 2.35 x 10 -6 s-1 with XO in the absence of xanthine. 10. Willson,R.L., (1970), J.Chem.Soc.Chem.Commun., 1005. 11. Kuthan,H.; Tsuji,H.; Graf, H.; UIIrich,U.; Werringloer,J.; Estabrook, R.W., (1978), FEBS Lett., 91,343-346.
851
