- Email: [email protected]
On the mechanism of oxidative phosphorylation IX. Energy-dependent reduction of nicotinamide adenine dinucleotide by ascorbate and ubiquinone or menadione
367
SHORT COMMUNICATIONS
sc 63042
On the mechanism of oxidative phosphorylation IX. Energy-dependent reduction of nicotinamide adenine dinucleotide by ascorbate and ubiquinone or menadione The reversal of oxidative phosphorylation originally observed by CI~ANCE AND HOLLUNGER1 in mitochondria m a y now be considered a fully established phenomenon. Recent work on this system has been carried out using succinate as the electron donor and NAD as the acceptor in submitchondrial particles in the presence of added substrate amounts of ATP z. Both segments of the electron transport chain, one originating from succinate, the' other from NADH~, are implicated in this reaction. This communication describes a system of reversed electron transfer using ascorbate as electron donor and probably mediated only b y the NADH~ segment of the respiratory chain. Since ascorbate reacts with the electron transport chain very slowly s, if at all, it was necessary to use an intermediate carrier which could transfer the electrons from ascorbate to the respiratory chain. Ubiquinone-5 (UQ1 or Coenzyme Q1) or menadione (vitamin K s or 2-methyl-I,4-naphthaquinone ) proved to be satisfactory intermediate carriers. The probable reactions involved in the system are shown in Eqns. I and 2. Ascorbate + quinone--> d e h y d r o a s c o r b a t e + h y d r o q u i n o n e H y d r o q u i n o n e + N A D + A T P - - > NADH2 + quinone + A D P + Pi Ascorbate + N A D + A T P - - > dehydroascorbate + N A D H 2 + A D P + Pj
(I) (2) (3)
Eqn. i is the non-enzymic reduction of the quinone b y ascorbate 4. Reversed electron transport, shown in Eqn. 2, consists of the ATP-requiring reduction of NAD b y the hydroquinone, via the respiratory chain. In the routine assay, 0.05 M Tris sulfate, 3-3 mM MgC12, I mM NAD, 2 mg crystalline bovine serum albumin, I mM potassium cyanide, 0.02 M ascorbate, o.I mM menadione or o.I mM UQ1 (added in IO/A of methanol solution) and mitochondrial particles 5 (0.75 mg protein) in a final volume of 2.9 ml at p H 7.3 were incubated for 3 rain at 3 o°. The reaction was started b y the addition of o.I ml of 0.06 M ATP, and absorbancy readings at 34 ° m# were taken every 2 mill beginning with the first minute after the final addition. The inhibitor, when used, was present in the incubation TABLE I INHIBITION OF N A D REDUCTION BY UNCOUPLING COMPOUNDS AND OLIGOMXrCIN E x p e r i m e n t s as in the t e x t using menadione as the intermediate carrier. Inhibitor None 2,4-Dinitrophenol, o.i mM 2,4-Dinitrophenol, 0.2 mM None Dicumarol, io #M Oligomycin, o. i / z g / m l Oligomycin, o. 3/~g/ml
miemoleslmin/mg Inhibition (%) 49.5 12. 3 o.o 42.9 1.7 15.4 o.o
7° ioo 96 64 ioo
Biochim. Biophys. Acta, 89 (I964) 367-369
368
SHORT COMMUNICATIONS
medium. If it was added in ethanol solution, a similar amount of ethanol was used in the control assay. Each assay was carried out concurrently with a blank containing no ATP in order to correct for a slow, non-enzymic increase in absorbancy. The data are expressed as m/zmoles of NAD reduced/min/mg mitochondrial particle protein. In a preliminary communication 4, it was shown that both menadione and UQ1 were equally efficient as intermediate carriers in the ATP-dependent reduction of NAD by ascorbate. The rate of NAD reduction was nearly the same as with succinate in the first 3 min and declined slowly. When ATP, quinone, ascorbate, NAD, or enzyme was left out of the medium, the rate of NAD reduction was quite small. The corrected increase at 34 ° m# in the complete enzymic system was almost entirely due to NAD TABLE Ii INHIBITION OF N A D REDUCTION BY ELECTRON TRANSPORT INHIBITORS E x p e r i m e n t s as in t h e t e x t w i t h m e n a d i o n e as t h e i n t e r m e d i a t e carrier.
Inhibitor
ml~moles/min[rng
Inhibition (%)
None A m y t a l , o. 5 mM i . o mM 1. 5 mM
54.o 25.1 lO.5 8.6
53 81 84
4,4,4-Trifluoro- i (2-thienyl)-1,3-butanedione, o.o 5 mM o . i mM
26.6 1.2
41 48
51.9 39.0 27.1
4 28 5°
34.2 5-4 2-3
84 93
A n t i m y c i n A,
i #g/mg io/,g/rag 15/~g/mg
None R o t e n o n e , o.o 5/uM o. i o / ~ M
reduction, since addition of lactate and lactate dehydrogenase resulted in a rapid decrease of absorbancy to nearly the original value. No careful search has been made for the phosphorylated NAD derivative reported to be accumulated under somewhat similar conditions 6. The concentrations of ascorbate and menadione were selected for maximum rates. Nearly the same activity was obtained by reducing the concentrations of both to onethird the indicated levels. Serum albumin was included routinely since the activity was 30% higher in its presence. The reaction rate was optimal at pH 7.3 ; the decrease in activity on either side of it (from p H 6.8 to pH 8.5) was only slight. This contrasts with the rather high pH optimum of 8.5 in the energy-dependent NAD reduction by succinateS,L The ATP-requiring reduction of NAD by ascorbate-menadione was inhibited almost completely b y 2,4-dinitrophenol and dicumarol at concentrations which uncouple oxidative phosphorylation in mitochondria (Table I). Olygomycin, an inhibitor of energy-coupling reactions, also inhibited NAD reduction. The respiration inhibitors, Amytal, rotenon@ and 4,4,4-trifluoro-i (2-thienyl)-i,3butanedione also inhibited Reaction 3 (Table II) as in the ATP-dependent reduction Biochim. Biophys. Acta, 89 (1964) 367-369
369
SHORT COMMUNICATIONS
of NAD by succinateZ, 5. We have recently observed that rotenone strongly inhibits the oxidation of NADH~ by UQ10, both in particles and with a highly purified enzyme at similar low concentrations 9. These results would implicate the NADH~-UQ reductase (oxidoreductase) in the energy-dependent reduction of NAD by ascorbatemenadione. NAD reduction by ascorbate-menadione was inhibited by antimycin, but only at unusually high levels (Table II), as was NAD reduction by succinate in the presence of added ATP~, 5 and NADHz oxidation by fumarate 5. On the other hand, NAD reduction by ascorbate in the presence of tetramethyl-p-phenylenediamine in the same particles and under identical conditions was highly sensitive to antimycin (see ref. IO). Thus, two distinct effects of antimycin have been detected. Our results would suggest that reduced menadione reacts with an electron carrier located on the substrate side of the classical antimycin sensitive region (between cytochromes b and c). Since 1.7 mM malonate had no effect on the system while the same level of malonate completely inhibited succinate-mediated NAD reduction in parallel experiments, the participation of the succinate dehydrogenase flavoprotein is excluded from the reaction. These studies were carried out with the competent technical assistance of Mrs. C. WASHINGTON. UQ1 was obtained through the courtesy of Dr. K. FOLKERS and of Dr. O. ISLER.
Gerontology Branch, National Heart Institute, National Institutes of Health, and Baltimore City Hospitals, Baltimore, Md. (U.S.A.)
D. R. SANADI
1 B. CHANCE AND G. HOLLUNGER,Nature, 185 (196o) 666. H. L o w AND I. VALLIN, Biochim. Biophys. Acta, 69 (1963) 361. a E. E. JACOBS AND D. R. SANADI, Biochim. Biophys. •cta, 38 (196o) 12. 4 D. R. SANADI, J. Biol. Chem., 238 (1963) PC482. 5 D. R. SANADI AND A. L. FLUHARTY, Biochemistry, 2 (1963) 523 . 6 D. E. GRIFFITH, Federation Proc., 22 (1963) lO64. F. A. HOMM~S, Biochim. Biophys. ,4cta, 77 (1963) 173. s L. ERNSTER, G. DALLNER AND G. FELICE, J. Biol. Chem., 238 (I963) 1124. 9 T. E. ANDREOLI R. L. PHARO AND D. R. SANADI, Biochim. Biophys. Acta, 90 (1964) 16. 10 H. L o w ANn I. VALLIN, Biochem. Biophys. Res. Commun., 9 (1962) 3o7.
Received March I2th, 1964 Note added in proof: C o n t r a r y to our earlier p r e l i m i n a r y r e p o r t 9, it h a s b e e n f o u n d t h a t N A D H t o x i d a t i o n b y UQ10, c a t a l y z e d b y t h e a b o v e particles or b y t h e purified soluble N A D H ~ UQ r e d u c t a s e , is n o t i n h i b i t e d b y t r i f l u o r o t h i e n y l b u t a n e d i o n e . Since N A D r e d u c t i o n m e d i a t e d b y a s c o r b a t e - m e n a d i o n e or succinate, as well as aerobic o x i d a t i o n of succinate, are i n h i b i t e d in t h e s a m e particles, it w o u l d a p p e a r t h a t t h e p o i n t of e n t r y o f electrons f r o m r e d u c e d m e n a d i o n e in t h e p r e s e n t e x p e r i m e n t s lies on t h e s u c c i n a t e - b r a n c h of t h e r e s p i r a t o r y chain. (Received J u l y Ioth, 1964). Biochim. Biophys. Acta, 89 (1964) 367-369
SHORT COMMUNICATIONS
sc 63042
On the mechanism of oxidative phosphorylation IX. Energy-dependent reduction of nicotinamide adenine dinucleotide by ascorbate and ubiquinone or menadione The reversal of oxidative phosphorylation originally observed by CI~ANCE AND HOLLUNGER1 in mitochondria m a y now be considered a fully established phenomenon. Recent work on this system has been carried out using succinate as the electron donor and NAD as the acceptor in submitchondrial particles in the presence of added substrate amounts of ATP z. Both segments of the electron transport chain, one originating from succinate, the' other from NADH~, are implicated in this reaction. This communication describes a system of reversed electron transfer using ascorbate as electron donor and probably mediated only b y the NADH~ segment of the respiratory chain. Since ascorbate reacts with the electron transport chain very slowly s, if at all, it was necessary to use an intermediate carrier which could transfer the electrons from ascorbate to the respiratory chain. Ubiquinone-5 (UQ1 or Coenzyme Q1) or menadione (vitamin K s or 2-methyl-I,4-naphthaquinone ) proved to be satisfactory intermediate carriers. The probable reactions involved in the system are shown in Eqns. I and 2. Ascorbate + quinone--> d e h y d r o a s c o r b a t e + h y d r o q u i n o n e H y d r o q u i n o n e + N A D + A T P - - > NADH2 + quinone + A D P + Pi Ascorbate + N A D + A T P - - > dehydroascorbate + N A D H 2 + A D P + Pj
(I) (2) (3)
Eqn. i is the non-enzymic reduction of the quinone b y ascorbate 4. Reversed electron transport, shown in Eqn. 2, consists of the ATP-requiring reduction of NAD b y the hydroquinone, via the respiratory chain. In the routine assay, 0.05 M Tris sulfate, 3-3 mM MgC12, I mM NAD, 2 mg crystalline bovine serum albumin, I mM potassium cyanide, 0.02 M ascorbate, o.I mM menadione or o.I mM UQ1 (added in IO/A of methanol solution) and mitochondrial particles 5 (0.75 mg protein) in a final volume of 2.9 ml at p H 7.3 were incubated for 3 rain at 3 o°. The reaction was started b y the addition of o.I ml of 0.06 M ATP, and absorbancy readings at 34 ° m# were taken every 2 mill beginning with the first minute after the final addition. The inhibitor, when used, was present in the incubation TABLE I INHIBITION OF N A D REDUCTION BY UNCOUPLING COMPOUNDS AND OLIGOMXrCIN E x p e r i m e n t s as in the t e x t using menadione as the intermediate carrier. Inhibitor None 2,4-Dinitrophenol, o.i mM 2,4-Dinitrophenol, 0.2 mM None Dicumarol, io #M Oligomycin, o. i / z g / m l Oligomycin, o. 3/~g/ml
miemoleslmin/mg Inhibition (%) 49.5 12. 3 o.o 42.9 1.7 15.4 o.o
7° ioo 96 64 ioo
Biochim. Biophys. Acta, 89 (I964) 367-369
368
SHORT COMMUNICATIONS
medium. If it was added in ethanol solution, a similar amount of ethanol was used in the control assay. Each assay was carried out concurrently with a blank containing no ATP in order to correct for a slow, non-enzymic increase in absorbancy. The data are expressed as m/zmoles of NAD reduced/min/mg mitochondrial particle protein. In a preliminary communication 4, it was shown that both menadione and UQ1 were equally efficient as intermediate carriers in the ATP-dependent reduction of NAD by ascorbate. The rate of NAD reduction was nearly the same as with succinate in the first 3 min and declined slowly. When ATP, quinone, ascorbate, NAD, or enzyme was left out of the medium, the rate of NAD reduction was quite small. The corrected increase at 34 ° m# in the complete enzymic system was almost entirely due to NAD TABLE Ii INHIBITION OF N A D REDUCTION BY ELECTRON TRANSPORT INHIBITORS E x p e r i m e n t s as in t h e t e x t w i t h m e n a d i o n e as t h e i n t e r m e d i a t e carrier.
Inhibitor
ml~moles/min[rng
Inhibition (%)
None A m y t a l , o. 5 mM i . o mM 1. 5 mM
54.o 25.1 lO.5 8.6
53 81 84
4,4,4-Trifluoro- i (2-thienyl)-1,3-butanedione, o.o 5 mM o . i mM
26.6 1.2
41 48
51.9 39.0 27.1
4 28 5°
34.2 5-4 2-3
84 93
A n t i m y c i n A,
i #g/mg io/,g/rag 15/~g/mg
None R o t e n o n e , o.o 5/uM o. i o / ~ M
reduction, since addition of lactate and lactate dehydrogenase resulted in a rapid decrease of absorbancy to nearly the original value. No careful search has been made for the phosphorylated NAD derivative reported to be accumulated under somewhat similar conditions 6. The concentrations of ascorbate and menadione were selected for maximum rates. Nearly the same activity was obtained by reducing the concentrations of both to onethird the indicated levels. Serum albumin was included routinely since the activity was 30% higher in its presence. The reaction rate was optimal at pH 7.3 ; the decrease in activity on either side of it (from p H 6.8 to pH 8.5) was only slight. This contrasts with the rather high pH optimum of 8.5 in the energy-dependent NAD reduction by succinateS,L The ATP-requiring reduction of NAD by ascorbate-menadione was inhibited almost completely b y 2,4-dinitrophenol and dicumarol at concentrations which uncouple oxidative phosphorylation in mitochondria (Table I). Olygomycin, an inhibitor of energy-coupling reactions, also inhibited NAD reduction. The respiration inhibitors, Amytal, rotenon@ and 4,4,4-trifluoro-i (2-thienyl)-i,3butanedione also inhibited Reaction 3 (Table II) as in the ATP-dependent reduction Biochim. Biophys. Acta, 89 (1964) 367-369
369
SHORT COMMUNICATIONS
of NAD by succinateZ, 5. We have recently observed that rotenone strongly inhibits the oxidation of NADH~ by UQ10, both in particles and with a highly purified enzyme at similar low concentrations 9. These results would implicate the NADH~-UQ reductase (oxidoreductase) in the energy-dependent reduction of NAD by ascorbatemenadione. NAD reduction by ascorbate-menadione was inhibited by antimycin, but only at unusually high levels (Table II), as was NAD reduction by succinate in the presence of added ATP~, 5 and NADHz oxidation by fumarate 5. On the other hand, NAD reduction by ascorbate in the presence of tetramethyl-p-phenylenediamine in the same particles and under identical conditions was highly sensitive to antimycin (see ref. IO). Thus, two distinct effects of antimycin have been detected. Our results would suggest that reduced menadione reacts with an electron carrier located on the substrate side of the classical antimycin sensitive region (between cytochromes b and c). Since 1.7 mM malonate had no effect on the system while the same level of malonate completely inhibited succinate-mediated NAD reduction in parallel experiments, the participation of the succinate dehydrogenase flavoprotein is excluded from the reaction. These studies were carried out with the competent technical assistance of Mrs. C. WASHINGTON. UQ1 was obtained through the courtesy of Dr. K. FOLKERS and of Dr. O. ISLER.
Gerontology Branch, National Heart Institute, National Institutes of Health, and Baltimore City Hospitals, Baltimore, Md. (U.S.A.)
D. R. SANADI
1 B. CHANCE AND G. HOLLUNGER,Nature, 185 (196o) 666. H. L o w AND I. VALLIN, Biochim. Biophys. Acta, 69 (1963) 361. a E. E. JACOBS AND D. R. SANADI, Biochim. Biophys. •cta, 38 (196o) 12. 4 D. R. SANADI, J. Biol. Chem., 238 (1963) PC482. 5 D. R. SANADI AND A. L. FLUHARTY, Biochemistry, 2 (1963) 523 . 6 D. E. GRIFFITH, Federation Proc., 22 (1963) lO64. F. A. HOMM~S, Biochim. Biophys. ,4cta, 77 (1963) 173. s L. ERNSTER, G. DALLNER AND G. FELICE, J. Biol. Chem., 238 (I963) 1124. 9 T. E. ANDREOLI R. L. PHARO AND D. R. SANADI, Biochim. Biophys. Acta, 90 (1964) 16. 10 H. L o w ANn I. VALLIN, Biochem. Biophys. Res. Commun., 9 (1962) 3o7.
Received March I2th, 1964 Note added in proof: C o n t r a r y to our earlier p r e l i m i n a r y r e p o r t 9, it h a s b e e n f o u n d t h a t N A D H t o x i d a t i o n b y UQ10, c a t a l y z e d b y t h e a b o v e particles or b y t h e purified soluble N A D H ~ UQ r e d u c t a s e , is n o t i n h i b i t e d b y t r i f l u o r o t h i e n y l b u t a n e d i o n e . Since N A D r e d u c t i o n m e d i a t e d b y a s c o r b a t e - m e n a d i o n e or succinate, as well as aerobic o x i d a t i o n of succinate, are i n h i b i t e d in t h e s a m e particles, it w o u l d a p p e a r t h a t t h e p o i n t of e n t r y o f electrons f r o m r e d u c e d m e n a d i o n e in t h e p r e s e n t e x p e r i m e n t s lies on t h e s u c c i n a t e - b r a n c h of t h e r e s p i r a t o r y chain. (Received J u l y Ioth, 1964). Biochim. Biophys. Acta, 89 (1964) 367-369