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[4] Resolution of complex I and isolation of NADH dehydrogenase and an iron-sulfur protein
[4]
RESOLUTION OF COMPLEX I
15
[4] R e s o l u t i o n o f C o m p l e x I a n d I s o l a t i o n o f N A D H Dehydrogenase and an Iron-Sulfur Protein By YVES M. GALANTE and YOUSSEF HATEFI
The mitochondrial N A D H dehydrogenase was first isolated in soluble form by Edelhoch et al. 1 and Mahler and co-workers. 2 The method used involved extraction of heart muscle homogenates with about 10% aqueous ethanol at pH 4.8-5.0 and 43o-45 °, a procedure originally used for isolation of the Straub diaphorase (lipoyl dehydrogenase). 3 Modifications of the above, 4-7 as well as other procedures, a have been used by others for isolation of NADH dehydrogenase from submitochondrial particles at various stages of purity. However, because NADH dehydrogenase is an iron-sulfur flavoprotein, the use of acid pH at elevated temperatures and other harsh conditions often resulted in preparations with low levels of nonheme iron and labile sulfide, variable amounts of flavin, and poor activities. The preparation described below involves the resolution of complex I at pH 8.0 with an appropriate chaotropic salt, followed by fractionation of the soluble fraction of complex I with ammonium sulfate. 9-'' An intermediate fraction obtained in this process is an ironsulfur protein component of complex I. Purification Method Reagents
1. NaCIO4, 8 M, pH 7-8 2. Saturated, neutralized ammonium sulfate (saturated at 20°-25 °) 3. IA-Dithiothreitol, 0.1 M, freshly made ' H. Edelhoch, O. Hayaishi, and L. J. Tepley, J. Biol. Chem. 197, 97 (1952). 2 H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 (1952). 3 F. B. Straub, Biochem. J. 33, 787 (1939). 4 B. de Bernard, Biochim. Biophys. Acta 23, 510 (1957). B. Mackler, Biochim. Biophys. Acta 50, 141 (1961). S. A. Kumar, N. A. Rao, S. P. Felton, F. M. Huennekens, and B. Mackler, Arch. Biochem. Biophys. 125, 436 (1968). 7 R. L. Pharo, L. A. Sordahl, S. R, Vyas, and D. R. Sanadi, J. Biol. Chem. 241, 4771 (1966). T. E. King and R. L. Howard, J. Biol. Chem. 237, 1686 (1962). 9 y . Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969). ,0 y. Hatefi, K. E. Stempel, and W. G. Hanstein, J. Biol. Chem. 244, 2358 (1969). 1, K. A. Davis and Y. Hatefi, Biochemistr>, 8, 3355 (1969).
16
ELECTRONTRANSFERCOMPLEXES
[4]
4. Tris-chloride, 50 raM, pH 7.5-8.0, adjusted at 200-25 °, then cooled to 0°-5 ° Isolation Procedure Step 1. Complex I is suspended in Tris-chloride containing 5 mM dithiothreitol, and adjusted to a protein concentration (biuret) of 10 mg/ m1.12 Sodium perchlorate is added from the 8 M solution to a concentration of 0.5 M, and the mixture is incubated for 10 min at 38 ° under an argon atmosphere. The suspension is cooled in an ice bath, and centrifuged for 20-30 min at 30,000 rpm in the No. 30 (or No. 40) rotor of Spinco ultracentrifuge. The amber supernatant is collected, and the gray residue is discarded. Step 2. The supernatant is brought to 0.275 saturation with saturated ammonium sulfate solution, allowed to stand in an ice bath for 5-10 min, then centrifuged for 10 min as before. The supernatant is collected and further fractionated as described below. The tightly packed brown residue is suspended in a small volume of 50 mM Tris-chloride containing 5 mM dithiothreitol, and carefully homogenized to avoid foaming. It is allowed to stand in an ice bath under an argon atmosphere for about 1 hr, then centrifuged for 10 min at 30,000 rom. The residue is discarded. The dark brown supernatant is a preparation of a complex I iron-sulfur protein with electron paramagnetic resonance characteristics of iron-sulfur center 2 of complex 1.13 For the composition and the absorption spectrum of this preparation, see Hatefi et al. 14 Step 3. The supernatant from the previous step is brought to 0.364 saturation with the saturated ammonium sulfate solution, allowed to stand in an ice bath for 5-10 min, then centrifuged as before. The residue is discarded, the supernatant is brought to 0.529 saturation with the ammonium sulfate solution, allowed to stand in an ice bath and centrifuged as before. The supernatant is discarded, and the residue, which constitutes the preparation of NADH dehydrogenase, is immediately frozen in liquid nitrogen and stored at - 7 0 °. The yield of this preparation is equivalent to 5-6% of the protein and 50-60% of the FMN content of complex I. This preparation will be referred to below as preparation A. 12 Five to ten microliters of 5 m M dithiothreitol added together with the protein sample to a 3-ml biuret reaction mixture c a u s e s a color change, which will dissipate completely w h e n the test tube containing the biuret mixture is placed in boiling water for 60-90 sec. 13 N. R. O r m e - J o h n s o n , R. E. H a n s e n , and H. Beinert, J. Biol. Chem. 249, 1922 (1974). 14 y . Hatefi, Y. M. Galante, D. L. Stiggall, and L. Djavadi-Ohaniance, in " T h e Structural Basis of M e m b r a n e F u n c t i o n " (Y. Hatefi and L. Djavadi-Ohaniance, eds.), p. 169. A c a d e m i c Press, N e w York, 1976.
[4]
RESOLUTION OF COMPLEX I
17
A cleaner and more active NADH dehydrogenase can be prepared by bringing the supernatant of step 2 to 0.42 saturation with the ammonium sulfate solution and discarding the resultant residue, then increasing the salt saturation of the supernatant from this step to 0.51 saturation and collecting the precipitated NADH dehydrogenase as before. 15The protein yield of this preparation is about 3% of complex I, but the preparation moves as a single band upon chromatography on Sephadex G-100 and gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). This preparation will be referred to below as preparation B. Preparations of NADH dehydrogenase are stable when kept frozen at - 7 0 ° as ammonium sulfate-precipitated pellets. In solution they are stable for hours at 0 °, but lose considerable activity when frozen and stored at - 7 0 ° or subjected to repeated freeze-thawing. Properties
Composition. The composition of the NADH dehydrogenase preparation A is shown in Table I. The purer preparation B described above has essentially the same amount of acid-extractable flavin, but a somewhat higher ratio of nonheme iron and labile sulfide to flavin. 15 All the flavin of NADH dehydrogenase is acid-extractable FMN. The preparation is completely free of cytochromes. Enzymic Properties. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH in the presence of appropriate electron acceptors. It also catalyzes transhydrogenation from NADH and NADPH to NAD or 3-acetylpyridine adenine dinucleotide (AcPyAD). 16 For NADH or AcPyADH as substrate, the pH optimum is close to pH 8.0, while for NADPH as substrate the activity increases with lowering of pH (tested
TABLE I COMPOSITION OF N A D H DEHYDROGENASE a
Component
Amount [nmol (ng-atoms) per mg protein]
FMN N o n h e m e iron Acid-labile sulfide
13.5-14.5 60-65 58-60
a For preparation A, from Y. Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969). ,5 y . M. Galante and Y. Hatefi, in preparation. 16 y . Hatefi and Y. M. Galante, Proc. Natl. Acad. Sci. U.S.A. 74, 846 (1977).
18
ELECTRON TRANSFER COMPLEXES
[4]
to pH 5.0). The enzyme reacts with quinoid structures [e.g., 2-methylnaphthoquinone (menadione, vitamin K3), the ubiquinone isoprenologs, 2,6-dichloroindophenol, methylene blue] and ferric compounds (e.g., ferricyanide, cytochrome c) as electron acceptors. Among these, ferricyanide, menadione, and ubiquinone-I are the most efficient acceptors, whereas cytochrome c reacts rapidly only at very high concentrations ( g m = 0 . 6 m M ) . Table II gives a summary of the enzymic activities of NADH dehydrogenase.
Molecular Properties. Chromatography of NADH dehydrogenase (preparation B) on Sephadex G-100 showed a single symmetrical band with an elution volume very close to that of bovine serum albumin, and indicated a molecular weight of 69,000 --- 5%. This value agrees with the minimum molecular weight calculated from the flavin content of the preparation (13.5-14.0 nmol per milligram of protein). When subjected to polyacrylamide gel electrophoresis in the presence of SDS and mercaptoethanol according to the method of Weber and Osborn,lZ the above preparation showed three polypeptide bands, stained with Coomassie blue, with mobilities corresponding to molecular weights of 51,000, TABLE
II
A C T I V I T I E S OF THE S O L U B L E N A D H
Reaction NADH NADH NADH NADH NADH NADPH NADPH NADH NADPH
~ ~ --* ~ ~ ~ --~ --~ --~
menadione ubiquinone-I ferricyanide cytochrome c DCIP menadione ferricyanide AcPyAD s AcPyAD
DEHYDROGENASE
pH
Specific activity a
8.0 8.0 7.5 8.0 8.0 5.0 5.5 8.0
40q-550 b 2 5 0 - 3 3 ~b 4 0 0 - 4 5 0 c,d 220-250 c 125-150 b 13 e 21 e 12-15 1.5 e
5.5
a Micromoles of N A D ( P ) H oxidized min 1 mg protein -1 at 38 °. Data are from references 9, 15, a n d 16, cited in text footnotes. The higher values for N A D H as substrate, and
all the values for N A D P H as substrate, were obtained with the N A D H dehydrogenase preparation B . acceptor c Wma, ax
1 m M , fcrricyanide inhibits the oxidation of N A D H by the soluble dehydrogenase. VN~°H at 1 m M ferricyanide is 3 3 0 - 3 8 0 . e I n the presence of 75 m M g u a n i d i n e . H C l . ± 3-Acetylpyridine adenine dinucleotide. ,/Above
17 K . Weber and M. Osborn, J. Biol. Chem. 244, 4 4 0 6 (1969).
[4]
RESOLUTION OF COMPLEX 1
19
24,000, and 10,000. Thus, there is a discrepancy of about 10% between the sum of the above molecular weights and the values obtained from Sephadex gel filtration and the flavin content of the enzyme. Whether all the three polypeptides found upon SDS gel electrophoresis are subunits of NADH dehydrogenase is difficult to decide. All three are present in chromatographically clean preparations of the enzyme iso!ated from complex I with the use of different chaotropes. Also when the Coomassie blue-stained SDS gels were scanned at 625 nm, total absorbance of each of the three bands divided by its molecular weight as given above indicated a'molar ratio of 1 : 1 : 1.
Spectral Properties. Shown in Fig. 1 are the absorption spectra of NADH dehydrogenase analyzed to show the contributions of flavin (trace 4) and the iron-sulfur chromophore(s) (trace 3), and its substrate and dithionite reduced forms. It is seen that about 50% of the absorbance of the enzyme at 450 nm is bleached with NADH (trace 2), and dithionite causes considerable additional bleaching (trace 5). Destruction of the iron-sulfur centers with high concentrations of mersalyl followed by reduction of the flavin with dithionite (trace 6) showed that the enzyme so treated had no other absorption in the visible region of the spectrum.
0.7~ i 06 1 0.5
0.4 ~0.3 0.2 0.1
'
4 ~ 4~ s& 6&
Wovelength,nm
FIG. 1. Spectral characteristics of the soluble NADH dehydrogenase (1.6 mg/ml) isolated from complex 1. Traces: 1, spectrum of oxidized enzyme; 2, NADH-reduced enzyme; 5, dithionite-reduced enzyme; 4, flavin contribution to 1 after destruction of iron-sulfur chromophore with sodium mersalyl; 3, iron-sulfur contribution to 1 obtained by subtraction of 4 from 1; 6, 4 plus dithionite showing that after destruction of the iron-sulfur chromophore with mersalyl and reduction of flavin with dithionite the enzyme has no absoption in the visible region. From Y. Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969).
20
ELECTRON TRANSFER COMPLEXES
[4]
Activators and Inhibitors. All the reactions catalyzed by NADH dehydrogenase, especially those involving NADPH as substrate, are activated by guanidine, arginine, arginyl methyl ester, and other alkylguanidines. Phosphoarginine is ineffective, indicating the importance of the positive charge on the guanido moiety of the above compounds. On a molar basis, guanidine is the most potent activator. Also, this activation is not due to the chaotropic property of the guanidinium ion, since various chaotropic anions do not show a similar effect. Analysis of the guanidine effect on the NADH-ferricyanide reductase activity of the enzyme has shown that the activation by guanidine involves a decrease of K m NADH and an increase of VN~AxDH.For an interpretation of the effect of guanidine, see Hatefi and Galante.16 NADH dehydrogenase is inhibited by mercurials (p-chloromercuribenzoate, p-chloromercuriphenylsulfonate, mersalyl), but not by N-ethylmaleimide. The quinone reductase activity, but not the ferricyanide reductase activity, of the enzyme is also strongly inhibited by NADH concentrations >0.25 mM. Piericidin A and rotenone, which are potent inhibitors of ubiquinone reduction by complex I, have little or no effect on the activities of the soluble enzyme. Incubation of NADH dehydrogenase at acid pH (e.g., 4.8) or in the presence of ophenanthroline results in destruction of activity. Both effects are correlated with the loss of labile sulfide from the enzyme.
Assay Methods Assays for the reduction of various dyes and cytochrome c, for the oxidation of NADPH, and for transhydrogenation from NADH and NADPH to NAD (or AcPyAD) have been detailed elsewhere.l~' 16 The following description will be limited to the most efficient reactions catalyzed by NADH dehydrogenase, namely the oxidation of NADH by 2methylnaphthoquinone, ubiquinone-1, 2,6-dichloroindophenol, and ferricyanide as electron acceptors. Reagents 1. Tris-chloride, 1 M, pH 8.0 (pH adjusted at 200-25 °) 2. Potassium phosphate, 1 M, pH 7.5 (pH adjusted at 20°-250) 3. NADH, 15 mM 4. 2-Methylnaphthoquinone (menadione), 20 mM in ethanol 5. Ubiquinone-1, 1 mM (see this volume [3]) 6. 2,6-Dichloroindophenol (DCIP), 1.3 mM 7. Potassium ferricyanide, 10 mM Oxidation of N A D H by Menadione, Ubiquinone-1, or DCIP. To each of two 1-ml quartz cuvettes are added 50 ~1 of Tris-chloride buffer, 10/zl of menadione, or 0.4 ml of ubiquinone-1 (when DCIP is the electron
[S]
PREPARATION AND PROPERTIES OF SUCCINATE
21
acceptor, add 50 /zl to the sample cuvette and 20 /xl to the reference cuvette), and water to a final volume of 1.0 ml. The cuvettes are placed in the thermostatted (30 ° or 38 °) chamber of a spectrophotometer for temperature equilibration. Shortly before addition of enzyme, 10/zl of NADH are added to the sample cuvette (when DCIP is the acceptor, a higher concentration of NADH, e.g., 50/xl, may be used). The reaction is started by the addition of 0.5-1.0/zg of enzyme (dissolved in 50 mM Tris-chloride, pH 8.0) in a small volume (e.g., 1-2/xl) to avoid excessive dilution of the stock enzyme solution. When menadione or ubiquinone-I is the acceptor, the reaction is followed as a function of time at 340 nm (NADH oxidation), using an extinction coefficient of E = 6.22 mM -~ cm -~ for calculation of activity. When DCIP is the acceptor, the reaction is monitored at 600 nm (DCIP reduction), and the extinction coefficient to be used is e = 21 mM -~ cm-k
Oxidation of NADH by Ferricyanide. To each of two 1-ml cuvettes are added 40 tzl of phosphate buffer, ferricyanide (100/zl to the sample cuvette and 20/zl to the reference cuvette), and water to a final volume of 1.0 ml. The cuvettes are placed in the thermostatted (30 ° or 38 °) chamber of a spectrophotometer for temperature equilibration. One minute prior to addition of enzyme, 30-50 tzl of NADH are added to the sample cuvette, and the nonenzymic reduction of ferricyanide by NADH is monitored at 410 nm. At pH 7.5 and in the presence of phosphate buffer, this rate is very slow. The reaction is started by the addition of 1-2 ~g of enzyme in a small volume, and the reduction of ferricyanide is monitored as before. Activity is calculated using an extinction coefficient of c = 1.0 mM -~ cm -~ for ferricyanide at 410 nm. Since ferricyanide is a single electron acceptor, the calculated activity must be halved in order to express the result in terms of moles of NADH oxidized per unit time.
[5] P r e p a r a t i o n a n d P r o p e r t i e s o f S u c c i n a t e : U b i q u i n o n e Oxidoreductase (Complex II)
By
YOUSSEF H A T E F I a n d D I A N A L . STIGGALL
Succinate + ubiquinone (Q) ~ fumarate + dihydroubiquinone (QH0
Complex II is the segment of the respiratory chain that catalyzes the transfer of reducing equivalents from succinate to ubiquinone. Succinate dehydrogenase constitutes approximately 50% of the protein weight of the complex. Complex II reacts with ubiquinone, ferricyanide, and phen-
RESOLUTION OF COMPLEX I
15
[4] R e s o l u t i o n o f C o m p l e x I a n d I s o l a t i o n o f N A D H Dehydrogenase and an Iron-Sulfur Protein By YVES M. GALANTE and YOUSSEF HATEFI
The mitochondrial N A D H dehydrogenase was first isolated in soluble form by Edelhoch et al. 1 and Mahler and co-workers. 2 The method used involved extraction of heart muscle homogenates with about 10% aqueous ethanol at pH 4.8-5.0 and 43o-45 °, a procedure originally used for isolation of the Straub diaphorase (lipoyl dehydrogenase). 3 Modifications of the above, 4-7 as well as other procedures, a have been used by others for isolation of NADH dehydrogenase from submitochondrial particles at various stages of purity. However, because NADH dehydrogenase is an iron-sulfur flavoprotein, the use of acid pH at elevated temperatures and other harsh conditions often resulted in preparations with low levels of nonheme iron and labile sulfide, variable amounts of flavin, and poor activities. The preparation described below involves the resolution of complex I at pH 8.0 with an appropriate chaotropic salt, followed by fractionation of the soluble fraction of complex I with ammonium sulfate. 9-'' An intermediate fraction obtained in this process is an ironsulfur protein component of complex I. Purification Method Reagents
1. NaCIO4, 8 M, pH 7-8 2. Saturated, neutralized ammonium sulfate (saturated at 20°-25 °) 3. IA-Dithiothreitol, 0.1 M, freshly made ' H. Edelhoch, O. Hayaishi, and L. J. Tepley, J. Biol. Chem. 197, 97 (1952). 2 H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 (1952). 3 F. B. Straub, Biochem. J. 33, 787 (1939). 4 B. de Bernard, Biochim. Biophys. Acta 23, 510 (1957). B. Mackler, Biochim. Biophys. Acta 50, 141 (1961). S. A. Kumar, N. A. Rao, S. P. Felton, F. M. Huennekens, and B. Mackler, Arch. Biochem. Biophys. 125, 436 (1968). 7 R. L. Pharo, L. A. Sordahl, S. R, Vyas, and D. R. Sanadi, J. Biol. Chem. 241, 4771 (1966). T. E. King and R. L. Howard, J. Biol. Chem. 237, 1686 (1962). 9 y . Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969). ,0 y. Hatefi, K. E. Stempel, and W. G. Hanstein, J. Biol. Chem. 244, 2358 (1969). 1, K. A. Davis and Y. Hatefi, Biochemistr>, 8, 3355 (1969).
16
ELECTRONTRANSFERCOMPLEXES
[4]
4. Tris-chloride, 50 raM, pH 7.5-8.0, adjusted at 200-25 °, then cooled to 0°-5 ° Isolation Procedure Step 1. Complex I is suspended in Tris-chloride containing 5 mM dithiothreitol, and adjusted to a protein concentration (biuret) of 10 mg/ m1.12 Sodium perchlorate is added from the 8 M solution to a concentration of 0.5 M, and the mixture is incubated for 10 min at 38 ° under an argon atmosphere. The suspension is cooled in an ice bath, and centrifuged for 20-30 min at 30,000 rpm in the No. 30 (or No. 40) rotor of Spinco ultracentrifuge. The amber supernatant is collected, and the gray residue is discarded. Step 2. The supernatant is brought to 0.275 saturation with saturated ammonium sulfate solution, allowed to stand in an ice bath for 5-10 min, then centrifuged for 10 min as before. The supernatant is collected and further fractionated as described below. The tightly packed brown residue is suspended in a small volume of 50 mM Tris-chloride containing 5 mM dithiothreitol, and carefully homogenized to avoid foaming. It is allowed to stand in an ice bath under an argon atmosphere for about 1 hr, then centrifuged for 10 min at 30,000 rom. The residue is discarded. The dark brown supernatant is a preparation of a complex I iron-sulfur protein with electron paramagnetic resonance characteristics of iron-sulfur center 2 of complex 1.13 For the composition and the absorption spectrum of this preparation, see Hatefi et al. 14 Step 3. The supernatant from the previous step is brought to 0.364 saturation with the saturated ammonium sulfate solution, allowed to stand in an ice bath for 5-10 min, then centrifuged as before. The residue is discarded, the supernatant is brought to 0.529 saturation with the ammonium sulfate solution, allowed to stand in an ice bath and centrifuged as before. The supernatant is discarded, and the residue, which constitutes the preparation of NADH dehydrogenase, is immediately frozen in liquid nitrogen and stored at - 7 0 °. The yield of this preparation is equivalent to 5-6% of the protein and 50-60% of the FMN content of complex I. This preparation will be referred to below as preparation A. 12 Five to ten microliters of 5 m M dithiothreitol added together with the protein sample to a 3-ml biuret reaction mixture c a u s e s a color change, which will dissipate completely w h e n the test tube containing the biuret mixture is placed in boiling water for 60-90 sec. 13 N. R. O r m e - J o h n s o n , R. E. H a n s e n , and H. Beinert, J. Biol. Chem. 249, 1922 (1974). 14 y . Hatefi, Y. M. Galante, D. L. Stiggall, and L. Djavadi-Ohaniance, in " T h e Structural Basis of M e m b r a n e F u n c t i o n " (Y. Hatefi and L. Djavadi-Ohaniance, eds.), p. 169. A c a d e m i c Press, N e w York, 1976.
[4]
RESOLUTION OF COMPLEX I
17
A cleaner and more active NADH dehydrogenase can be prepared by bringing the supernatant of step 2 to 0.42 saturation with the ammonium sulfate solution and discarding the resultant residue, then increasing the salt saturation of the supernatant from this step to 0.51 saturation and collecting the precipitated NADH dehydrogenase as before. 15The protein yield of this preparation is about 3% of complex I, but the preparation moves as a single band upon chromatography on Sephadex G-100 and gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). This preparation will be referred to below as preparation B. Preparations of NADH dehydrogenase are stable when kept frozen at - 7 0 ° as ammonium sulfate-precipitated pellets. In solution they are stable for hours at 0 °, but lose considerable activity when frozen and stored at - 7 0 ° or subjected to repeated freeze-thawing. Properties
Composition. The composition of the NADH dehydrogenase preparation A is shown in Table I. The purer preparation B described above has essentially the same amount of acid-extractable flavin, but a somewhat higher ratio of nonheme iron and labile sulfide to flavin. 15 All the flavin of NADH dehydrogenase is acid-extractable FMN. The preparation is completely free of cytochromes. Enzymic Properties. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH in the presence of appropriate electron acceptors. It also catalyzes transhydrogenation from NADH and NADPH to NAD or 3-acetylpyridine adenine dinucleotide (AcPyAD). 16 For NADH or AcPyADH as substrate, the pH optimum is close to pH 8.0, while for NADPH as substrate the activity increases with lowering of pH (tested
TABLE I COMPOSITION OF N A D H DEHYDROGENASE a
Component
Amount [nmol (ng-atoms) per mg protein]
FMN N o n h e m e iron Acid-labile sulfide
13.5-14.5 60-65 58-60
a For preparation A, from Y. Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969). ,5 y . M. Galante and Y. Hatefi, in preparation. 16 y . Hatefi and Y. M. Galante, Proc. Natl. Acad. Sci. U.S.A. 74, 846 (1977).
18
ELECTRON TRANSFER COMPLEXES
[4]
to pH 5.0). The enzyme reacts with quinoid structures [e.g., 2-methylnaphthoquinone (menadione, vitamin K3), the ubiquinone isoprenologs, 2,6-dichloroindophenol, methylene blue] and ferric compounds (e.g., ferricyanide, cytochrome c) as electron acceptors. Among these, ferricyanide, menadione, and ubiquinone-I are the most efficient acceptors, whereas cytochrome c reacts rapidly only at very high concentrations ( g m = 0 . 6 m M ) . Table II gives a summary of the enzymic activities of NADH dehydrogenase.
Molecular Properties. Chromatography of NADH dehydrogenase (preparation B) on Sephadex G-100 showed a single symmetrical band with an elution volume very close to that of bovine serum albumin, and indicated a molecular weight of 69,000 --- 5%. This value agrees with the minimum molecular weight calculated from the flavin content of the preparation (13.5-14.0 nmol per milligram of protein). When subjected to polyacrylamide gel electrophoresis in the presence of SDS and mercaptoethanol according to the method of Weber and Osborn,lZ the above preparation showed three polypeptide bands, stained with Coomassie blue, with mobilities corresponding to molecular weights of 51,000, TABLE
II
A C T I V I T I E S OF THE S O L U B L E N A D H
Reaction NADH NADH NADH NADH NADH NADPH NADPH NADH NADPH
~ ~ --* ~ ~ ~ --~ --~ --~
menadione ubiquinone-I ferricyanide cytochrome c DCIP menadione ferricyanide AcPyAD s AcPyAD
DEHYDROGENASE
pH
Specific activity a
8.0 8.0 7.5 8.0 8.0 5.0 5.5 8.0
40q-550 b 2 5 0 - 3 3 ~b 4 0 0 - 4 5 0 c,d 220-250 c 125-150 b 13 e 21 e 12-15 1.5 e
5.5
a Micromoles of N A D ( P ) H oxidized min 1 mg protein -1 at 38 °. Data are from references 9, 15, a n d 16, cited in text footnotes. The higher values for N A D H as substrate, and
all the values for N A D P H as substrate, were obtained with the N A D H dehydrogenase preparation B . acceptor c Wma, ax
1 m M , fcrricyanide inhibits the oxidation of N A D H by the soluble dehydrogenase. VN~°H at 1 m M ferricyanide is 3 3 0 - 3 8 0 . e I n the presence of 75 m M g u a n i d i n e . H C l . ± 3-Acetylpyridine adenine dinucleotide. ,/Above
17 K . Weber and M. Osborn, J. Biol. Chem. 244, 4 4 0 6 (1969).
[4]
RESOLUTION OF COMPLEX 1
19
24,000, and 10,000. Thus, there is a discrepancy of about 10% between the sum of the above molecular weights and the values obtained from Sephadex gel filtration and the flavin content of the enzyme. Whether all the three polypeptides found upon SDS gel electrophoresis are subunits of NADH dehydrogenase is difficult to decide. All three are present in chromatographically clean preparations of the enzyme iso!ated from complex I with the use of different chaotropes. Also when the Coomassie blue-stained SDS gels were scanned at 625 nm, total absorbance of each of the three bands divided by its molecular weight as given above indicated a'molar ratio of 1 : 1 : 1.
Spectral Properties. Shown in Fig. 1 are the absorption spectra of NADH dehydrogenase analyzed to show the contributions of flavin (trace 4) and the iron-sulfur chromophore(s) (trace 3), and its substrate and dithionite reduced forms. It is seen that about 50% of the absorbance of the enzyme at 450 nm is bleached with NADH (trace 2), and dithionite causes considerable additional bleaching (trace 5). Destruction of the iron-sulfur centers with high concentrations of mersalyl followed by reduction of the flavin with dithionite (trace 6) showed that the enzyme so treated had no other absorption in the visible region of the spectrum.
0.7~ i 06 1 0.5
0.4 ~0.3 0.2 0.1
'
4 ~ 4~ s& 6&
Wovelength,nm
FIG. 1. Spectral characteristics of the soluble NADH dehydrogenase (1.6 mg/ml) isolated from complex 1. Traces: 1, spectrum of oxidized enzyme; 2, NADH-reduced enzyme; 5, dithionite-reduced enzyme; 4, flavin contribution to 1 after destruction of iron-sulfur chromophore with sodium mersalyl; 3, iron-sulfur contribution to 1 obtained by subtraction of 4 from 1; 6, 4 plus dithionite showing that after destruction of the iron-sulfur chromophore with mersalyl and reduction of flavin with dithionite the enzyme has no absoption in the visible region. From Y. Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969).
20
ELECTRON TRANSFER COMPLEXES
[4]
Activators and Inhibitors. All the reactions catalyzed by NADH dehydrogenase, especially those involving NADPH as substrate, are activated by guanidine, arginine, arginyl methyl ester, and other alkylguanidines. Phosphoarginine is ineffective, indicating the importance of the positive charge on the guanido moiety of the above compounds. On a molar basis, guanidine is the most potent activator. Also, this activation is not due to the chaotropic property of the guanidinium ion, since various chaotropic anions do not show a similar effect. Analysis of the guanidine effect on the NADH-ferricyanide reductase activity of the enzyme has shown that the activation by guanidine involves a decrease of K m NADH and an increase of VN~AxDH.For an interpretation of the effect of guanidine, see Hatefi and Galante.16 NADH dehydrogenase is inhibited by mercurials (p-chloromercuribenzoate, p-chloromercuriphenylsulfonate, mersalyl), but not by N-ethylmaleimide. The quinone reductase activity, but not the ferricyanide reductase activity, of the enzyme is also strongly inhibited by NADH concentrations >0.25 mM. Piericidin A and rotenone, which are potent inhibitors of ubiquinone reduction by complex I, have little or no effect on the activities of the soluble enzyme. Incubation of NADH dehydrogenase at acid pH (e.g., 4.8) or in the presence of ophenanthroline results in destruction of activity. Both effects are correlated with the loss of labile sulfide from the enzyme.
Assay Methods Assays for the reduction of various dyes and cytochrome c, for the oxidation of NADPH, and for transhydrogenation from NADH and NADPH to NAD (or AcPyAD) have been detailed elsewhere.l~' 16 The following description will be limited to the most efficient reactions catalyzed by NADH dehydrogenase, namely the oxidation of NADH by 2methylnaphthoquinone, ubiquinone-1, 2,6-dichloroindophenol, and ferricyanide as electron acceptors. Reagents 1. Tris-chloride, 1 M, pH 8.0 (pH adjusted at 200-25 °) 2. Potassium phosphate, 1 M, pH 7.5 (pH adjusted at 20°-250) 3. NADH, 15 mM 4. 2-Methylnaphthoquinone (menadione), 20 mM in ethanol 5. Ubiquinone-1, 1 mM (see this volume [3]) 6. 2,6-Dichloroindophenol (DCIP), 1.3 mM 7. Potassium ferricyanide, 10 mM Oxidation of N A D H by Menadione, Ubiquinone-1, or DCIP. To each of two 1-ml quartz cuvettes are added 50 ~1 of Tris-chloride buffer, 10/zl of menadione, or 0.4 ml of ubiquinone-1 (when DCIP is the electron
[S]
PREPARATION AND PROPERTIES OF SUCCINATE
21
acceptor, add 50 /zl to the sample cuvette and 20 /xl to the reference cuvette), and water to a final volume of 1.0 ml. The cuvettes are placed in the thermostatted (30 ° or 38 °) chamber of a spectrophotometer for temperature equilibration. Shortly before addition of enzyme, 10/zl of NADH are added to the sample cuvette (when DCIP is the acceptor, a higher concentration of NADH, e.g., 50/xl, may be used). The reaction is started by the addition of 0.5-1.0/zg of enzyme (dissolved in 50 mM Tris-chloride, pH 8.0) in a small volume (e.g., 1-2/xl) to avoid excessive dilution of the stock enzyme solution. When menadione or ubiquinone-I is the acceptor, the reaction is followed as a function of time at 340 nm (NADH oxidation), using an extinction coefficient of E = 6.22 mM -~ cm -~ for calculation of activity. When DCIP is the acceptor, the reaction is monitored at 600 nm (DCIP reduction), and the extinction coefficient to be used is e = 21 mM -~ cm-k
Oxidation of NADH by Ferricyanide. To each of two 1-ml cuvettes are added 40 tzl of phosphate buffer, ferricyanide (100/zl to the sample cuvette and 20/zl to the reference cuvette), and water to a final volume of 1.0 ml. The cuvettes are placed in the thermostatted (30 ° or 38 °) chamber of a spectrophotometer for temperature equilibration. One minute prior to addition of enzyme, 30-50 tzl of NADH are added to the sample cuvette, and the nonenzymic reduction of ferricyanide by NADH is monitored at 410 nm. At pH 7.5 and in the presence of phosphate buffer, this rate is very slow. The reaction is started by the addition of 1-2 ~g of enzyme in a small volume, and the reduction of ferricyanide is monitored as before. Activity is calculated using an extinction coefficient of c = 1.0 mM -~ cm -~ for ferricyanide at 410 nm. Since ferricyanide is a single electron acceptor, the calculated activity must be halved in order to express the result in terms of moles of NADH oxidized per unit time.
[5] P r e p a r a t i o n a n d P r o p e r t i e s o f S u c c i n a t e : U b i q u i n o n e Oxidoreductase (Complex II)
By
YOUSSEF H A T E F I a n d D I A N A L . STIGGALL
Succinate + ubiquinone (Q) ~ fumarate + dihydroubiquinone (QH0
Complex II is the segment of the respiratory chain that catalyzes the transfer of reducing equivalents from succinate to ubiquinone. Succinate dehydrogenase constitutes approximately 50% of the protein weight of the complex. Complex II reacts with ubiquinone, ferricyanide, and phen-