- Email: [email protected]
Oxidation-reduction components of a reduced diphosphopyridine nucleotide dehydrogenase
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
436-448
125,
Oxidation-Reduction
(1968)
Components
Diphosphopyridine
of a Reduced
Nucleotide
Dehydrogenase’ S. A. KUMAR,2 Department
N. APPAJI
of Biochemistry,
Scripps
RAO,2 S. P. FELTON: Clinic
and Research
Foundation,
F. M. HUENNEKENS La Jolla,
California
Washington
98106
9%‘037
AND
Department
of Pediatrics, Received
November
BRUCE
MACKLER4
University
of Washington,
23, 1967;
accepted
Seattle, December
19, 1967
DPNH dehydrogenase, solubilieed from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43”, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c a-,tivity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as a:ceptors.
Previous reports from these laboratories (l-4) have described some of the properties of a DPNH dehydrogenase that is solubilized by treatment of beef heart DPNH oxidase (5) with acid-ethanol according to a modification of the procedure originally devised by iLIahler et al. (6). The enzyme was found to be reasonably homogeneous when examined electrophoretically (2, 7) or To Richard S. Schweet, pioneer in the field of peptide synthesis-in memoriam. 1 Paper X in the series “Flavin Nucleotides and Flavoproteins.” This work was supported by grants from the American JIeart Association (6% 436
in the ultracentrifuge (4, 7), but the molecular weight varied from 70,000 to 90,000, based on sedimentation measurements (4, 7) to 100,000 on the basis of the flavin 796) and the National Heart Institute (I-I-5457), National Institutes of Health. A preliminary report of this work has appeared elsewhere (1). 2 Present address: Ijepartment of Biochemistry, Indian Institute of Science, Bangalore, India. 3 Present address: Children’s Orthopedic Hospital, Seattle, Washington. 4 Research Career Development Awardee of the National Inst,itutes of Health.
COhlPONENTS
OF
J>PNH
content (7). The enzyme contained FMN and nonheme iron in the ratio of 1: 2 (2, 7). Both the DPNH dehydrogenase and the parent DPNH oxidase are specific for the B-form of DPNH and have a K, value of 1 X 1O-5 M for DPNH (8). The present report describes an improved purification procedure in which the acidethanol extracted enzyme is subjected to ammonium sulfate fractionation and filtration through Sephadex G-25. Electrophoretic criteria are presented for the homogeneity of the enzyme, and the molecular weight is shown to be 70,000 by Whitaker’s method (9). Analytical data are reported for various components (FMN, nonheme iron, and labile sulfide and thiol groups) that could be involved in the electron transfer mechanisms. Studies involving inhibition or deletion of these oxidation-reduction components provide a basis for formulating a tentative sequence of electron transfer when the dehydrogenation of DPNH is coupled to the various artificial electron acceptors (ferricyanide, 2,6-dichlorophenolindophenol, menadione, and cytochrome c). EXPERIMENTAL
PROCEDURE
l\fateri&. Chemicals were obtained from the following sources: DPNH, DPN, FMN, pCM8, pCMB,5 DTNB, bovine hemoglobin, trypsin, beef liver catalase, and cytochrome c from the Sigma Chemical Co.; bovine serum albumin from ilrmour and Co.; potassium ferricyanide from Fisher Scientific Co. ; 2, F-dichlorophenolindophenol from LaMotte Chemical Products Co.; acetylpgridine-DPN from P-L Biochemicals, Inc.; menadione from Merck & Co., Inc.; N,lV-dimethyl-p-phenylenediamiIle-HCl from Matheson, Coleman and Bell; Amido Black from K & K Laboratories; o-phenanthroline from G. Frederick Smit,h Chemical Co.; Sephadexes and Blue J)extran 2000 from Pharmacia; Bio-Gel (stored in 0.1 .n phosphate bufler, pH 6.8, for 3 months at 4”) and Chelex resin from Bio-Rad Laboratories; and DEAE-cellulose from Schleicher & Schuell Co. Coenzyme Q, was a gift from l)r. Y. Hatefi. Beef hearts were obtained from Talone Packing co. Methods. Absorbance changes at a single wave5 Abbreviations used: pCMB, p-chloromercuribenzoate; pCMS, p-chloromercuriphenylsul5,5’-dithiobis-(2.nitrobenzoic fonate; IjTNB, acid); ETP, electron transport particle.
DEHYDROGENASE
437
length were followed using a Beckman spectrophotometer, model DU. Complete spectra (220600 rnb) were measured with a Cary recording spectrophotometer, model 14, or a Beckman UK-2A recording spectrophotometer. Protein was determined by the biuret met.hod (10) with crystalline bovine serum albumin as the standard. Nonheme iron (ll), labile sulfide (ll), and sialic acid (12) were measured by published methods. After being released by heat- and acid-denaturation of the protein (7), FMN was quantitated by the decrease in absorbance at 450 rnp upon treatment with hydrosulfite; a millimolar extinction coefficient of 9.8 was used for the difference of oxidized minus reduced FMN (13). Electrophoresis was performed on horizontal starch-gel plates (13) or vertical polyacrylamide tubes (15); in each instance, protein bands were stained with Amido Black. The molecular weight of the protein was determined by the Andrews’ modification (16) of Whitaker’s method (9). Amino acid analyses were carried out by the method of Spackman et ul. (17). Thiol groups on the enzyme were assayed spectrophotometrically by the following procedure, based upon the DTNB method of Ellman (18). The following were added to a 1.5-ml ruvette having a light path of 1 cm: 0.5 ml of 0.1 M phosphate buffer, pH 8.0; l-2 mg of the protein; and 0.2 ml of a DTNB solution (40 mg of DTNB dissolved in 1.0 ml of 95y0 ethanol and diluted to 10 ml with 0.1 M phosphate buffer, pIi 8.0); water was used to adjust the volume to 1 ml. In some instances, ruea was also present at a final concentration of 4 M. The solution was allowed to stand at room temperature for 15 minutes and the absorbancy at 412 m,u was measured against a blank containing all components except the DTNB. A correction was made for the small initial absorbancy of DTNB. The thiol contentj of the protein was calculated by using a millimolar extinction coefficient of 13.G for the DTNB chromogen (17). Occasionally, the results were checked by Boyer’s method by using pCMB (19). Fluorescence of the enzyme was measured with a recording Amino-Bowman speetrophotofluxometer, eqllipped with a 150-W Hanovia Xenon light source and an RCA 1 P 28 photomultiplier tube. The exciting wavelength was 300 mH, and emission spectra were recorded with the Aminco 1620-809 X-Y recorder. Enzyme asscrys. DPNH dehydrogenase activity with the following acceptors was mea.sured spectrophotometrically by minor modifications of previously published methods: ferricyanide (7) ; 2, G-dichlorophenolindophenol (20) ; cytochrome
438
KUMAR
c (5) ; and menadione or coenzyme &I (21). Specific activities are expressed as micromoles of DPNH oxidized per minute per milligram of protein at 37”. Transhydrogenase activities were measured by the procedure of Stein el al. (22). Preparation of DPNH dehydrogenase. DPNH oxidase was prepared from beef heart mitochondria by the procedure of Mackler (23), except that the pH was taken only to 8.8, rather than to 10.0, in the initial step. DPNH dehydrogenase was solubilized and purified by the following modification of earlier procedures (2, 6, 24). Frozen DPNH oxidase (20 mg/ml in 5% sucrose) was thawed and diluted with one volume of cold water. The mixture was homogenized gently and centrifuged for 30 minutes at 40,000 rpm (Spinco, No. 40 head). The residue was suspended in cold water and the volume was adjusted to give a protein concentration of about 30 mg/ml. The pH was adjusted to 4.8 with 1 M acetic acid. Upon the addition of acetic acid, the mitochondrial suspension became less fluid. Ethanol at -10” was added dropwise to a final concentration of 9%. The beaker containing the mitochondrial suspension was then transferred to a water bath a 5&55”. The temperature of the suspension was allowed to rise as quickly as possible (no more than 15 minutes) to 43”. The temperature was maintained at 43” for 15 minutes, whereupon the beaker was transferred to a Dry-Ice and methoxyethanol bath, and the pH of the suspension was returned to 7.0 by the addition of 6 N KOH. After centrifugation for 15 minutes at 35,000 rpm (Spinco, No. 40 head), the residue was discarded and the supernatant fluid was lyophilized. The lyophilized powder was dissolved in 25 ml of cold wat,er (pH 7.0), and a saturated solution of ammonium sulfate, adjusted to pH 7.8 with concentrated NH,OH and kept previously at 4”, was added slowly (1 ml/5 minutes) until 44% saturation was reached (20 ml of ammonium sulfate per 25 ml of protein solution). The precipitate was removed by centrifugation for 20 minutes at 40,ooO rpm (Spinco, No. 40 head). For each 25 ml of original protein solution an additional 18 ml of ammonium sulfate was now added to the supernatant fluid to raise the concentration to 6Ooj, saturation. The precipitate was collected by centrifugation, dissolved in a minimum volume of water, and passed through a 2.5 X 35.cm column of Sephadex G-25 with water as the eluant. The yellow protein fraction immediately following the void volume was lyophilized and stored at - 20”. Removal of FMN from the dehydrogenase. Method A: Florisil was washed thoroughly with 1 M acetic acid, followed by water, and then packed by de-
ET
AL.
cantation into a 1 X IO-cm column. One liter of cold water was passed through the column. Two ml of a solution containing about 10 mg DPNH dehydrogenase/ml was passed through the column, which was then eluted with cold water. The pale yellow eluate (about 10 ml) was passed again through the column and the protein fraction (indicated by absorbance at 280 rnp) was collected and lyophilized. Recovery of protein after two cycles was usually about 8O-857o. Method B: Two ml of a solution containing about 12 mg of the dehydrogenase of 0.1 M phosphate buffer, pH 6.8, was passed through a 1.9 X 75-cm column of Bio-Gel P-200 that had been equilibrated with the same buffer. (Bio-Gel was deaerated before being packed in the column.) The void volume of the column, as determined with Blue Dextran 2000 (mol. wt. 2 X 10s), was 65 ml. DPNH dehydrogenase was eluted after g&120 ml of the above buffer had passed through the column. The eluate was lyophilized and the residue was dissolved in 2 ml of water (pH adjusted to 7.0, if necessary). The solution was passed through a 2 X 34-cm column of Sephadex G-25 to remove salt. This procedure was repeated several times. Recovery of protein after three cycles was usually about 65%. RESIJLTS
Homogeneity and molecular weight of the DPNH clehydrogenase. As purified by our
previous method (a), the DPNH drogenase
preparations
usually
dehy-
contained
5-10% extraneous protein, as judged by ultracentrifugal or electrophoretic examination (2, 4, 7). One of the more troublesome contaminants was cytochrome c, which obscures the absorption spectrum of the enzyme (considerably more cytochrome c is found at this stage if the enzyme is extracted from mitochondria or ETP rather than DPNH oxidase). Also loosely associatedwith earlier preparations of the enzyme was a sialic acid-like material (25).6 However, by 6 As much as 5 moles of this material (determined calorimetrically with thiobarbituric acid using N-acetyl neuraminic acid as the standard) per mole of FMN has been found in preparations that were taken only through a O-SO% precipitation with ammonium sulfate. The final step in the present purification procedure, i.e., filtration of the enzyme through Sephadex G-25, completely removes this material without any concomitant loss of enzymic activity. The properties of this compound will be described in detail elsewhere (Felton, S. P., Rao, N. A., Mackler, B., and Huennekens, F. M., in preparation).
COMPONENTS
OF
DPNII
narrowing the range of the ammonium sulfate fractionation to the 44-60 70 precipitate, followed by filtration of the enzyme through Sephadex, these and other contaminants have been largely eliminated. Electrophoresis of the purified enzyme on starch gel (14) at pH 8.2 and 4.6 revealed in each instance only a single protein band migrating slowly toward the positive and negative electrodes, respectively. A similar result was obtained by electrophoresis at pH 8.0 in a polyacrylamide column (15), except that the band was slightly diffuse. In our previous studies, the molecular weight of the enzyme, calculated from the flavin content (7), was higher than the value obtained from sedimentation measurements (4). In view of the relative ease with which F&IN can be detached from the protein (2, 7, 26), it seemedlikely that earlier preparations contained variable, although small, amounts of the apoenzyme. By careful handling of t’he enzyme in the present procedure, this difficulty has been overcome. The molecular weight of the protein, estimated to be
Cytochrome ’
2.5-
Trypsin
DPNH \ DehydrogenaseO
2.0-
E
Hemoglobin Serum Albumin
1\‘”
l Catalase 1.5-
\
I
4
1
5
6
log Molecular Weight FIN. 1. Molecular weight of DPNH dehydrogenase determined by filtration through Sephadex G-200. Sephadex G-200 (2W300 mesh) that had been stored in water for 3-4 months was resuspended in 0.15 M KC1 for 6 days, deaerated, and packed under gravity into a 2 X 50-cm column. The column was equilibrated with 0.15 M KCl. Reference proteins and DPNH dehydrogenase (4-7 mg each) were dissolved in 1 ml of 0.15 M KC1 and passed through the column to determine their respective elution volumes (V). T’oid volume (V”), 54 ml. Flow rate, 0.1 ml/minute.
439
J)EHYI>ROGENASE TABLE AMINO DPNH Amino
ACID
I
COMPOSITION
OF
L)EHTDROGENASE~~
*
acid
Composition
(moles/mole proteinJc
of
29.26 8.05 20.79 7.28 39.06 32.13 21.21 42.42 25.69 35.98 29.61 26.60 11.69 17.64 32.97 13.79 15.82
LYS His Arg CYS Asp
Thr Ser Glu Pro GUY Ala \-al Met Ile Leu ‘WPhe
U Enzyme used for this analysis had specific activities of 30 and 2.6 pmoles DPNH oxidized/ minute/mg protein with ferricyanide and cytochrome c, respectively, and contained 1.04 moles of FMN and 0.91 mole of labile sulfide per mole of protein. * Analysis performed by Dr. Karl I>us, University of California at San Diego. c Based upon a molecular weight of 70,009.
70,000 (Fig. 1) by the Andrews’ modification (16) of Whitaker’s method (9), is in good agreement with the minimum molecular weight calculated from analytical data for various oxidation-reduction components (see below). The amino acid composition of the enzyme, kindly determined by Dr. I<. Dus, is given in Table I. Oxidation-reduction DPNH dehydroyenase.
components
of
the
We have reported previously (2) that the enzyme contains FMN and nonheme iron in the ratio of approximately 1:2. As an extension of this work, the present preparations have been analyzed for various potential oxidationreduction components, namely F&IN, nonheme iron, labile sulfide, and thiol groups. The data, given in Table II, are expressed as mpmoles/mg protein and as moles/mole protein, with an assumed molecular weight of 70,000. For t’hree separate preparations of
440
KUMAR TABLE OF DPNH
COMPONENTS
II DEHYDROGENSSE
FMN
Nonheme iron Labile fide
sul-
Thiol groups a Figures protein.
(moles/mole
of enzyme)
1
2
TABLE
in
0.95 (13.5)a
0.99 (14.2)
1.05 (15.0)
(1:::
2.05 (29.3)
2.23 (31 A)
1.82 (26.0)
2.03 (29.0)
0.91 (12.9)
0.95 (13.5)
0.93 (13.3)
0.93 (13.2)
7.14
7.28 W)
7.00 (100)
7.14
parentheses
are
Specificactivitya Enzymepreparation 1 2 3
Acceptor
3
-
III
ACTIVITY 0~ DPNH DEHYDROGENasE WITH VARIOUS ACCEPTORS
Avg.
-
(102)
AL.
SPECIFIC
Enzymeprepardiion
components -
ET
-
(102)
mpmoles/mg
Ferricyanide Dichlorophenolindopheno1 Coenzyme Q1 Menadione Cytochrome c Acetylpyridine-DPN a Micromoles protein.
of
DPNH
31 30
40 38.8
33 40
11 6 2.7 1
12.5 6.2 3.4 0.8
10 5.7 3.7 1.1
oxidized/minute/mg
shown by the data in Table III. The enzyme does not show any appreciable TPNH-DPN transhydrogenase activity, but it doeshave a low DPNH-acetyl pyridine DPN activity (about 1 hmole DPNH oxidized/minute/mg protein). Previous measurements (2) have shown that the Michaelis constant for DPNH with this enzyme (with indophenol as acceptor) is 1.1 X lop5 M, a value in close agreement with that obtained for the particulate DPNH oxidase (27) from which the dehydrogenase is derived. In this investigation, the interaction of DPNH with the enzyme was examined nonkinet’ically with a fluorimetric method. When the prot’ein was excited at 300 rnp, an emission maximum at 350 rnp was observed (curve 1 in Fig. 2). Upon the addition of small increments of DPNH, the band at 350 rnp decreased progressively and a new band at 454 rnp appeared (curves 2-9) ; the latter corresponds to the fluorescencemaximum of DPNH (28). Alternatively, addition of DPN quenched the fluorescence at 350 rnp without any concomitant appearance of the 454 rnp band. Both DPNH and DPN produced maximum quenching at about 1 X 1O-4 M. From the data in Fig. 2, a K, value of 1.1 X 1O-5 ;M could be calculated’ for interaction of DPNH with the enzyme.
the enzyme, the following average values, per mole of protein, were obtained: 1 mole of FMN; 2 atoms of nonheme iron; and 1 mole of labile sulfide. Lower values of FMN (about 10 mpmoles/mg protein), encountered in earlier preparations of the enzyme (7), were probably caused by the facile dissociation of this coenzyme during the isolation procedure. Thiol groups on the protein were quantitated by the DTNB method (18), and checked by the pCMB procedure (19); both gave essentially the same results. In the presence of 4 M urea, 7 thiol groups were found per mole of enzyme (Table II), a value in agreement with the cysteine content (see Table I). In the absence of urea, however, only two or three of these thiol groups could be detected immediately with DTNB, and the remainder were slowly titratable over a 90minute period. Contrary to our earlier report (3), there was no appreciable increase in thiol groups when FMN was removed from the protein by Method A (see Experimental section). Catalytic and kinetic properties of the enzyme. DPNH dehydrogenase can utilize ferricyanide, indophenol, cytochrome c, and menadione (or coenzyme Q1) as electron ’ Since these measurements acceptors. The specific activities with these aerobic conditions, DPNH acceptors (pmoles DPNH oxidized/minoxidation by oxygen. This ute/mg protein) are reasonably constant for K, measurement, however, different preparat’ions of the enzyme, as DPN have about the same
were made under was undergoing slow does not invalidate the since both DPNH and affinity for t,he protein.
COMPONENTS
OF
DPNH
441
DEHYDROGEXASE
400 500 600 700 Wavelength, mp
Fro. 2. Effect of DPNII on fluorescence of DPNH dehydrogenase. The fluorescence of DPNH dehydrogenase (48 rg of enzyme in 1 ml of 0.04 M phosphate buffer, pH 7.5) was recorded (curve 0) in an Aminco-Bowman spectrophotofluorometer (sensitivity, 26; meter multiplier, 0.03). Curves l-9 represent the fluorescence spectrum after successive additions of 5-J increments of UPXH (1.6 rnM in 0.04 phosphate bnffer, pH 7.5).
Reduction of enzyme-bound j&win by DPNH. As shown by curve 1 in Fig. 3, the DPXH dehydrogenase has a principal absorbance band at about 440 rnp with a pronounced shoulder on the short, wavelength side (approximately 420 mp). A band of lower absorbancy is seen at about 550 rnp. The 420 and 650 mp bands are not’ due to contamination by cytochrome c, but very probably to the presence of nonheme iron (see Ref. 29 for a discussion of the contribu-
FIG. 3. Changes in absorption spectrum of enzyme upon addition of DPNH under anaerobic conditions. DPNH dehydrogenase (1.8 mg) dissolved in 1 ml of 0.04 M phosphate buffer, pH 7.5, was placed in an anaerobic quart,z cuvette equipped with a rubber stopper. The cuvette was evacuated and filled with Nz. The spectrum (curve 1) was recorded with a Beckman spectrophotometer, model DK-2A, against a blank containing only buffer. Small increments of 1.6 X 10-S M DPNH in 0.04 M phosphate buffer, pH 7.5, were added successively through the stopper by using a microsyringe. After each addition, the spectrum was again recorded (curve 2, total of 10 ~1; curve 3, total of 20 ~1; curve 4, total of 35 ~1; and curve 5, total of 45 ~1). At the end of the titration, 5 11 of a lyc solution of sodium dithionite was added (dashed curve) and the final spect.rlIm was recorded.
duced st’ate, rat’her t’han the semiquinone, as judged by the absence of long wavelength bands characteristic of the latter (30), and by t’he fact that further addition of dithionite tion of nonheme iron to the spectra of aft’er curve 5 did not appreciably change the flavoferroproteins). spectrum. During the transformation from When DPNH, even in a lo-fold excess curves 1 through 5, 0.072 pmole of DPNH relative to the flavin, was added to the enzyme under aerobic conditions, the de- was added. This may be compared with the amount of enzyme present (0.026 pmole, if a crease in absorbance at 450 rnp n-as about molecular weight of 70,000 is assumed). In so (2 of that produced by dithionite. cont,rast, addit,ion of small increments of Thus, approximately a 3-fold molal excess DPKH under anaerobic conditions caused of DPKH is required to reduce the enzymethe 450 rnp absorbance to decrease mark- bound FMN. Titration of DPNH by the enzyme. In the edly, the 420 rnp absorbancy lessso, and Dhe 550 peak not at all (curves 2-5 in Fig. 3). above experiment (Fig. 3) there was some about the exact amount, of The flavin appeared to be in the full>- re- uncertainty
442
KUMAR
ET
AL.
DPNH required for the complete reduction of the enzyme-bound flavin. Accordingly, the amount of DPNH capable of being oxidized by 1 mole of enzyme was measured in a different way. A known amount of enzyme was added anaerobically to an excess of DPNH, and the decrease in absorbancy at 340 rnl.c was followed. In the experiment shown in Fig. 4, 0.029 pmole of DPNH was oxidized by 0.009 pmole of enzyme. No oxidation of DPNH was observed, however, when the experiment was repeated by using the FMN-free apoenzyme (prepared by Method A). Attempted removal of nonheme iron from the enzyme. Based upon the observation (31) that the enzyme must be reduced with dithionite before any g = 1.94 signal is seen by electron paramagnetic resonance measurements, the nonheme iron is assumed to be in the $3 oxidation state. It has been shown Tube Number
’1 0.340fj--E’
Time, minutes 4. Stoichiometry of oxidation of DPNH by DPNH dehydrogenase. An anaerobic cuvette contained 0.1 ml of 1.66 X W3 M DPNH; 0.2 ml of 0.2 M phosphate buffer, pH 7.5; and 2.65 ml of water in the main compartment. DPNH dehydrogenase, 0.64 mg (FMN content, 16 mpmoles/mg protein), in 0.05 ml was present in the side arm. The cuvette was evacuated and filled with Nz. After the initial absorbance at 340 m/* was recorded, the enzyme was tipped in and the decrease in absorbance was measured at 30-second intervals against a blank containing only buffer. FIG.
5. Filtration of DPNH dehydrogenase through Bio-Gel. Bio-Gel P-200 was deaerated, packed under gravity into a 1.9 X 75-cm column, and equilibrated with 0.1 M phosphate buffer, pH 6.8. Flow rate, 30 ml/hour. Void volume (determined with Blue Dextran ZOOO), 65 ml. A solution containing 12.8 mg of enzyme in 2 ml of 0.1 M phosphate buffer, pH 6.8, was passed through the column and eluted with the same buffer. One-ml fractions were collected automatically and monitored for absorbancy at 280 rnp. Pooled fractions 85-98 (total volume, 14 ml), 99-108 (10 ml), and 109-125 (17 ml) were assayed for DPNH-ferricyanide reductase activity. Total protein recovered, 11.4 mg. FIG.
previously (2) that dialysis against Versene did not remove the iron. In the present study, treatment of the enzyme with dithionite and (Y,a-dipyridyl (which causes the protein to become red), followed by dialysis for 3 days against water, failed to remove any of the bound nonheme iron. Likewise, pretreatment of the enzyme with DPNH, followed by passage of the protein through a Chelex resin, did not affect the iron content. However, treatment with the resin was useful for removing extraneous iron. For example, two somewhat atypical enzyme preparations, having 2.80 and 2.27 atoms of nonheme iron per mole of protein, were passed through Chelex, whereupon the val-
COMPONENTS
OF
DPNH
ues dropped to 2.03 and 2.05, respectively, and remained constant even aft’er further recycling of the enzyme through the resin. Removal of FMN from the enzyme. It has been shown that FMN was readily removed when the enzyme was subjected to prolonged dialysis (2, 7) or passed through a Florisil column (7). Loss of FMN can also be achieved by f&ration of the enzyme through Bio-Gel (Fig. 5). Table IV presents typical data that compare the effectiveness of the Florisil and Bio-Gel treatments. In addition to removing most of the FMN, the Florisil procedure also resulted in the loss of about one-half of the nonheme iron and labile sulfide. The Bio-Gel procedure, which was about as effective as Florisil for removing FMN, caused a selective loss of nonheme iron with respect’ t)o labile sulfide. Neither of t,hese treatments affected the total thiol content of the protein. Table IV also records the enzymic activit’ies before and after these treatments. In confirmation of our previous results (7), removal of FMN (even accomTABLE TREATMENT
OF DPNH
443
DEHYDROGENASE
panied by considerable loss of nonheme iron and labile sulfide) did not depress the ferricyanide activity (see also Fig. 5 for data on the DPNH-ferricyanide activity of the Bio-Geltreated enzyme). However, loss of the flavin by either method greatly reduced the activity with indophenol or cytochrome c, and only the latter could be restored by the addition of FMN. Chromatography of the enzyme on DEAE-cellulose at pH 6.S, using a gradient of low ionic skength, yielded a symmetrical elution profile (Fig. 6) with a 96% recovery of protein. This procedure led to removal of essentially all of the flak and labile sulfide and about, two-thirds of the nonheme iron. Following this treatment, the enzyme had no activity with any of the acceptors, even in the presence of added FMN. Efects of thiol reagents on the enzyme. In an earlier study (3), fluorometric measurements were used to demonstrate that pCMS at 10e5 M caused the release of enzyme-bound IV
DEHYDROGENASE
WITH
FLORISIL
AND BIO-GEL
Florisil
A. Components” FMN Nonheme iron Labile sulfide Thiol groups
After
Before
After
1.03 1.86 0.92 7.14
0.098 1.15 0.53 7.21
0.95 2.05 0.91 7.20
0.13 1.36 0.87 7.00
wtolesDPNH/min~mg
B. Activity with acceptors” Ferricyanide Dichlorophenolindophenol Cytochrome c
Bio-Gel
Before
32.8 34.6 3.4
$rolein
26.5 4.74 0.18-2.4”
.umoles DPNH/min’nry
29.3 30.4 2.5
protein
29.6 6.5 0.9-1.7”
0 (A) Treatment with Florid. A solution containing 20.8 mg of DPNH dehydrogenase in 2 ml of water was passed through a 1 X lo-cm column of Florisil with water. The first 4 ml in the eluate were discarded and the next 10 ml containing the protein were recycled through the column as before. One-ml fractions were monitored at 280 rnp, and those containing protein were pooled and lyophilized. Recovery, 17 mg protein. 6 (B) Treatment with Bio-Gel. A solution containing 12.8 mg of DPNH dehydrogenase in 2 ml of 0.1 M phosphate buffer, pH 6.8, was passed through a 1.9 X 75.cm column of Bio-Gel P-200; elution was carried out with the same buffer. One-ml fractions were collected (flow rate, 0.5 ml/min) and monitored at 280 mp. The protein appeared in fractions 90-120. These fractions were lyophilized, and desalted by passage through Sephadex G-25 The entire procedure was repeated three times. Recovery of protein after three cycles, 8.2 mg. c Value before and after reactivation with 5 X 1W M FMN.
444
KUMAR
‘.“r-----l
ET AL. Ferricyanide
Dichlorop’wolindophenol
Cytcchrome
4
r
08.
log WMS),
M
7. Effect of preincubation with DPNH upon sensitivity of enzyme to pCMB. No preincubation ( l ) . The experimental cuvette contained 0.1 ml of 2.48 X lO+ M enzyme, pCMB (pH 7) at the indicated concentrations, and water to make 0.25 ml; the blank w&s identical except for pCMB. After standing at room temperature (about 22”) for 15 minutes, other components were added, and DPNH dehydrogenase assays, as described in the Experimental section, were performed. Preincubation with DPNH (A). Procedure same as above except that 0.1 ml of enzyme and 0.025 ml of 1OW M DPNH were preincubated at room temperature for 15 minutes before addition of pCMB . FIG.
Tube
number
6. Chromatography of DPNH dehydrogenase on DEAE-cellulose. DEAE-Cellulose was packed under gravity into a 1.5 X 27-cm column and equilibrated with lo+ M phosphate buffer, pH 6.8. A solution containing 61.6 mg of enzyme in 10 ml of W3 M phosphate buffer, pH 6.8, was adsorbed onto the column. Gradient elution was performed using 500 ml of the initial buffer in the mixing flask and 500 ml of 0.1 M phosphate buffer, pH 6.8, in the reservoir. Flow rate, 36 ml/hour. Three-ml fractions were collected automatically, and monitored for absorbancy at 280 mp. Total protein recovered, 59.5 mg. FIG.
centration of pCMB about one order of magnitude lower (10m4 M). Preincubation with DPNH markedly affected the sensitivity of the enzyme to mercurials. Under these conditions, the ferricyanide and cytochrome c activities were inhibited at much lower levels of the mercurial, but the indoFMN; the process required about 1.5 min- phenol curve remained essentially unutes at 25”. Dissociation of the flavin could changed. Complete inhibition of the ferricyalso be achieved with Cu2+, Ag+, and Cd2+ anide activity was now achieved at about a at loe3 M. 1: 1 ratio of inhibitor to enzyme, and the A more detailed study has now been made curve was no longer biphasic. The present of the effect of pCMB concentration upon findings are of interest in view of the report the various catalytic activities of the enzyme by Tyler et al. (32) of an “occult” thiol (Fig. 7). In each instance, the enzyme con- group in ETP-like preparations that becomes centration was 1 X lo+ M, and inhibition more sensitive to mercurials when the system curves for the enzyme preincubated with is pretreated with DPNH. DPNH were compared with those in which DISCUSSION DPNH was added after the inhibitor. In the controls (i.e., enzyme not preincubated wit,h If the mitochondrial electron transport DPNH), the activity with each acceptor de- chain linking DPNH to oxygen is assumed clined in a sigmoid curve as the concentra- to consist of a linear sequenceof oxidationtion of the mercurial was increased; in the reduction carriers (A, B, C, D, etc. in the case of the ferricyanide activity, the curve diagram below), it should be possible, was biphasic. Complete inhibition of the cytochrome c and indophenol activities was DPNH ---f rIC-D....( + 01 achieved at about 1O-3 RZpCMB, while the SCHEME 1 ferricyanide activity was abolished at a con-
COMPONENTS
OF
DPNII
in principle, to derive several different “DPNH dehydrogenases” from this chain. These enzymeswould differ in their content of carriers (A, AB, ABC, etc.), but each would be capable of oxidizing DPNH provided that an art’ificial electron acceptor could be found to react with the reduced form of the terminal carrier. It is not surprising, therefore, that the various soluble DPNH dehydrogenases, which have been extracted from part’iculate systems by an assortment of methods (acid-ethanol, thiourea, urea, snake venoms), should differ somewhat in molecular weight, mole ratios of bound oxidationreduction components (FMN, nonheme iron and labile sulfide), and specificity toward artificial acceptors [see (33) for a comprehensive review of these enzymes]. The properties of these soluble DPNH dehydrogenases are best understood in terms of the particulate DPNH-coenzyme Q reductase (34). The lat’ter complex has a molecular weight of about 509,000 and contains, per milligram of protein, 1.5 mpmoles of FAIN, 26 mpatoms of nonheme iron, a comparable amount of labile sulfide, and considerable lipid and structural protein. Recently, Hatefi and Stempel (35) have resolved the DPNH-Q reductase by treatment with urea into (a) a soluble flavoferroprotein (FMN, nonheme iron, and labile sulfide in the mole ratio of 1:4:4); (6) a ferroprotein (nonheme iron and labile sulfide in a ratio of 1: 1) ; and (c) an insoluble component similar to mitochondrial structural protein (36). The ability of the ferroprotein to serve as the oxidant for the DPNH-reduced flavoferroprotein suggests, as predicted previously by several investigators (34, 37, 38), that the DPNH --+ Q sequence in mitochondria may be formulated as follows: DPNH + flavoferroprotein + ferroprotein -+ Q. The above experiments of Hatefi and Stempel (35) help to clarify the relationship between the various DPNH dehydrogenases (2, 6, 21, 24, 33, 39, 40) that have been obtained from mitochondria, ETP, DPNH oxidase, or similar particulate precursors. Thus, the high molecular weight (> 300,000) particulate or quasi-soluble dehydrogenases with a high (> 10: 1) nonheme iron-to-flavin ratio (33, 40) appear to be closely related to the DPNH-Q reductase except for some loss of lipid, struct’ural protein, and a small
DEHYDROGENASE
445
amount of nonheme iron (see, for example, Table I in Ref. 41). Alternatively, the various low molecular weight (< lOO,OOO), soluble dehydrogenases (2, 6, 21, 24, 39) having nonheme iron-to-flavin ratios of 1:2 or 1:4 are similar to the flavoferroprotein of Hatefi and Stempel (35). This interpretation is consistent with the demonstration by Singer’s group that their high molecular weight DPNH dehydrogenase preparation (40, 42) can be transformed by a variety of methods [treatment with proteolytic enzymes (43), urea (43), thiourea (44)) or acid-ethanol (45)] into the smaller molecular weight dehydrogenases. It is rather remarkable that such a wide variety of agents as those listed above are able to extract soluble DPNH dehydrogenases from particulate precursors. Since urea, thiourea, and acid-ethanol very probably exert their effects without breaking covalent bonds, it appears as if the dehydrogenase pre-exists as a discrete entity encased more or less mechanically within the particle. If “solubilization” results from disrupting the particle and detaching the loosely bound dehydrogenase, it is understandable why there is considerable reproducibility in the preparation of any given soluble dehydrogenase and why further purification of these enzymes involves only the removal of other soluble proteins that may have been released adventitiously during the xolubilization process. The soluble DPNH dehydrogenases should be considered, therefore, as having been “released” from a particulate precursor, rather than having arisen by “degradation” of the latter (41). The present study demonstrates that treatment of the particulate DPNH oxidase by an acid-ethanol procedure leads to soluble flavoferroprotein, which, after two additional purification steps, is essentially homogeneous. The preparation is quite reproducible and only a single form of the enzyme is obtained, in contrast to the chromatographitally separable DPNH dehydrogenases that are obtained when Singer’s DPNH dehydrogenase is treated with acid-ethanol (45). The molecular weight of the enzyme is 70,000 as determined by filtration through Sephadex, and this value is in good agreement with the minimum molecular weight calculated
446
KUMAR
from the content of various oxidation-reduction components. Provided that care is taken during the isolation procedure, the enzyme contains almost exactly 1 mole of FMN per mole of protein. The identification of FMN as the prosthetic group of the DPNH portion of the mitochondrial electron transport chain has now been recorded by a number of investigators (7, 26, 35, 38, 42, 46, 47). The functional nature of FMN in the electron transport pattern. of the dehydrogenase is shown by (a) reduction of the flavin by DPNH, and (b) loss of DPNH-cytochrome c activity by the apoenzyme and its restoration by added FMN. The dehydrogenase contains 2 atoms of nonheme iron, probably in the Fe3+ state, per mole of protein. These are rather firmly attached and cannot be removed, even after reduction of the iron, by treatment with various chelating agents or by passage of the enzyme through Chelex resin. The nonheme iron contributes to the absorption spectrum of the enzyme (Fig. 3), and, if the contribution of FMN is subtracted, the spectrum of a ferroprotein (29) is obtained. The functional nature of the nonheme iron has not yet been demonstrated. Reduction of the iron nonenzymically (e.g., with dithionite) leads both to absorbancy changes in the 400-500 rnp region and to the appearance of a g = 1.94 signal (31), but these effects cannot be achieved by the addition of DPNH. The g = 1.94 signal seen in DPNH-Q reductase (48) and in 1similar preparations (49) probably arises, therefore, from the ferroprotein portion of these enzymes; none of the soluble flavoferroprotein dehydrogenases, containing 2 or 4 iron atoms per mole, show this signal during reduction of DPNH (33). The present DPNH dehydrogenase contains 1 mole of labile sulfide per mole of protein. The fact that this value is only one-half of the nonheme iron content is unusual and may reflect the loss of labile sulfide during exposure to the slightly acidic pH during the extraction procedure. If so, this finding implies that (a) the labile sulfide groups are not equivalent; (6) loss of labile sulfide does not automatically lead to loss of
ET AL.
nonheme iron; and (c) loss of part of the labile sulfide does not abolish catalytic activity, although it may account for the lower activity of the present preparation compared, for example, to the HatefiStempel enzyme (35). As shown by the data in Table IV, treatments that result in the loss of additional labile sulfide do not appreciably affect the DPNH-ferricyanide activity or the reconstructed DPNH-cytochrome c activity. Thus, the role of labile sulfide, like that of nonheme iron, is still obscure in the dehydrogenase. As determined by the DTNB or pCMB methods, the enzyme contains approximately 7 thiol groups per mole of protein. This value is in good agreement with the total number of cysteine residues determined by amino acid analysis. Addition of mercurials leads to the displacement of FMN (3), but it is not yet clear whether one or more of the thiol groups is directly involved in binding FMN to the protein or whether derivatization of these groups causes conformational changes in the protein that weaken ionic or hydrogen bonds to the coenzyme. In view of the lability of the enzymeFMN linkage toward mercurials, it is interesting that the electron acceptors commonly used with the enzyme do not appear to cause any release of FMN, although all of them are capable of oxidizing thiol groups. Inhibition by mercurials of the various catalytic activities of the dehydrogenase (Fig. 7) occurs at pCMB-to-enzyme ratios somewhat lower than those required to displace bound FMN. Also, in the case of the DPNH-ferricyanide activity, release of FMN cannot be the basis for inhibition by pCMB, since it has been shown previously (7) and in Table IV of this paper that the ferricyanide-reducing activity is retained by the flavin-free enzyme. Alternatively, the increased sensitivity toward mercurials when the enzyme is preincubated with DPNH (Fig. 7) suggeststhat thiol groups are participating directly in the electron transport mechanism. (This line of reasoning would be invalid, of course, if the mercurial were reacting with some other group other than a thiol group.) Alternatives would include a
COMPONENTS
vicinal dithiol group whose would be a cyclic disulfide
OF
oxidized
DPNH
form
447
DEHYDROGENSSE
lated as follows: ferricyanide
SH R’
\
*
DPNH
R’
SH
‘s
+
ei
--+ ferroprotein
T
or by a single thiol group whose form would be a thioketone
oxidized
L. indophenol, cyt . c, quinones SCHEME
2
ACKNOWLEDGMENTS H-
+ -SH
+
+.
Preincubation of the enzyme with DPNH in the absence of an external acceptor would be expected, therefore, to maximize the probability of the mercurial interacting with, and inactivating, the reduced form of a thiol component. The failure of the indophenol activity (middle panel of Fig. 7) to show a DPNH-dependent sensitivity toward mercurials is present’ly unexplained. The relative insensitivity of the DPNH dehydrogenase toward arsenite or Cd2+ would seem to rule out a vicinal dithiol grouping, and a single thiol group of the type described above has relatively few precedents (see, for example, Ref. 50) in enzymology. Alternatively, the catalytically active thiol group might be part of an ironlabile sulfide complex, different from that in ferredoxin, inasmuch as the sulfur rather t,han the iron would he t,he oxidationreduction component. Evidence presented in this paper (Figs. 3 and 4) indicates that each mole of enzyme can oxidize approximately 3 moles of DPNH. Two equivalents of oxidizing power could be accommodated by the FMN and ones ironsulfur moiety (Fe-S). If these groups form a linear sequence for electron transport, (Fe-S) would appear more likely to be the component adjacent’ to DPXH, whereupon the electron transfer sequence could be formu8 The determinations of labile sulfide reported in this paper (Table II) may be low since recent work in one of our laboratories (31) has shown a ratio of more nearly 1:2:2 for FMN, nonheme iron, and labile sulfide in the enzyme.
The authors ire indebted to Dr. Y. Hatefi for helpful discussions of this problem, to Mr. Calisto Munoz for the preparation of mitochondria, to Dr. K. Dus for carrying out the amino acid analyses, and to Dr. W. L. Yu for assistance in the starch-gel electrophoretic measurements. REFERENCES 1. KUMAR, S. A., R-10, N. A., FELTON, Y. P., HUENNEKENS, F. M., AND MACKLER, B., Federation Proc. 26, 738 (1966). 2. MACKLER, B., Biochim. Biophys. Acta 60, 141 (1961). 3. MACKLER, B., in “Nm-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 421. Antioch Press, Yellow Springs, Ohio (1965). -i. M~CKLER, B., in “Flavins and Flavoproteins” (E. C. Slater, ed.), p. 427. Elsevier, Amsterdam (1966). 5. MACKLER, B., .~ND GREEN, 1). E., Biochim. Biophys. Acta 21, 1 (1956). 6. MAHLER, H. R., SARKAR, N. K., VERNON, L. P., AND ALBERTY, R. A., J. Biol. Chem. 199, 585 (1952). 7. Rao, N. A., FELTON, S. P., HUENNEKENS, F. M., AND MACKLER, B., J. Biol. Chem. 238, 449 (1963). 8. ERNSTER, L., HOBERUAN, H. D., HOWARD, R. L., KING, T. E., LEE, C. P., MACKLER, B., AND SOTTOCASA, G., -vuture 207, 940 (1965). 9. WHIT~~KER, J. R., ilnd. Chem. 85, 1950 (1963). 10. GORNALL, A. B., B.iRDAWILL, C. J., .IND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 11. LOVENBERG, W., BUCHAN~~N, B. B., AND R~BIN~~ITZ, J. C., J. Biol. Chem. 238, 3899 (1963). 12. WARSVDEKAR, '\'. S., AND SASL.4W, L. I)., J. Biol. Chem. 234, 1945 (1959). 13. MASSEY,TT.,AND SWOBODA, B.E.P., Biochem. 2. 338, 474 (1963).
448
KUMAR
14. SMITHIES, O., Advan. Protein (1959). 15. DAVIS, B. J., Ann. N.Y. Acad.
Chem. Sci.
14,
65
121, 404
(1964). 16. ANDREW, P., Biochem. J. 96, 595 (1965). 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 18. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 19. BOYER, P. D., J. Am. Chem. Sot. 76, 4331
(1954). 20. HBTEFI, Y., HAAVIK, A. G., AND JURTSHUK, P., Biochim. Biophys. Acta 52, 106 (1961). 21. PH~RO, R. L., SORDAHL, L. A., VYAS, S. R., AND S~NADI, D. R., J. Biol. Chem. 241,
4771 (1966). 22. STEIN, A. M., KAPLAN, N. O., AND CIOTTI, M. M., J. Biol. Chem. 234, 979 (1959). 23. MXKLER, B., in “Biochemical Preparations” (M. J. Coon, ed.), Vol. 9, p. 40. Wiley, New York (1962). 24. DEBERNARD, B., Biochim. Biophys. Acta 23, 510 (1957). 25. R-40, N. A., AND FELTON, S. P., Federation Proc. 22, 411 (1963). 26. HUENNEKENS, F. M., FELTON, S. P., RAO, N. A., AND MACKLER, B., J. Biol. Chem. 236, PC57 (1961). 27. MACKLER, B., AND GREEN, D. E., Biochim. Biophys. Acta 21, 6 (1956). 28. DUYSENS, L. N. M., AND AMESZ, J., Biochim. Biophys. Acta 24, 19 (1957). 29. ALEMAN, v., SMITH, S. T., RAJAGOPALAN, K. V., AND HANDLER, P., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 327. Antioch Press, Yellow Springs, Ohio (1965). 30. M.\SSEY, J-., ~~~~~~~~ G., WILLUMS, C. H., SWOBOD.~, B. E. P., AND SANDS, R. H., in “Flavins and Flavoproteins” (E. C. Slater, ed.), p. 133. Elsevier, Amsterdam (1966). 31. XICKLER, B., ERICKSON, R. J., Davis, S. D., MEHL, J. T., SHARP, C., WEDGWOOD, R. J., PBLMER, G., AND KING, T. E., drch. Biochem. Biophys. in press. 32. TYLER, D. D., B~TOW, R. A., GONZE, J., .\ND EST;LBROOK, R. W., Biochem. Biophys. Res. Commun. 19, 551 (1965).
ET AL. 33. KING,
T. E.,
HEDGEKAR, K. S., AND
HOWARD,
R.
B. M.,
KuBOYAM~,
L.,
KETTMAN,
J.,
M., NICKEL, “Flavins and ed.), p. 441.
POSSEHL, E. A., in Flavoproteins” (E. C. Slater, Elsevier, Amsterdam (1966). 34. HATEFI, Y., in “Comprehensive Biochemistry” (M. Florkin and E. Stotz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam (1966). 35. HATEFI, Y., END STEMPEL, K. E., Biochem. Biophys. Res. Commun. 26, 301 (1967). 36. CRIDDLE, R. S., BOCK, R. M., GREEN, D. E., AND TISDALE, H. D., Biochemistry 1, 287
(1962). 37. REDFEARN, E. R., AND KING, T. E., Nature 209, 1313 (1964). 38. SANADI, D. R., PHARO, R., AND SORDAHL, L., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 429. Antioch Press, Yellow Springs, Ohio. (1965). 39. KING, T. E., AND HOWARD, R. L., Biochim. Biophys. Acta 37, 557 (1960). 40. RINGLER, R. L.,. MIN.~K~MI, S., AND SINGER, T. P., J. Biol. Chem. 238, 801 (1963). 41. SINGER, T. P., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 349. Antioch Press, Yellow Springs, Ohio (1965). 42. CREMONB, T., BND KEBRNEY, E. B., J. Biol. Chem. 239, 2328 (1964). 43. CREMON.4, T., KEbRNEY, E. B., T’ILL~vICENCIO, M., AND SINGER, T. P., Biochem. Z. 338, 407 (1963). 44. CREMONA, T., KEARNEY, E. B., AND VALENTINE, G., Nature 200, 673 (1963). 45. W.~URI, H., KEARNEY, E. B., AND SINGER, T. P., J. Biol. Chem. 238, 4063 (1963). 46. KING, T. E., Proc. Intern. Congr. Biochem., 5th, 1961, yol. 5, p. 207. Pergamon, New York (1961). 47. MEROL~, A. J., COLEMAN, R., AND HANSEN, R., Biochim. Biophys. Acta 73, 638 (1963). 48. HATEFI, Y., HUVII~ A. G., AND GRIFFITHS, D. E., J. Biol. Chem. 237, 1676 (1962). 49. BEINERT, H., PALMER, G., CREMONA, T., AND SINGER, T. P., J. Biol. Chem. 240, 475 (1965). 50. M~PSON, L. W., AND ISHERWOOD, F. A., Biochem. J. 86, 173 (1963).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
436-448
125,
Oxidation-Reduction
(1968)
Components
Diphosphopyridine
of a Reduced
Nucleotide
Dehydrogenase’ S. A. KUMAR,2 Department
N. APPAJI
of Biochemistry,
Scripps
RAO,2 S. P. FELTON: Clinic
and Research
Foundation,
F. M. HUENNEKENS La Jolla,
California
Washington
98106
9%‘037
AND
Department
of Pediatrics, Received
November
BRUCE
MACKLER4
University
of Washington,
23, 1967;
accepted
Seattle, December
19, 1967
DPNH dehydrogenase, solubilieed from a particulate DPNH oxidase by treatment of the latter with ethanol at pH 4.8 and 43”, has been further purified by precipitation with ammonium sulfate at pH 7.8 and by filtration through Sephadex G-25. The enzyme migrates as a single band when subjected to electrophoresis on starch gel or polyacrylamide. Based upon a molecular weight of 70,000 (determined by filtration through Sephadex G-200), each molecule of protein contains 1 FMN, 2 nonheme irons, 1 labile sulfide group, and 7 thiol groups. The latter are determined by titration of the protein with DTNB in the presence of 4 M urea. When the enzyme is reduced by DPNH under anaerobic conditions and in the absence of an external electron acceptor, the flavin appears to be fully reduced rather than at the semiquinone level; under these conditions, each mole of enzyme oxidizes approximately 3 moles of DPNH. Removal of the bound FMN by passage of the enzyme through Florisil or Bio-Gel does not affect the DPNH-ferricyanide activity while completely abolishing activity with cytochrome c or 2,6-dichlorophenolindophenol; cytochrome c a-,tivity can be largely restored by the addition of FMN. p-Chloromercuribenzoate (pCMB) inhibits the dehydrogenation of DPNH coupled to ferricyanide, indophenol, or cytochrome c; inhibition of the DPNH-ferricyanide activity is a biphasic function of pCMB concentration. Preincubation of the enzyme with DPNH increases the sensitivity toward pCMB when ferricyanide and cytochrome c are used as a:ceptors.
Previous reports from these laboratories (l-4) have described some of the properties of a DPNH dehydrogenase that is solubilized by treatment of beef heart DPNH oxidase (5) with acid-ethanol according to a modification of the procedure originally devised by iLIahler et al. (6). The enzyme was found to be reasonably homogeneous when examined electrophoretically (2, 7) or To Richard S. Schweet, pioneer in the field of peptide synthesis-in memoriam. 1 Paper X in the series “Flavin Nucleotides and Flavoproteins.” This work was supported by grants from the American JIeart Association (6% 436
in the ultracentrifuge (4, 7), but the molecular weight varied from 70,000 to 90,000, based on sedimentation measurements (4, 7) to 100,000 on the basis of the flavin 796) and the National Heart Institute (I-I-5457), National Institutes of Health. A preliminary report of this work has appeared elsewhere (1). 2 Present address: Ijepartment of Biochemistry, Indian Institute of Science, Bangalore, India. 3 Present address: Children’s Orthopedic Hospital, Seattle, Washington. 4 Research Career Development Awardee of the National Inst,itutes of Health.
COhlPONENTS
OF
J>PNH
content (7). The enzyme contained FMN and nonheme iron in the ratio of 1: 2 (2, 7). Both the DPNH dehydrogenase and the parent DPNH oxidase are specific for the B-form of DPNH and have a K, value of 1 X 1O-5 M for DPNH (8). The present report describes an improved purification procedure in which the acidethanol extracted enzyme is subjected to ammonium sulfate fractionation and filtration through Sephadex G-25. Electrophoretic criteria are presented for the homogeneity of the enzyme, and the molecular weight is shown to be 70,000 by Whitaker’s method (9). Analytical data are reported for various components (FMN, nonheme iron, and labile sulfide and thiol groups) that could be involved in the electron transfer mechanisms. Studies involving inhibition or deletion of these oxidation-reduction components provide a basis for formulating a tentative sequence of electron transfer when the dehydrogenation of DPNH is coupled to the various artificial electron acceptors (ferricyanide, 2,6-dichlorophenolindophenol, menadione, and cytochrome c). EXPERIMENTAL
PROCEDURE
l\fateri&. Chemicals were obtained from the following sources: DPNH, DPN, FMN, pCM8, pCMB,5 DTNB, bovine hemoglobin, trypsin, beef liver catalase, and cytochrome c from the Sigma Chemical Co.; bovine serum albumin from ilrmour and Co.; potassium ferricyanide from Fisher Scientific Co. ; 2, F-dichlorophenolindophenol from LaMotte Chemical Products Co.; acetylpgridine-DPN from P-L Biochemicals, Inc.; menadione from Merck & Co., Inc.; N,lV-dimethyl-p-phenylenediamiIle-HCl from Matheson, Coleman and Bell; Amido Black from K & K Laboratories; o-phenanthroline from G. Frederick Smit,h Chemical Co.; Sephadexes and Blue J)extran 2000 from Pharmacia; Bio-Gel (stored in 0.1 .n phosphate bufler, pH 6.8, for 3 months at 4”) and Chelex resin from Bio-Rad Laboratories; and DEAE-cellulose from Schleicher & Schuell Co. Coenzyme Q, was a gift from l)r. Y. Hatefi. Beef hearts were obtained from Talone Packing co. Methods. Absorbance changes at a single wave5 Abbreviations used: pCMB, p-chloromercuribenzoate; pCMS, p-chloromercuriphenylsul5,5’-dithiobis-(2.nitrobenzoic fonate; IjTNB, acid); ETP, electron transport particle.
DEHYDROGENASE
437
length were followed using a Beckman spectrophotometer, model DU. Complete spectra (220600 rnb) were measured with a Cary recording spectrophotometer, model 14, or a Beckman UK-2A recording spectrophotometer. Protein was determined by the biuret met.hod (10) with crystalline bovine serum albumin as the standard. Nonheme iron (ll), labile sulfide (ll), and sialic acid (12) were measured by published methods. After being released by heat- and acid-denaturation of the protein (7), FMN was quantitated by the decrease in absorbance at 450 rnp upon treatment with hydrosulfite; a millimolar extinction coefficient of 9.8 was used for the difference of oxidized minus reduced FMN (13). Electrophoresis was performed on horizontal starch-gel plates (13) or vertical polyacrylamide tubes (15); in each instance, protein bands were stained with Amido Black. The molecular weight of the protein was determined by the Andrews’ modification (16) of Whitaker’s method (9). Amino acid analyses were carried out by the method of Spackman et ul. (17). Thiol groups on the enzyme were assayed spectrophotometrically by the following procedure, based upon the DTNB method of Ellman (18). The following were added to a 1.5-ml ruvette having a light path of 1 cm: 0.5 ml of 0.1 M phosphate buffer, pH 8.0; l-2 mg of the protein; and 0.2 ml of a DTNB solution (40 mg of DTNB dissolved in 1.0 ml of 95y0 ethanol and diluted to 10 ml with 0.1 M phosphate buffer, pIi 8.0); water was used to adjust the volume to 1 ml. In some instances, ruea was also present at a final concentration of 4 M. The solution was allowed to stand at room temperature for 15 minutes and the absorbancy at 412 m,u was measured against a blank containing all components except the DTNB. A correction was made for the small initial absorbancy of DTNB. The thiol contentj of the protein was calculated by using a millimolar extinction coefficient of 13.G for the DTNB chromogen (17). Occasionally, the results were checked by Boyer’s method by using pCMB (19). Fluorescence of the enzyme was measured with a recording Amino-Bowman speetrophotofluxometer, eqllipped with a 150-W Hanovia Xenon light source and an RCA 1 P 28 photomultiplier tube. The exciting wavelength was 300 mH, and emission spectra were recorded with the Aminco 1620-809 X-Y recorder. Enzyme asscrys. DPNH dehydrogenase activity with the following acceptors was mea.sured spectrophotometrically by minor modifications of previously published methods: ferricyanide (7) ; 2, G-dichlorophenolindophenol (20) ; cytochrome
438
KUMAR
c (5) ; and menadione or coenzyme &I (21). Specific activities are expressed as micromoles of DPNH oxidized per minute per milligram of protein at 37”. Transhydrogenase activities were measured by the procedure of Stein el al. (22). Preparation of DPNH dehydrogenase. DPNH oxidase was prepared from beef heart mitochondria by the procedure of Mackler (23), except that the pH was taken only to 8.8, rather than to 10.0, in the initial step. DPNH dehydrogenase was solubilized and purified by the following modification of earlier procedures (2, 6, 24). Frozen DPNH oxidase (20 mg/ml in 5% sucrose) was thawed and diluted with one volume of cold water. The mixture was homogenized gently and centrifuged for 30 minutes at 40,000 rpm (Spinco, No. 40 head). The residue was suspended in cold water and the volume was adjusted to give a protein concentration of about 30 mg/ml. The pH was adjusted to 4.8 with 1 M acetic acid. Upon the addition of acetic acid, the mitochondrial suspension became less fluid. Ethanol at -10” was added dropwise to a final concentration of 9%. The beaker containing the mitochondrial suspension was then transferred to a water bath a 5&55”. The temperature of the suspension was allowed to rise as quickly as possible (no more than 15 minutes) to 43”. The temperature was maintained at 43” for 15 minutes, whereupon the beaker was transferred to a Dry-Ice and methoxyethanol bath, and the pH of the suspension was returned to 7.0 by the addition of 6 N KOH. After centrifugation for 15 minutes at 35,000 rpm (Spinco, No. 40 head), the residue was discarded and the supernatant fluid was lyophilized. The lyophilized powder was dissolved in 25 ml of cold wat,er (pH 7.0), and a saturated solution of ammonium sulfate, adjusted to pH 7.8 with concentrated NH,OH and kept previously at 4”, was added slowly (1 ml/5 minutes) until 44% saturation was reached (20 ml of ammonium sulfate per 25 ml of protein solution). The precipitate was removed by centrifugation for 20 minutes at 40,ooO rpm (Spinco, No. 40 head). For each 25 ml of original protein solution an additional 18 ml of ammonium sulfate was now added to the supernatant fluid to raise the concentration to 6Ooj, saturation. The precipitate was collected by centrifugation, dissolved in a minimum volume of water, and passed through a 2.5 X 35.cm column of Sephadex G-25 with water as the eluant. The yellow protein fraction immediately following the void volume was lyophilized and stored at - 20”. Removal of FMN from the dehydrogenase. Method A: Florisil was washed thoroughly with 1 M acetic acid, followed by water, and then packed by de-
ET
AL.
cantation into a 1 X IO-cm column. One liter of cold water was passed through the column. Two ml of a solution containing about 10 mg DPNH dehydrogenase/ml was passed through the column, which was then eluted with cold water. The pale yellow eluate (about 10 ml) was passed again through the column and the protein fraction (indicated by absorbance at 280 rnp) was collected and lyophilized. Recovery of protein after two cycles was usually about 8O-857o. Method B: Two ml of a solution containing about 12 mg of the dehydrogenase of 0.1 M phosphate buffer, pH 6.8, was passed through a 1.9 X 75-cm column of Bio-Gel P-200 that had been equilibrated with the same buffer. (Bio-Gel was deaerated before being packed in the column.) The void volume of the column, as determined with Blue Dextran 2000 (mol. wt. 2 X 10s), was 65 ml. DPNH dehydrogenase was eluted after g&120 ml of the above buffer had passed through the column. The eluate was lyophilized and the residue was dissolved in 2 ml of water (pH adjusted to 7.0, if necessary). The solution was passed through a 2 X 34-cm column of Sephadex G-25 to remove salt. This procedure was repeated several times. Recovery of protein after three cycles was usually about 65%. RESIJLTS
Homogeneity and molecular weight of the DPNH clehydrogenase. As purified by our
previous method (a), the DPNH drogenase
preparations
usually
dehy-
contained
5-10% extraneous protein, as judged by ultracentrifugal or electrophoretic examination (2, 4, 7). One of the more troublesome contaminants was cytochrome c, which obscures the absorption spectrum of the enzyme (considerably more cytochrome c is found at this stage if the enzyme is extracted from mitochondria or ETP rather than DPNH oxidase). Also loosely associatedwith earlier preparations of the enzyme was a sialic acid-like material (25).6 However, by 6 As much as 5 moles of this material (determined calorimetrically with thiobarbituric acid using N-acetyl neuraminic acid as the standard) per mole of FMN has been found in preparations that were taken only through a O-SO% precipitation with ammonium sulfate. The final step in the present purification procedure, i.e., filtration of the enzyme through Sephadex G-25, completely removes this material without any concomitant loss of enzymic activity. The properties of this compound will be described in detail elsewhere (Felton, S. P., Rao, N. A., Mackler, B., and Huennekens, F. M., in preparation).
COMPONENTS
OF
DPNII
narrowing the range of the ammonium sulfate fractionation to the 44-60 70 precipitate, followed by filtration of the enzyme through Sephadex, these and other contaminants have been largely eliminated. Electrophoresis of the purified enzyme on starch gel (14) at pH 8.2 and 4.6 revealed in each instance only a single protein band migrating slowly toward the positive and negative electrodes, respectively. A similar result was obtained by electrophoresis at pH 8.0 in a polyacrylamide column (15), except that the band was slightly diffuse. In our previous studies, the molecular weight of the enzyme, calculated from the flavin content (7), was higher than the value obtained from sedimentation measurements (4). In view of the relative ease with which F&IN can be detached from the protein (2, 7, 26), it seemedlikely that earlier preparations contained variable, although small, amounts of the apoenzyme. By careful handling of t’he enzyme in the present procedure, this difficulty has been overcome. The molecular weight of the protein, estimated to be
Cytochrome ’
2.5-
Trypsin
DPNH \ DehydrogenaseO
2.0-
E
Hemoglobin Serum Albumin
1\‘”
l Catalase 1.5-
\
I
4
1
5
6
log Molecular Weight FIN. 1. Molecular weight of DPNH dehydrogenase determined by filtration through Sephadex G-200. Sephadex G-200 (2W300 mesh) that had been stored in water for 3-4 months was resuspended in 0.15 M KC1 for 6 days, deaerated, and packed under gravity into a 2 X 50-cm column. The column was equilibrated with 0.15 M KCl. Reference proteins and DPNH dehydrogenase (4-7 mg each) were dissolved in 1 ml of 0.15 M KC1 and passed through the column to determine their respective elution volumes (V). T’oid volume (V”), 54 ml. Flow rate, 0.1 ml/minute.
439
J)EHYI>ROGENASE TABLE AMINO DPNH Amino
ACID
I
COMPOSITION
OF
L)EHTDROGENASE~~
*
acid
Composition
(moles/mole proteinJc
of
29.26 8.05 20.79 7.28 39.06 32.13 21.21 42.42 25.69 35.98 29.61 26.60 11.69 17.64 32.97 13.79 15.82
LYS His Arg CYS Asp
Thr Ser Glu Pro GUY Ala \-al Met Ile Leu ‘WPhe
U Enzyme used for this analysis had specific activities of 30 and 2.6 pmoles DPNH oxidized/ minute/mg protein with ferricyanide and cytochrome c, respectively, and contained 1.04 moles of FMN and 0.91 mole of labile sulfide per mole of protein. * Analysis performed by Dr. Karl I>us, University of California at San Diego. c Based upon a molecular weight of 70,009.
70,000 (Fig. 1) by the Andrews’ modification (16) of Whitaker’s method (9), is in good agreement with the minimum molecular weight calculated from analytical data for various oxidation-reduction components (see below). The amino acid composition of the enzyme, kindly determined by Dr. I<. Dus, is given in Table I. Oxidation-reduction DPNH dehydroyenase.
components
of
the
We have reported previously (2) that the enzyme contains FMN and nonheme iron in the ratio of approximately 1:2. As an extension of this work, the present preparations have been analyzed for various potential oxidationreduction components, namely F&IN, nonheme iron, labile sulfide, and thiol groups. The data, given in Table II, are expressed as mpmoles/mg protein and as moles/mole protein, with an assumed molecular weight of 70,000. For t’hree separate preparations of
440
KUMAR TABLE OF DPNH
COMPONENTS
II DEHYDROGENSSE
FMN
Nonheme iron Labile fide
sul-
Thiol groups a Figures protein.
(moles/mole
of enzyme)
1
2
TABLE
in
0.95 (13.5)a
0.99 (14.2)
1.05 (15.0)
(1:::
2.05 (29.3)
2.23 (31 A)
1.82 (26.0)
2.03 (29.0)
0.91 (12.9)
0.95 (13.5)
0.93 (13.3)
0.93 (13.2)
7.14
7.28 W)
7.00 (100)
7.14
parentheses
are
Specificactivitya Enzymepreparation 1 2 3
Acceptor
3
-
III
ACTIVITY 0~ DPNH DEHYDROGENasE WITH VARIOUS ACCEPTORS
Avg.
-
(102)
AL.
SPECIFIC
Enzymeprepardiion
components -
ET
-
(102)
mpmoles/mg
Ferricyanide Dichlorophenolindopheno1 Coenzyme Q1 Menadione Cytochrome c Acetylpyridine-DPN a Micromoles protein.
of
DPNH
31 30
40 38.8
33 40
11 6 2.7 1
12.5 6.2 3.4 0.8
10 5.7 3.7 1.1
oxidized/minute/mg
shown by the data in Table III. The enzyme does not show any appreciable TPNH-DPN transhydrogenase activity, but it doeshave a low DPNH-acetyl pyridine DPN activity (about 1 hmole DPNH oxidized/minute/mg protein). Previous measurements (2) have shown that the Michaelis constant for DPNH with this enzyme (with indophenol as acceptor) is 1.1 X lop5 M, a value in close agreement with that obtained for the particulate DPNH oxidase (27) from which the dehydrogenase is derived. In this investigation, the interaction of DPNH with the enzyme was examined nonkinet’ically with a fluorimetric method. When the prot’ein was excited at 300 rnp, an emission maximum at 350 rnp was observed (curve 1 in Fig. 2). Upon the addition of small increments of DPNH, the band at 350 rnp decreased progressively and a new band at 454 rnp appeared (curves 2-9) ; the latter corresponds to the fluorescencemaximum of DPNH (28). Alternatively, addition of DPN quenched the fluorescence at 350 rnp without any concomitant appearance of the 454 rnp band. Both DPNH and DPN produced maximum quenching at about 1 X 1O-4 M. From the data in Fig. 2, a K, value of 1.1 X 1O-5 ;M could be calculated’ for interaction of DPNH with the enzyme.
the enzyme, the following average values, per mole of protein, were obtained: 1 mole of FMN; 2 atoms of nonheme iron; and 1 mole of labile sulfide. Lower values of FMN (about 10 mpmoles/mg protein), encountered in earlier preparations of the enzyme (7), were probably caused by the facile dissociation of this coenzyme during the isolation procedure. Thiol groups on the protein were quantitated by the DTNB method (18), and checked by the pCMB procedure (19); both gave essentially the same results. In the presence of 4 M urea, 7 thiol groups were found per mole of enzyme (Table II), a value in agreement with the cysteine content (see Table I). In the absence of urea, however, only two or three of these thiol groups could be detected immediately with DTNB, and the remainder were slowly titratable over a 90minute period. Contrary to our earlier report (3), there was no appreciable increase in thiol groups when FMN was removed from the protein by Method A (see Experimental section). Catalytic and kinetic properties of the enzyme. DPNH dehydrogenase can utilize ferricyanide, indophenol, cytochrome c, and menadione (or coenzyme Q1) as electron ’ Since these measurements acceptors. The specific activities with these aerobic conditions, DPNH acceptors (pmoles DPNH oxidized/minoxidation by oxygen. This ute/mg protein) are reasonably constant for K, measurement, however, different preparat’ions of the enzyme, as DPN have about the same
were made under was undergoing slow does not invalidate the since both DPNH and affinity for t,he protein.
COMPONENTS
OF
DPNH
441
DEHYDROGEXASE
400 500 600 700 Wavelength, mp
Fro. 2. Effect of DPNII on fluorescence of DPNH dehydrogenase. The fluorescence of DPNH dehydrogenase (48 rg of enzyme in 1 ml of 0.04 M phosphate buffer, pH 7.5) was recorded (curve 0) in an Aminco-Bowman spectrophotofluorometer (sensitivity, 26; meter multiplier, 0.03). Curves l-9 represent the fluorescence spectrum after successive additions of 5-J increments of UPXH (1.6 rnM in 0.04 phosphate bnffer, pH 7.5).
Reduction of enzyme-bound j&win by DPNH. As shown by curve 1 in Fig. 3, the DPXH dehydrogenase has a principal absorbance band at about 440 rnp with a pronounced shoulder on the short, wavelength side (approximately 420 mp). A band of lower absorbancy is seen at about 550 rnp. The 420 and 650 mp bands are not’ due to contamination by cytochrome c, but very probably to the presence of nonheme iron (see Ref. 29 for a discussion of the contribu-
FIG. 3. Changes in absorption spectrum of enzyme upon addition of DPNH under anaerobic conditions. DPNH dehydrogenase (1.8 mg) dissolved in 1 ml of 0.04 M phosphate buffer, pH 7.5, was placed in an anaerobic quart,z cuvette equipped with a rubber stopper. The cuvette was evacuated and filled with Nz. The spectrum (curve 1) was recorded with a Beckman spectrophotometer, model DK-2A, against a blank containing only buffer. Small increments of 1.6 X 10-S M DPNH in 0.04 M phosphate buffer, pH 7.5, were added successively through the stopper by using a microsyringe. After each addition, the spectrum was again recorded (curve 2, total of 10 ~1; curve 3, total of 20 ~1; curve 4, total of 35 ~1; and curve 5, total of 45 ~1). At the end of the titration, 5 11 of a lyc solution of sodium dithionite was added (dashed curve) and the final spect.rlIm was recorded.
duced st’ate, rat’her t’han the semiquinone, as judged by the absence of long wavelength bands characteristic of the latter (30), and by t’he fact that further addition of dithionite tion of nonheme iron to the spectra of aft’er curve 5 did not appreciably change the flavoferroproteins). spectrum. During the transformation from When DPNH, even in a lo-fold excess curves 1 through 5, 0.072 pmole of DPNH relative to the flavin, was added to the enzyme under aerobic conditions, the de- was added. This may be compared with the amount of enzyme present (0.026 pmole, if a crease in absorbance at 450 rnp n-as about molecular weight of 70,000 is assumed). In so (2 of that produced by dithionite. cont,rast, addit,ion of small increments of Thus, approximately a 3-fold molal excess DPKH under anaerobic conditions caused of DPKH is required to reduce the enzymethe 450 rnp absorbance to decrease mark- bound FMN. Titration of DPNH by the enzyme. In the edly, the 420 rnp absorbancy lessso, and Dhe 550 peak not at all (curves 2-5 in Fig. 3). above experiment (Fig. 3) there was some about the exact amount, of The flavin appeared to be in the full>- re- uncertainty
442
KUMAR
ET
AL.
DPNH required for the complete reduction of the enzyme-bound flavin. Accordingly, the amount of DPNH capable of being oxidized by 1 mole of enzyme was measured in a different way. A known amount of enzyme was added anaerobically to an excess of DPNH, and the decrease in absorbancy at 340 rnl.c was followed. In the experiment shown in Fig. 4, 0.029 pmole of DPNH was oxidized by 0.009 pmole of enzyme. No oxidation of DPNH was observed, however, when the experiment was repeated by using the FMN-free apoenzyme (prepared by Method A). Attempted removal of nonheme iron from the enzyme. Based upon the observation (31) that the enzyme must be reduced with dithionite before any g = 1.94 signal is seen by electron paramagnetic resonance measurements, the nonheme iron is assumed to be in the $3 oxidation state. It has been shown Tube Number
’1 0.340fj--E’
Time, minutes 4. Stoichiometry of oxidation of DPNH by DPNH dehydrogenase. An anaerobic cuvette contained 0.1 ml of 1.66 X W3 M DPNH; 0.2 ml of 0.2 M phosphate buffer, pH 7.5; and 2.65 ml of water in the main compartment. DPNH dehydrogenase, 0.64 mg (FMN content, 16 mpmoles/mg protein), in 0.05 ml was present in the side arm. The cuvette was evacuated and filled with Nz. After the initial absorbance at 340 m/* was recorded, the enzyme was tipped in and the decrease in absorbance was measured at 30-second intervals against a blank containing only buffer. FIG.
5. Filtration of DPNH dehydrogenase through Bio-Gel. Bio-Gel P-200 was deaerated, packed under gravity into a 1.9 X 75-cm column, and equilibrated with 0.1 M phosphate buffer, pH 6.8. Flow rate, 30 ml/hour. Void volume (determined with Blue Dextran ZOOO), 65 ml. A solution containing 12.8 mg of enzyme in 2 ml of 0.1 M phosphate buffer, pH 6.8, was passed through the column and eluted with the same buffer. One-ml fractions were collected automatically and monitored for absorbancy at 280 rnp. Pooled fractions 85-98 (total volume, 14 ml), 99-108 (10 ml), and 109-125 (17 ml) were assayed for DPNH-ferricyanide reductase activity. Total protein recovered, 11.4 mg. FIG.
previously (2) that dialysis against Versene did not remove the iron. In the present study, treatment of the enzyme with dithionite and (Y,a-dipyridyl (which causes the protein to become red), followed by dialysis for 3 days against water, failed to remove any of the bound nonheme iron. Likewise, pretreatment of the enzyme with DPNH, followed by passage of the protein through a Chelex resin, did not affect the iron content. However, treatment with the resin was useful for removing extraneous iron. For example, two somewhat atypical enzyme preparations, having 2.80 and 2.27 atoms of nonheme iron per mole of protein, were passed through Chelex, whereupon the val-
COMPONENTS
OF
DPNH
ues dropped to 2.03 and 2.05, respectively, and remained constant even aft’er further recycling of the enzyme through the resin. Removal of FMN from the enzyme. It has been shown that FMN was readily removed when the enzyme was subjected to prolonged dialysis (2, 7) or passed through a Florisil column (7). Loss of FMN can also be achieved by f&ration of the enzyme through Bio-Gel (Fig. 5). Table IV presents typical data that compare the effectiveness of the Florisil and Bio-Gel treatments. In addition to removing most of the FMN, the Florisil procedure also resulted in the loss of about one-half of the nonheme iron and labile sulfide. The Bio-Gel procedure, which was about as effective as Florisil for removing FMN, caused a selective loss of nonheme iron with respect’ t)o labile sulfide. Neither of t,hese treatments affected the total thiol content of the protein. Table IV also records the enzymic activit’ies before and after these treatments. In confirmation of our previous results (7), removal of FMN (even accomTABLE TREATMENT
OF DPNH
443
DEHYDROGENASE
panied by considerable loss of nonheme iron and labile sulfide) did not depress the ferricyanide activity (see also Fig. 5 for data on the DPNH-ferricyanide activity of the Bio-Geltreated enzyme). However, loss of the flavin by either method greatly reduced the activity with indophenol or cytochrome c, and only the latter could be restored by the addition of FMN. Chromatography of the enzyme on DEAE-cellulose at pH 6.S, using a gradient of low ionic skength, yielded a symmetrical elution profile (Fig. 6) with a 96% recovery of protein. This procedure led to removal of essentially all of the flak and labile sulfide and about, two-thirds of the nonheme iron. Following this treatment, the enzyme had no activity with any of the acceptors, even in the presence of added FMN. Efects of thiol reagents on the enzyme. In an earlier study (3), fluorometric measurements were used to demonstrate that pCMS at 10e5 M caused the release of enzyme-bound IV
DEHYDROGENASE
WITH
FLORISIL
AND BIO-GEL
Florisil
A. Components” FMN Nonheme iron Labile sulfide Thiol groups
After
Before
After
1.03 1.86 0.92 7.14
0.098 1.15 0.53 7.21
0.95 2.05 0.91 7.20
0.13 1.36 0.87 7.00
wtolesDPNH/min~mg
B. Activity with acceptors” Ferricyanide Dichlorophenolindophenol Cytochrome c
Bio-Gel
Before
32.8 34.6 3.4
$rolein
26.5 4.74 0.18-2.4”
.umoles DPNH/min’nry
29.3 30.4 2.5
protein
29.6 6.5 0.9-1.7”
0 (A) Treatment with Florid. A solution containing 20.8 mg of DPNH dehydrogenase in 2 ml of water was passed through a 1 X lo-cm column of Florisil with water. The first 4 ml in the eluate were discarded and the next 10 ml containing the protein were recycled through the column as before. One-ml fractions were monitored at 280 rnp, and those containing protein were pooled and lyophilized. Recovery, 17 mg protein. 6 (B) Treatment with Bio-Gel. A solution containing 12.8 mg of DPNH dehydrogenase in 2 ml of 0.1 M phosphate buffer, pH 6.8, was passed through a 1.9 X 75.cm column of Bio-Gel P-200; elution was carried out with the same buffer. One-ml fractions were collected (flow rate, 0.5 ml/min) and monitored at 280 mp. The protein appeared in fractions 90-120. These fractions were lyophilized, and desalted by passage through Sephadex G-25 The entire procedure was repeated three times. Recovery of protein after three cycles, 8.2 mg. c Value before and after reactivation with 5 X 1W M FMN.
444
KUMAR
‘.“r-----l
ET AL. Ferricyanide
Dichlorop’wolindophenol
Cytcchrome
4
r
08.
log WMS),
M
7. Effect of preincubation with DPNH upon sensitivity of enzyme to pCMB. No preincubation ( l ) . The experimental cuvette contained 0.1 ml of 2.48 X lO+ M enzyme, pCMB (pH 7) at the indicated concentrations, and water to make 0.25 ml; the blank w&s identical except for pCMB. After standing at room temperature (about 22”) for 15 minutes, other components were added, and DPNH dehydrogenase assays, as described in the Experimental section, were performed. Preincubation with DPNH (A). Procedure same as above except that 0.1 ml of enzyme and 0.025 ml of 1OW M DPNH were preincubated at room temperature for 15 minutes before addition of pCMB . FIG.
Tube
number
6. Chromatography of DPNH dehydrogenase on DEAE-cellulose. DEAE-Cellulose was packed under gravity into a 1.5 X 27-cm column and equilibrated with lo+ M phosphate buffer, pH 6.8. A solution containing 61.6 mg of enzyme in 10 ml of W3 M phosphate buffer, pH 6.8, was adsorbed onto the column. Gradient elution was performed using 500 ml of the initial buffer in the mixing flask and 500 ml of 0.1 M phosphate buffer, pH 6.8, in the reservoir. Flow rate, 36 ml/hour. Three-ml fractions were collected automatically, and monitored for absorbancy at 280 mp. Total protein recovered, 59.5 mg. FIG.
centration of pCMB about one order of magnitude lower (10m4 M). Preincubation with DPNH markedly affected the sensitivity of the enzyme to mercurials. Under these conditions, the ferricyanide and cytochrome c activities were inhibited at much lower levels of the mercurial, but the indoFMN; the process required about 1.5 min- phenol curve remained essentially unutes at 25”. Dissociation of the flavin could changed. Complete inhibition of the ferricyalso be achieved with Cu2+, Ag+, and Cd2+ anide activity was now achieved at about a at loe3 M. 1: 1 ratio of inhibitor to enzyme, and the A more detailed study has now been made curve was no longer biphasic. The present of the effect of pCMB concentration upon findings are of interest in view of the report the various catalytic activities of the enzyme by Tyler et al. (32) of an “occult” thiol (Fig. 7). In each instance, the enzyme con- group in ETP-like preparations that becomes centration was 1 X lo+ M, and inhibition more sensitive to mercurials when the system curves for the enzyme preincubated with is pretreated with DPNH. DPNH were compared with those in which DISCUSSION DPNH was added after the inhibitor. In the controls (i.e., enzyme not preincubated wit,h If the mitochondrial electron transport DPNH), the activity with each acceptor de- chain linking DPNH to oxygen is assumed clined in a sigmoid curve as the concentra- to consist of a linear sequenceof oxidationtion of the mercurial was increased; in the reduction carriers (A, B, C, D, etc. in the case of the ferricyanide activity, the curve diagram below), it should be possible, was biphasic. Complete inhibition of the cytochrome c and indophenol activities was DPNH ---f rIC-D....( + 01 achieved at about 1O-3 RZpCMB, while the SCHEME 1 ferricyanide activity was abolished at a con-
COMPONENTS
OF
DPNII
in principle, to derive several different “DPNH dehydrogenases” from this chain. These enzymeswould differ in their content of carriers (A, AB, ABC, etc.), but each would be capable of oxidizing DPNH provided that an art’ificial electron acceptor could be found to react with the reduced form of the terminal carrier. It is not surprising, therefore, that the various soluble DPNH dehydrogenases, which have been extracted from part’iculate systems by an assortment of methods (acid-ethanol, thiourea, urea, snake venoms), should differ somewhat in molecular weight, mole ratios of bound oxidationreduction components (FMN, nonheme iron and labile sulfide), and specificity toward artificial acceptors [see (33) for a comprehensive review of these enzymes]. The properties of these soluble DPNH dehydrogenases are best understood in terms of the particulate DPNH-coenzyme Q reductase (34). The lat’ter complex has a molecular weight of about 509,000 and contains, per milligram of protein, 1.5 mpmoles of FAIN, 26 mpatoms of nonheme iron, a comparable amount of labile sulfide, and considerable lipid and structural protein. Recently, Hatefi and Stempel (35) have resolved the DPNH-Q reductase by treatment with urea into (a) a soluble flavoferroprotein (FMN, nonheme iron, and labile sulfide in the mole ratio of 1:4:4); (6) a ferroprotein (nonheme iron and labile sulfide in a ratio of 1: 1) ; and (c) an insoluble component similar to mitochondrial structural protein (36). The ability of the ferroprotein to serve as the oxidant for the DPNH-reduced flavoferroprotein suggests, as predicted previously by several investigators (34, 37, 38), that the DPNH --+ Q sequence in mitochondria may be formulated as follows: DPNH + flavoferroprotein + ferroprotein -+ Q. The above experiments of Hatefi and Stempel (35) help to clarify the relationship between the various DPNH dehydrogenases (2, 6, 21, 24, 33, 39, 40) that have been obtained from mitochondria, ETP, DPNH oxidase, or similar particulate precursors. Thus, the high molecular weight (> 300,000) particulate or quasi-soluble dehydrogenases with a high (> 10: 1) nonheme iron-to-flavin ratio (33, 40) appear to be closely related to the DPNH-Q reductase except for some loss of lipid, struct’ural protein, and a small
DEHYDROGENASE
445
amount of nonheme iron (see, for example, Table I in Ref. 41). Alternatively, the various low molecular weight (< lOO,OOO), soluble dehydrogenases (2, 6, 21, 24, 39) having nonheme iron-to-flavin ratios of 1:2 or 1:4 are similar to the flavoferroprotein of Hatefi and Stempel (35). This interpretation is consistent with the demonstration by Singer’s group that their high molecular weight DPNH dehydrogenase preparation (40, 42) can be transformed by a variety of methods [treatment with proteolytic enzymes (43), urea (43), thiourea (44)) or acid-ethanol (45)] into the smaller molecular weight dehydrogenases. It is rather remarkable that such a wide variety of agents as those listed above are able to extract soluble DPNH dehydrogenases from particulate precursors. Since urea, thiourea, and acid-ethanol very probably exert their effects without breaking covalent bonds, it appears as if the dehydrogenase pre-exists as a discrete entity encased more or less mechanically within the particle. If “solubilization” results from disrupting the particle and detaching the loosely bound dehydrogenase, it is understandable why there is considerable reproducibility in the preparation of any given soluble dehydrogenase and why further purification of these enzymes involves only the removal of other soluble proteins that may have been released adventitiously during the xolubilization process. The soluble DPNH dehydrogenases should be considered, therefore, as having been “released” from a particulate precursor, rather than having arisen by “degradation” of the latter (41). The present study demonstrates that treatment of the particulate DPNH oxidase by an acid-ethanol procedure leads to soluble flavoferroprotein, which, after two additional purification steps, is essentially homogeneous. The preparation is quite reproducible and only a single form of the enzyme is obtained, in contrast to the chromatographitally separable DPNH dehydrogenases that are obtained when Singer’s DPNH dehydrogenase is treated with acid-ethanol (45). The molecular weight of the enzyme is 70,000 as determined by filtration through Sephadex, and this value is in good agreement with the minimum molecular weight calculated
446
KUMAR
from the content of various oxidation-reduction components. Provided that care is taken during the isolation procedure, the enzyme contains almost exactly 1 mole of FMN per mole of protein. The identification of FMN as the prosthetic group of the DPNH portion of the mitochondrial electron transport chain has now been recorded by a number of investigators (7, 26, 35, 38, 42, 46, 47). The functional nature of FMN in the electron transport pattern. of the dehydrogenase is shown by (a) reduction of the flavin by DPNH, and (b) loss of DPNH-cytochrome c activity by the apoenzyme and its restoration by added FMN. The dehydrogenase contains 2 atoms of nonheme iron, probably in the Fe3+ state, per mole of protein. These are rather firmly attached and cannot be removed, even after reduction of the iron, by treatment with various chelating agents or by passage of the enzyme through Chelex resin. The nonheme iron contributes to the absorption spectrum of the enzyme (Fig. 3), and, if the contribution of FMN is subtracted, the spectrum of a ferroprotein (29) is obtained. The functional nature of the nonheme iron has not yet been demonstrated. Reduction of the iron nonenzymically (e.g., with dithionite) leads both to absorbancy changes in the 400-500 rnp region and to the appearance of a g = 1.94 signal (31), but these effects cannot be achieved by the addition of DPNH. The g = 1.94 signal seen in DPNH-Q reductase (48) and in 1similar preparations (49) probably arises, therefore, from the ferroprotein portion of these enzymes; none of the soluble flavoferroprotein dehydrogenases, containing 2 or 4 iron atoms per mole, show this signal during reduction of DPNH (33). The present DPNH dehydrogenase contains 1 mole of labile sulfide per mole of protein. The fact that this value is only one-half of the nonheme iron content is unusual and may reflect the loss of labile sulfide during exposure to the slightly acidic pH during the extraction procedure. If so, this finding implies that (a) the labile sulfide groups are not equivalent; (6) loss of labile sulfide does not automatically lead to loss of
ET AL.
nonheme iron; and (c) loss of part of the labile sulfide does not abolish catalytic activity, although it may account for the lower activity of the present preparation compared, for example, to the HatefiStempel enzyme (35). As shown by the data in Table IV, treatments that result in the loss of additional labile sulfide do not appreciably affect the DPNH-ferricyanide activity or the reconstructed DPNH-cytochrome c activity. Thus, the role of labile sulfide, like that of nonheme iron, is still obscure in the dehydrogenase. As determined by the DTNB or pCMB methods, the enzyme contains approximately 7 thiol groups per mole of protein. This value is in good agreement with the total number of cysteine residues determined by amino acid analysis. Addition of mercurials leads to the displacement of FMN (3), but it is not yet clear whether one or more of the thiol groups is directly involved in binding FMN to the protein or whether derivatization of these groups causes conformational changes in the protein that weaken ionic or hydrogen bonds to the coenzyme. In view of the lability of the enzymeFMN linkage toward mercurials, it is interesting that the electron acceptors commonly used with the enzyme do not appear to cause any release of FMN, although all of them are capable of oxidizing thiol groups. Inhibition by mercurials of the various catalytic activities of the dehydrogenase (Fig. 7) occurs at pCMB-to-enzyme ratios somewhat lower than those required to displace bound FMN. Also, in the case of the DPNH-ferricyanide activity, release of FMN cannot be the basis for inhibition by pCMB, since it has been shown previously (7) and in Table IV of this paper that the ferricyanide-reducing activity is retained by the flavin-free enzyme. Alternatively, the increased sensitivity toward mercurials when the enzyme is preincubated with DPNH (Fig. 7) suggeststhat thiol groups are participating directly in the electron transport mechanism. (This line of reasoning would be invalid, of course, if the mercurial were reacting with some other group other than a thiol group.) Alternatives would include a
COMPONENTS
vicinal dithiol group whose would be a cyclic disulfide
OF
oxidized
DPNH
form
447
DEHYDROGENSSE
lated as follows: ferricyanide
SH R’
\
*
DPNH
R’
SH
‘s
+
ei
--+ ferroprotein
T
or by a single thiol group whose form would be a thioketone
oxidized
L. indophenol, cyt . c, quinones SCHEME
2
ACKNOWLEDGMENTS H-
+ -SH
+
+.
Preincubation of the enzyme with DPNH in the absence of an external acceptor would be expected, therefore, to maximize the probability of the mercurial interacting with, and inactivating, the reduced form of a thiol component. The failure of the indophenol activity (middle panel of Fig. 7) to show a DPNH-dependent sensitivity toward mercurials is present’ly unexplained. The relative insensitivity of the DPNH dehydrogenase toward arsenite or Cd2+ would seem to rule out a vicinal dithiol grouping, and a single thiol group of the type described above has relatively few precedents (see, for example, Ref. 50) in enzymology. Alternatively, the catalytically active thiol group might be part of an ironlabile sulfide complex, different from that in ferredoxin, inasmuch as the sulfur rather t,han the iron would he t,he oxidationreduction component. Evidence presented in this paper (Figs. 3 and 4) indicates that each mole of enzyme can oxidize approximately 3 moles of DPNH. Two equivalents of oxidizing power could be accommodated by the FMN and ones ironsulfur moiety (Fe-S). If these groups form a linear sequence for electron transport, (Fe-S) would appear more likely to be the component adjacent’ to DPXH, whereupon the electron transfer sequence could be formu8 The determinations of labile sulfide reported in this paper (Table II) may be low since recent work in one of our laboratories (31) has shown a ratio of more nearly 1:2:2 for FMN, nonheme iron, and labile sulfide in the enzyme.
The authors ire indebted to Dr. Y. Hatefi for helpful discussions of this problem, to Mr. Calisto Munoz for the preparation of mitochondria, to Dr. K. Dus for carrying out the amino acid analyses, and to Dr. W. L. Yu for assistance in the starch-gel electrophoretic measurements. REFERENCES 1. KUMAR, S. A., R-10, N. A., FELTON, Y. P., HUENNEKENS, F. M., AND MACKLER, B., Federation Proc. 26, 738 (1966). 2. MACKLER, B., Biochim. Biophys. Acta 60, 141 (1961). 3. MACKLER, B., in “Nm-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 421. Antioch Press, Yellow Springs, Ohio (1965). -i. M~CKLER, B., in “Flavins and Flavoproteins” (E. C. Slater, ed.), p. 427. Elsevier, Amsterdam (1966). 5. MACKLER, B., .~ND GREEN, 1). E., Biochim. Biophys. Acta 21, 1 (1956). 6. MAHLER, H. R., SARKAR, N. K., VERNON, L. P., AND ALBERTY, R. A., J. Biol. Chem. 199, 585 (1952). 7. Rao, N. A., FELTON, S. P., HUENNEKENS, F. M., AND MACKLER, B., J. Biol. Chem. 238, 449 (1963). 8. ERNSTER, L., HOBERUAN, H. D., HOWARD, R. L., KING, T. E., LEE, C. P., MACKLER, B., AND SOTTOCASA, G., -vuture 207, 940 (1965). 9. WHIT~~KER, J. R., ilnd. Chem. 85, 1950 (1963). 10. GORNALL, A. B., B.iRDAWILL, C. J., .IND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 11. LOVENBERG, W., BUCHAN~~N, B. B., AND R~BIN~~ITZ, J. C., J. Biol. Chem. 238, 3899 (1963). 12. WARSVDEKAR, '\'. S., AND SASL.4W, L. I)., J. Biol. Chem. 234, 1945 (1959). 13. MASSEY,TT.,AND SWOBODA, B.E.P., Biochem. 2. 338, 474 (1963).
448
KUMAR
14. SMITHIES, O., Advan. Protein (1959). 15. DAVIS, B. J., Ann. N.Y. Acad.
Chem. Sci.
14,
65
121, 404
(1964). 16. ANDREW, P., Biochem. J. 96, 595 (1965). 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 18. ELLMAN, G. L., Arch. Biochem. Biophys. 82, 70 (1959). 19. BOYER, P. D., J. Am. Chem. Sot. 76, 4331
(1954). 20. HBTEFI, Y., HAAVIK, A. G., AND JURTSHUK, P., Biochim. Biophys. Acta 52, 106 (1961). 21. PH~RO, R. L., SORDAHL, L. A., VYAS, S. R., AND S~NADI, D. R., J. Biol. Chem. 241,
4771 (1966). 22. STEIN, A. M., KAPLAN, N. O., AND CIOTTI, M. M., J. Biol. Chem. 234, 979 (1959). 23. MXKLER, B., in “Biochemical Preparations” (M. J. Coon, ed.), Vol. 9, p. 40. Wiley, New York (1962). 24. DEBERNARD, B., Biochim. Biophys. Acta 23, 510 (1957). 25. R-40, N. A., AND FELTON, S. P., Federation Proc. 22, 411 (1963). 26. HUENNEKENS, F. M., FELTON, S. P., RAO, N. A., AND MACKLER, B., J. Biol. Chem. 236, PC57 (1961). 27. MACKLER, B., AND GREEN, D. E., Biochim. Biophys. Acta 21, 6 (1956). 28. DUYSENS, L. N. M., AND AMESZ, J., Biochim. Biophys. Acta 24, 19 (1957). 29. ALEMAN, v., SMITH, S. T., RAJAGOPALAN, K. V., AND HANDLER, P., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 327. Antioch Press, Yellow Springs, Ohio (1965). 30. M.\SSEY, J-., ~~~~~~~~ G., WILLUMS, C. H., SWOBOD.~, B. E. P., AND SANDS, R. H., in “Flavins and Flavoproteins” (E. C. Slater, ed.), p. 133. Elsevier, Amsterdam (1966). 31. XICKLER, B., ERICKSON, R. J., Davis, S. D., MEHL, J. T., SHARP, C., WEDGWOOD, R. J., PBLMER, G., AND KING, T. E., drch. Biochem. Biophys. in press. 32. TYLER, D. D., B~TOW, R. A., GONZE, J., .\ND EST;LBROOK, R. W., Biochem. Biophys. Res. Commun. 19, 551 (1965).
ET AL. 33. KING,
T. E.,
HEDGEKAR, K. S., AND
HOWARD,
R.
B. M.,
KuBOYAM~,
L.,
KETTMAN,
J.,
M., NICKEL, “Flavins and ed.), p. 441.
POSSEHL, E. A., in Flavoproteins” (E. C. Slater, Elsevier, Amsterdam (1966). 34. HATEFI, Y., in “Comprehensive Biochemistry” (M. Florkin and E. Stotz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam (1966). 35. HATEFI, Y., END STEMPEL, K. E., Biochem. Biophys. Res. Commun. 26, 301 (1967). 36. CRIDDLE, R. S., BOCK, R. M., GREEN, D. E., AND TISDALE, H. D., Biochemistry 1, 287
(1962). 37. REDFEARN, E. R., AND KING, T. E., Nature 209, 1313 (1964). 38. SANADI, D. R., PHARO, R., AND SORDAHL, L., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 429. Antioch Press, Yellow Springs, Ohio. (1965). 39. KING, T. E., AND HOWARD, R. L., Biochim. Biophys. Acta 37, 557 (1960). 40. RINGLER, R. L.,. MIN.~K~MI, S., AND SINGER, T. P., J. Biol. Chem. 238, 801 (1963). 41. SINGER, T. P., in “Non-Heme Iron Proteins: Role in Energy Conversion” (A. San Pietro, ed.), p. 349. Antioch Press, Yellow Springs, Ohio (1965). 42. CREMONB, T., BND KEBRNEY, E. B., J. Biol. Chem. 239, 2328 (1964). 43. CREMON.4, T., KEbRNEY, E. B., T’ILL~vICENCIO, M., AND SINGER, T. P., Biochem. Z. 338, 407 (1963). 44. CREMONA, T., KEARNEY, E. B., AND VALENTINE, G., Nature 200, 673 (1963). 45. W.~URI, H., KEARNEY, E. B., AND SINGER, T. P., J. Biol. Chem. 238, 4063 (1963). 46. KING, T. E., Proc. Intern. Congr. Biochem., 5th, 1961, yol. 5, p. 207. Pergamon, New York (1961). 47. MEROL~, A. J., COLEMAN, R., AND HANSEN, R., Biochim. Biophys. Acta 73, 638 (1963). 48. HATEFI, Y., HUVII~ A. G., AND GRIFFITHS, D. E., J. Biol. Chem. 237, 1676 (1962). 49. BEINERT, H., PALMER, G., CREMONA, T., AND SINGER, T. P., J. Biol. Chem. 240, 475 (1965). 50. M~PSON, L. W., AND ISHERWOOD, F. A., Biochem. J. 86, 173 (1963).