Studies on dihydronicotinamide adenine dinucleotide ubiquinone reductase

ARCHIVES

OF

BIOCHEMISTRY

Studies

on

AND

BIOPHYSICS

1%.

416-828

Dihydronicotinamide

Adenine

Ubiquinone

L. PHARO,

LOUIS AND

Dinucleotide

Reductase

II. Purification RICHARD

(1968)

and

Properties

A. SORDAHL,I

HAR.OLD

EDELHOCH,

D. R. SANADI

Department of Bioenergetics Research, Institute of Biological and Medical Sciences, Retina Foundation, Boston, Massachusetts; and Gerontology Research Center, N.I.C.H.D., National Institutes of Health, P.H.S., Baltimore, Maryland Received

November

13, 1967

The ubiquinone reductase activity of a soluble extract of beef heart submitochondrial particles can be separated into two active fractions by chromatography on a hydroxylapatite column. Fraction I was further purified by ammonium sulfate fractionation and Fraction II by chromatography on DEAE-cellulose. Both fractions catalyze the oxidation of NADH by ubiquinone-10, ubiquinone-6, ubiquinone-1, menadione, cytochrome c, and ferricyanide at nearly equal rates. Both contain FMN, nonheme iron, and acid-labile sulfide in the approximate ratio of 1:4:5, and have nearly identical spectra. Sedimentation and electrophoretic analyses indicate that the fractions are better than 95% homogeneous. The molecular weights of the two preparations are very similar (near 90,000), although their electrophoretic mobilities differ. NADH causes anaerobic bleaching of the flavin (450 mp), but the absorption peak at 550 rnp is not altered. The reductases are inhibited by Amytal and rotenone, the latter showing an unusual biphasic curve with maximal inhibition at a rot.enone to enzyme flavin ratio of nearly one. Piericidin A, dicumarol, o-phenanthrolene, or Tiron do not inhibit reduct,ase activity.

It has been shown previously that, a soluble NADH ubiquinone reductase capable of utilizing the higher isoprenologs of ubiquinone as electron acceptors can be extracted from beef heart submitochondrial particles (1). The ubiquinone reductase activity can be separated into two flavoprotein enzymes on hydroxylapatite or by electrophoresis. This report describes the purification and properties of each of these To Richard S. Schweet, pioneer in the field of peptide synthesis-in memoriam. 1 Present address: Department of Pharmacology, Baylor University College of Medicine, Texas Medical Center, Houston, Texas. 2 Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. 41F

fractions. Preliminary reports of this work have appeared previously (2-8). MATERIALS

AND

METHODS

The isolation of submitochondrial particles, and extraction of reductase activity from these particles by acidic aqueous ethanol, have been reported earlier (1,9). The assay conditions necessary for measuring quinone reductase activity were described. All further purification procedures were carried out in an ice-bath or in a cold room at less than 4”. Separation of two jlavoproteins on hydroxylapatite. Several types of hydroxylapatite were tried to achieve a separation of the acid-ethanol extract into two flavoproteins. The gels which were prepared in this laboratory by the method of Tiselius (10) were fine a?d flocculent and packed very tightly in the column, allowing only slow

UBIQUINONE

REDUCTASE.

i TOTAL

Ferricyanide

ACTIVITY

UNITS

hfenodione

f-J

0.04M Phosphofl

417

II

1

O./M Phosphofe

3

40

I

i I

/ ... .J

FRACTION

50

60

70

NUMBER

FIG. 1. Hydroxylapatite column chromatography of submitochondrial particle extract. A total of 69 mg of lyophilized protein from an acid-ethanol extract of beef heart submitochondrial particles was applied to a 2.2 X lo-cm column of hydroxylapatite. After being washed with 0.01 M phosphate-l rnM EDTA buffer, pH 7.5, the first active fraction was eluted with 0.04 M phosphate buffer, and the second fraction with 0.10 M phosphate buffer. The volume of each fraction was about 4 ml. Each fraction was assayed individually for menadione reductase (-), Q6 reductase (- - -), and ferricyanide reductase (. . .) activity. Protein (-.-.-) was determined by the Lowry procedure (13). The menadione reductase specific activities of the extract,, peak Fraction I, and peak Fraction II were 63, 101, and 96, and the QI red&case specific activities were 8.1, 19.3, and 14.0, respectively. flow through the column even at pressures in excess of 25 psi. Mixing cellulose with the gel in a ratio as high as 4:l did not increase the flow rate, apparently because the gel filtered through the cellulose and settled at the bottom of the column. Of the two commercial hydroxylapatites, only one,“Bio-Gel HTP” (Bio-Rad Laboratories, Riehmond, California), gave sufficiently reproducible results from one lot to another to make it useful for routine separation. The Bio-Gel HTP, in the form of a dry powder, was suspended in 10 mM phosphate buffer, pH 7.5, at a concentration of 130 mg gel per milliliter buffer and allowed to stand overnight in the cold. The chromatography column was prepared by filling a glass column with an appropriate amount of this suspension and immediately packing the gel by forcing buffer through the column with a “finger” pump at a flow rate of 2.5 ml/minute. Once the pressure was applied, the flow through the column was not interrupted until the entire chromatographic procedure was completed. Excess liquid above the column could be forced through by interrupting liquid flow into the column and pumping only air until the liquid had reached the desired level for changing eluate. If

the pressure was released, or the flow was stopped, flow rates through t,he column became exceedingly slow. Figure 1 shows a typical chromatographic elution pattern. The lyophilized extract of submitochondrial particles, which contained 69 mg protein, was dissolved in 25 ml water. This was pumped on a 2.2 X lo-cm column of hydroxylapatite that had been previously equilibrated with 10 mM phosphate-l mM EDTA, pH 7.5. All buffers used subsequently in the purification contained 1 mM EDTA to reduce loss of enzymic activity. The effluent from the column was monitored at 280 rnb. A component having a spectrum similar to that’ of FMN, and having essentially no reductase aetivit.y, was washed through with the 10 rnM phosphate buffer. When no more ultraviolet-absorbing material appeared in the effluent, 0.04 M phosphate buffer, pH 7.5, was pumped through the column. This eluted the first reductase fraction in about 40 ml. When the ultraviolet absorption again reached the base line, 0.1 M phosphate buffer, pH 7.5, was introduced in the column, and the second reductase fraction was eluted in about 40 ml. At this point, each

418

PHARO

fract,ion was lyophilized and stored for short periods of time at -70” until further purified.3 Purijication of Fraction I by precipitation with ammonium sulfate. Lyophilized Fraction I was dissolved in about 5 ml distilled water and passed through a column of Sephadex G-25 equilibrated with 10 mM phosphate-l mM EDTA buffer, pH 7.5. The protein concentration was adjusted to 5 mg/ml, and 225 mg solid ammonium sulfate was added per milliliter protein solution. This solution was stirred for 20 minutes, and the resulting precipitate was eliminated by centrifugation. Sixt,y mg solid ammonium sulfate was added to each milliliter of t,he supernatant fluid. After 20 minutes the precipitate was collected by centrifugation. The pellet, containing the purified reductase, was dissolved in 10 mM phosphate-l mM EDTA buffer, pH 7.5, and the ammonium sulfate was removed on Sephadex G-25. The resulting Fraction I-P was used immediately for characterization and activity experiments. Flavin analysis was also carried out immediately. Purification of Fraction II by DEAE-cellulose column chromatography. Lyophilized Fraction II was dissolved in about 5 ml distilled water and equilibrated on Sephadex G-25 with 3 mM phosphate buffer, pH 8.0, with no EDTA. The EDTA was not added to this buffer because the enzyme binds to the DEAE at such low ionic strength that the addition of the 1 mM EDTA tends to interfere with the binding. The enzyme from the Sephadex column was then dialyzed for 3 hours against 1 liter of 3 mM buffer with hourly changes in buffer. DEAE-Cellulose was prepared in the phosphate form by prewashing with 1 M phosphate buffer, followed by extensive washing with 3 mM phosphate buffer, pH 8.0. A 1 X lo-cm column of the DEAE-cellulose was equilibrated with 3 mM phosphate buffer, pH 8.0. The dialyzed Fraction II was applied to the column with pumping, and after a protein contaminant was washed off with the 3 mM buffer, the active Fraction II was eluted with 15 mM phosphate buffer, pH 8.0, in a volume of about 20 ml. The enzyme could be eluted from the column with only 8 mM buffer; however, since there is no contaminant eluted at 15 mM buffer, 3 It had been observed early in this work that a significant loss of reductase activity occurred when the preparations were lyophilized. This was seen whenever the lyophilizing was done overnight. However, since then it has been noted that when the lyophilization is kept to a minimum time, just long enough to achieve dryness, there is little loss of activity. Presumably, exposure of the enzymes to room temperature, even when dry and under vacuum, can lead to loss of activity.

ET

BL.

this concentration was routinely used in order to recover the enzyme in a smaller volume. Fraction II was further purified by adding 360 mg solid ammonium sulfate per milliliter of the eluate from the DEAE column. After 20 minutes, the precipitate was recovered by centrifugation. The pellet was dissolved in 10 IIIM phosphate-l mM EDTA buffer, pH 7.5, and the ammonium sulfate was removed on Sephadex G-25. This purified enzyme preparation also was used immediately for all experiments including flavin analysis and inhibition studies. Iron determination. The assay for the nonheme iron content of the enzyme was a modification of the micro method of Mahler et al. (II). The enzyme sample was deproteinized with 57, trichloroacetic acid (TCA) (final concentration). After centrifugation, aliquots of the supernatant fluid, containing iron in the range of 10-100 nmoles, were made up to 0.4 ml volume with5% TCA. The following freshly prepared reagents were added in order: 0.5 ml of 1 M sodium acetat,e buffer, pH 4.0; 0.5 ml of 0.5% ophenanthroline in 10% ethanol; and 0.1 ml of 0.6 M ascorbate, pH 6.8. After the contents were mixed, the tube was placed in a 38” water bath for 30 minutes and then cooled, and the absorbance of the sample was determined at 500 rnp, using a reagent blank containing all reagents and 5’% TCA in place of the sample. Using either standard FeCl2 (Bio-Rad Laboratories) or ferrous ammonium sulfate, this assay yielded a millimolar extinction coefficient of 12 at 500 rnp under these experimental conditions. Sulfide defermination. The acid-labile sulfide cont,ent of the enzyme was determined by a modification of the procedure of Fogo (12). A sample of 0.85 ml was placed in a tightly capped test tube. The following freshly prepared reagents were added in order with adequate mixing betwsen each addition: 1.3 ml of 1% ZnAc ; 0.5 ml of 12% NaOH; 0.25 ml of 0.1% N,N-dimethyl-p-phenylenediamine in 5.5 M HCl; and 0.05 ml of 0.23 M FeC13 in 1.2 M HCl. The color was allowed to develop at room temperature for 30 minutes, and the absorbance at 670 m was read against a reagent blank. The assay was sensitive to 0.05-5 pg sulfide. Other materials and methods. Protein concentration was determined by the method of Lowry (13); crystalline bovine serum albumin dried over PZOS was used as standard. Other methods have been described (1). Nucleotides were obtained from the Sigma Chemical Co.; ubiquinone from Mann Research Laboratories; and rotenone from K&K Laboratories. We are indebted to Dr. Karl Folkers for the generous gift of Piericidin A.

UBTQUINOXE

REDUCTASE. TABLE

PURIFICATION EXTRXTS Fraction

Total

Acid-ethanol extract,

protein (md

OF VISIQCJIK~SE OF BEEF

319

II

I

REDLKXXSE FRMZTIOXS HEART MIT~CH~NDRI~YL

I ,~ND PIRTICLES

II

FROM

Menadione sp. act.a

Q6 Recovery

(70)

sp. act.”

.-

60

82

Recovery

16.9

Fraction Fraction

I II

18 20

107 102

39 41

24.8 20.3

45 39

Fraction Fraction

I-P II-P

10 9.6

146 178

29 36

26.4 28.1

25 27

11Micromoles were assayed matography.

NASH oxidized/minute/mg protein. Menadione reductase as described previously (1). Fractions I and II were assayed Fractions I-P and II-P were assayed after final purification RESULTS

f’ur@~~~ion of Fractions I and II. Table I indicates the purification of the two ubiquinone reductase fractions from the starting aqueous alcoholic extract of submit’ochondrial particles. The total menadione and Q6 reductase activities are divided almost equally bet#ween Fractions I and II. Menadione reductase specific activity is nearly doubled on purification over that of the starting extract. It is almost 700 times higher than the activity of the submit’ochondrial particles, m-hich have menadione reductase specific activity of about 0.2 (1). The Q6 reductase activity does not exhibit this extent of purification partly because of its instability (see also Ref. 1). In fact, t,he in&ability leads to a decrease in specific act)ivity, although inactive protein is removed during the final stages of purification (Table II). Many attempts have been made to overcome this instability, including storage of the enzyme anaerobically under nitrogen and in the presence of NAD, NADH, menadione, menadione plus NADH, thioglycerol, dithioerythritol, ethanolamine, mercaptoethanol, and FMN. None of these measures has been successful. Storage in the cold does retard some of the activity loss, and preparations may be kept at -70” for short periods of time, if necessary. In a preliminary report (2) we reported that the absorption spectrum of a partially

(o/o)

and Q6 reductase activities after hydroxylapatite chroas described in the text. TABLE

ANALYSES

OF UBIQUINONE

FMN Nonheme iron Acid-labile sulfide FMN: iron: sulfide Molecular weight Flavin basis Ultracentrifuge u Concentrations protein.

II REDUCTME

FRICTIONS

Fraction I-P

Fraction

10.3” 41.8 50 1:4.1:4.9

9.3” 42.1 53 1:4.5:5.7

98,000

are

reported

II-P

-

108,000 98,000 in

nmoles/mg

purified ubiquinone reductase preparation showed a peak at 410 mp. In the present procedure, which leads to separation of two reductase fractions on hydroxylapatite, the component with absorption at 410 rnw, presumably a hemoprotein, appears in Fraction II. Purification of Fraction II on DEAEcellulose yields a preparation free of the hemoprotein contaminant. Fractions I and II have been subjected to rechromatography on hydroxylapatite under conditions initially used for the separation. Each fraction was rechromatographed separately, and a mixture of the two was rechromatographed. In each case, the fraction was eluted from the column at the original concentration. Elution of the reductase from the column was determined both

420

PHARO

by measuring the reductase activity and monitoring the 280 rnp absorption of the column effluent. It was also possible to see a faint fluorescence associated with the reductases as they migrated down the column. Chemical analyses. Both reductase fractions contained FMN, nonheme iron, and acid-labile sulfide. The TCA extract contained a flavin that was identified as FXIN by using three solvent systems in paper chromatography (14), and by its high fluorescence compared to FAD. More rigorous characterization was considered unnecessary, since it is now generally accepted that FMN is the prosthetic group of the NADH oxidizing flavoproteins (15). Table II shows results of the chemical analyses. Both fractions contained roughly the same amount of each of these components, in an FMN-iron-sulfide molar ratio of approximately 1:4:5. The slightly lower FMN content of Fraction II could be due to dissociation and loss during isolation of the enzyme. Analysis in the ultracentrifuge. The sedimentation coefficients of the two reductase preparations were determined in the model E Spinco ultracentrifuge at 5-S”, and corrected to water at 20”. Figure 2 shows the sedimentation patterns. A small amount of a slower sedimenting component has been observed in all preparations of Fraction I-P. The preparation of Fraction II-P has a small amount of a more rapidly sedimenting contaminant. The szolu,values for Fractions I-P and II-P were 5.00 and 5.15, respectively, at a protein concentration of 0.3%. Molecular weights were determined by the Yphantis meniscus depletion procedure by using interference optics (16). The buffer used was 50 mM phosphate, pH 8.0. The temperature of the experiment was 25”. The solution column heights were 2.7 mm. Weight-average molecular weights were calculated from the slope of a plot of In c versus r2 according to the following equation :

Units

of c were in readings of y-coordinate

ET

AL.

FIG. 2. Sedimentation patterns of Fractions 1-P and II-P. Fraction I-P and Fraction II-P were purified as described in the text. Menadione and Q6 reductase specific activities at the time of the centrifugal analyses were 144 and 26, respectively, for Fraction I-P and 105 and 17 for Fraction II-P. Figure 2A represents the pattern of Fraction I-P after centrifugation at 56,000 rpm for 128 minutes and shows the slower trailing material from the origin. Figure 2B is the ultracentrifuge pattern of Fraction II-P after 108 minutes at the same speed. The enzymes were both dissolved in 50 mM phosphate buffer, pH 8.0.

7

displacement of the first fringe from the horizontal. Starting protein concentrations were 0.04%. The plot of In c versus r2 gave a linear relationship for Fraction II-P. A molecular weight of 98,000 was computed by assuming a value of 0.73 for p. A slightly curved line was found for Fraction I with a molecular weight near 70,000. Since this preparation was contaminated with slower sedimenting material (Fig. 2) that contributed rather strongly to the molecular weight, this value is considered as being too low and not representative of t,he major component.

UBIQUINONE

Electrophoresis. While both reductase fractions are similar in many respects, they differ in their electrical charge, as evidenced by at least two methods. Fraction I binds poorly, if at, all, to DEAE-cellulose in 3 m&i phosphate buffer, but Fraction II binds well. This difference can be used, in fact, to separate the two reductase fractions. Also significant is the difference in electrophoretic mobilities. Figure 3 shows the results of simultaneous electrophoresis of the two fractions and the crude extract. on cellulose acetate strips. The purified fractions mi-

t-

6.lcm.-4

-Jk ORIGIN 3. Cellulose acetate electrophoresis of ubiquinone reductase fractions. Electrophoresis of ethanolic extract, Fraction I-P, and Fraction II-P was carried out on cellulose acetate strips rising chilled 50 mM phosphate-l mM El)TA buffer, pI1 8.0, under a potent,ial of 150 V (334mA/strip) for 2 hours. Eight ~1 of the sample was applied at 1-574 protein concentration, to a 1 X (j-inch strip. At the end of t,he 2 hours, the strip was stained with Procion Blue M-RS in a 2% (v/v) HCl/MeOH solution. Excess stain was removed with MeOH, the st.rip was cleared with heat, and the resulting stained patteru was scanned with a Beckman Analytrol strip scanner. The anodic migration movement of each preparation was from the origin at the right toward the left. FIG.

REDUCTASE.

II

421

grated essentially as single bands with less than 10% contamination appearing as stainable protein outside of the major peak. Each fraction could be identified with one of the two major peaks in the acid-ethanol extract. If the two fractions were mixed together and subjected to electrophoresis, they could be readily separated, suggesting that there was no association when they were combined. Similar results were obtained with polyacrylamide-gel slab electrophoresis in both phosphate and Tris-borate buffer systems. In both gel and strip electrophoretic systems it was possible to demonstrate by using overlay techniques that the enzymic activity was associated with the major stainable peak. Duplicate strips (or channels) were run in the electrophoresis apparatus. One of them was removed and stained by the standard method. The other was removed and laid on top of a heavy piece of filter paper soaked in a solution of Tris-SOa buffer at pH 8.0, NADH, and menadione in the same proportions as used in t’he standard assay system. Enzymic activity was observed by the disappearance of the NADH fluorescence. In each case, the area of enzymic activity coincided with the position of the major stainable peak on the companion strip. The reductase fractions described in Fig. 3 were prepared fresh and run immediately after purification. If the fractions were allowed to stand, and there was significant loss in Q6 reductase activity, then a new faster-migrating band appeared in the electrophoresis. The loss in activity may be related t’o the formation of this new frontrunning component . Spectra of the reductases. Figure 4 shows the spectra of Fractions I and II. The primary absorption peaks at 550, 450, 333, and 276 rnp, and a shoulder at 360 ml, are common to both reductases. The difference in relat’ive absorption at 333 rnp is consistently found in all preparations. It should be noted that the absorption at, 550 rnp and the trough at 398 rnp vary somewhat relative to the peak at 450 rnp from one preparation to another. This is believed to be due to varying amounts of contaminating

422

PHARO

FIG. 4. Spectra of ubiquinone reductases. The absorption spectra of Fraction I-P (-) and Fraction II-P (---) were determined by using a Cary model 15 recording spectrophotometer. The Fraction II spectrum has been normalized at 450 rnp for comparison. The enzymes were in 10 mM phosphate-l rnM EDTA buffer, pH 7.5, and the protein concentrations for Fraction I and II were 0.50 and 0.73 mg/ml, respectively.

iron protein. Figures 4 and 5 show the curves with our better preparations. Figure 5 shows spectral changes that occur on addition of substrate to Fraction I ubiquinone reductase. The changes observed with Fraction II are similar. With the anaerobic addition of a 4-fold molar excessof NADH, there is a bleaching of the 450 rnp absorption peak, suggesting reduction of the FMN. Indeed, the difference spectrum is the same for oxidized minus reduced FMN in the 450 rnp region measured under similar conditions. The extent of bleaching of the 450 rnp peak is more than 90% of that obtained with the addition of dithionite, and is in agreement with the amount of extractable FMN in the enzyme. Figure 6 shows the results of an experiment designed to determine the role of the enzyme-bound iron in the oxidation of NADH by menadione. The spectrum of the reduced flavoprotein (curve 2) shows increased absorption on addition of o-phenanthroline (curve 3). The difference spectrum is similar to that of authentic ferro-o-phenanthroline complex obtained under the same experimental conditions. When menadiane is subsequently added to the solution, the spectrum changes to that shown by curve 4. The difference between curves 4

ET

AL.

and 3 is similar to that between curves 1 and 2 (not shown in this figure), indicating that the reduced flavin has been reoxidized. Also, th.e difference between curves 4 and 1 is similar to the spectrum of ferro-o-phenanthroline complex, indicating that the iron remains in the reduced stat’e while the reduced flavin is reoxidized by menadione. The enzyme preparation, reassayed after all th.ese manipulations, showed greater than 70 % of the initial specific activit(y. When the instability of the reductase is considered, this high recovery is a good indication that th.e flavin and iron behave differently in SADH oxidation by menadione. Similar experiments with Q6 proved difficult’ because of its low solubility.

WA VEL ENG TH, m/l

FIG. 5. Effect of NADH on the spectrum of Fraction II ubiquinone reductase. The reductase concentration was 1.2 mg protein/ml in 10 rnM phosphate-l mM EDTA buffer, pH 7.5. The Thunberg-type cuvettes were made anaerobic by alternate evacuation and flushing with nitrogen that had been purified over hot copper. Additions to the cuvette were made by injecting anaerobic solutions through a serum cap on the side arm of the cuvette. To the purified enzyme (-) was added 200 nmoles NADH. The reduced enzyme spectrum (---) shows bleaching at 450 rnp. Subsequent additiqn of dithionite in excess (-.-.-) produces little bleaching. The oxidized minus reduced spectrum of the enzyme (. . . ) is the same as the difference spectrum (oxidized minus reduced) of FMN (--) in the 450 rnp region (lower two curves).

UBIQUINO?;E

O-400

REDUCTASE.

II

423

nanthroline, secondary changes in the protein occur leading to lossof enzymic act,ivity. Inhibitors. Figure 7 shows t’he inhibit#ion of the reductases by rotenone. The inhibition curve is biphasic, the first stage of inhibition occurring when the concentration of rotenone is approximately equal to the concentration of FMS in the enzyme.4 On addition of more rotenone, at, :I concentration 510 times greater than the flavin concentration, the enzymic activity is restored. Ubiquinone reductase activity is more sensitive to rotenone than is the menadione reduct,ase activit,y; however, both activit,ies show the same type of response to rotenone. At concentrations of 450

500 WAVELENGTH,ap

550

600

FIG. 6. Spectra of ubiquinone reductase treated with o-phenanthroline. Fraction I was prepared in an anaerobic cuvette as described in Fig. 5. To the anaerobic enzyme (curve 1) was added a 5-fold molar excess of NAI)H. When flavin reduction was complete (curve 2), 0.01 ml of a 57, solution of o-phenanthrolene in 207, ethanol was added (curve 3). After a lo-minute incubation, 0.01 ml of 10 mM menadione was added (curve 4). The difference spectra of o-phenanthroline treated erlzyme and untreated enzyme were compared with the spectrum of authentic ferro-o-phenanthroline complex (----) obtained under similar experimental conditions, normalized at 500 rnp (upper block).

It has also been found that neither 10 rnM o-phenanthroline nor 8 rnN Tiron inhibit NADH-ubiquinone reductase activity with either Q6 or menadione under our assay conditions. These compounds were incubated with the reductase for 3 minutes before st’arting the reaction with the addition of NADH. However, Hatefi and St,emple (18) have recently reported that o-phenanthroline inhibits a similar flavoprotein preparation, although prolonged incubation was necessary (personal communication from Dr. Y. Hatefi). It is significant. that the flavoprotein with the iron corn plexed in the reduced state, as evidenced by the spectral changes shown in Fig. 6, is still enzymically active in catalyzing NADH oxidation by menadione. It is conceivable that on prolonged incubation wit.h o-phe-

4 iVote added in proof: In a recent cornmunication which appeared after this paper was submitted for pltblication, Horgan, Singer, and Casida (J. Biol. (Them., 243,834, 1968) have carried out similar studies on the inhibition of ubiqllinone reductase activity by rotenone on a soluble enzyme prepared in their laboratory. The binding of excess rotenone at nonspecific sites leading to reactivation of t,he inhibited enzyme was colifirmed. Maximal inhibition (abollt 25%) was obtained with 6 moles of rotenone per mole of flavin. With our preparation of ubiquinone reductase, maximal inhibitioll (abollt GO’%) was observed with 1 rnole of rotenone per rnole of enzyme-bolmd flavin. These results were consistently obtained with 14 preparations of the flavoprotein. The protein-bound flavin was determined in each case after passage through a column of Sephadex to remove free flavin. The considerably higher amount, of rotenone required for maximum inhibition with Horgan’s preparation suggests that their reductase had lower affinity for the inhibitor alid was not identical with the reductase prepared in our laboratory. IXfferences between preparations made in different laboratories are not unusual with mitochondrial enzymes and emphasize t,he need for caution of generalizations. They have also found the rotenone-lJC added to submitochondrial particles does not appear in the soluble prot)ein fraction contjaining the ubiquinone redllctase, and they concluded that the inhibitor is not bolmd to the flavoprotein. However, the possible dissociation of the rotenone during the relatively drastic extraction procedure (heating at, 43” for 15 mimites at pH 5.3 in 10% ethanol) and rebinding to t,he lipid fraction of the residue has not been excluded. This is not an ludikely event since even washing the particles with bovine serum albumin removes the hnllrrd rotellone.

424

PHARO ET AL.

MOLES

ROTENONE/MOLE

REDUCTASE

FLAVIN

FIG. 7. Inhibition of ubiquinone reductase by rotenone. A reaction medium containing 50 pmoles T&-SO*, pH 8.0, 0.5 rmoles menadione (or 0.3 pmole Q,), 4 pg (or 20 pg) purified ubiquinone reductase, and rotenone, in a final volume of 2.9 ml, was incubated for 2.5 minutes at 30”. The reaction was then initiated by the addition of 0.1 ml of 4 mM NADH, and the activity of the reductase was determined. The control sample was treated similarly except for the addition of ethanol in the same volume as the rotenone solution. Both the Q6 reductase activity and the menadione reductase activity show biphasic response to rotenone using either Fraction I-P (circles) or Fraction II-P (squares).

rotenone 50-100 times greater than the flavin concentration, there is a second stage of inhibition of both the Q6 and menadione reductase activities. The ubiquinone reductase activity of submitochondrial particles does not show this biphasic response to rotenone. This rather unusual response of the purified enzyme suggests the possibility that t’he purified enzyme may have multiple sites of binding for rotenone. At low rotenone concentrations, there may be a one-to-one binding of the inhibitor to the enzyme at the more sensitive site. It may be speculated that as the concentration of the inhibitor is increased, additional binding at other sites may occur and result in conformational changes in the protein and restoration of activity. The altered protein could also be sensitive to rotenone, although its affinity for the inhibitor may be reduced, as in the second stage of inhibiton. To demonstrate maximal inhibition by rotenone, it. is necessary to incubate the

enzyme, rotenone, and Q6 together in the assay medium for 2.5 minutes. During this time there is a loss of enzymic activity due to a nonspecific protein interaction (1). Therefore, it is the remaining activity that demonstrates the maximal sensitivity to rotenone. However, it is also possible to show a similar, but slightly lower, rotenone sensitivity under conditions that measure the maximum activity of the enzyme. In order to do this, the enzyme was incubated with rotenone in half the volume of assay medium, and the Q6 was incubated in the other half. It was convenient to have sufficient medium present in each of the incubations so that (a) the alcohol in which the roteneone was dissolved did not denature the enzyme, and (b) there was sufficient volume for the Q6 to form a stable suspension. Under these conditions, when the two preincubated volumes were combined, and the NADH was added, the reductase activity was not altered, compared with the routine assay conditions. When the effect of rotenone was measured under these conditions, the reductase exhibited approximately 40% inhibition, instead of 70 %, as demonstrated when the first method was used. However, the biphasic nature of the enzyme sensitivity, with maximal inhibition at 1 mole rotenone/mole FMN, is the same. Also, Fractions I and II show similar inhibition by rotenone. Chance and co-workers (21) have recently suggested that two flavoproteins may participate sequentially in the oxidation of NADH in the respiratory chain and that the rotenone inhibition site may be located between them. If this should be true, our preparations of Fractions I and II may be related to the first flavoprotein in view of their rotenone sensitivity. The Q6 reductase activity is inhibited more than 80% by 4 mM Amytal (Fig. 8). In the original submitochondrial particles, a similar degree of inhibition is obtained with approximately 1 mM Amytal. The menadione reductase activity of the soluble enzyme, however, is completely insensitive to Amytal under these conditions. Dicumarol has no effect on the reductase activities of either fraction at concentrations as high as 1 mM. This property, together

UBIQUINONE

P

Menadione

I

I I

/ 2

AM Y TA L CONCEN

I 3

REDUCTASE.

I 4 TRA T/ON,

I 5

I

42.3

II

(22). We have considered the possibility that substrate levels of Q6 present’ in the standard assay of the ubiquinone reductase may, in fact, prevent the inhibitor from acting. To rule out this possibility, the ubiquinone reduct’ase was assayed as described by Cunningham et al. (3). In this system, a cat)alytic amount of menadione act’s as an immediate electron acceptor, and the menadiol produced in the reaction undergoes nonenzymic reoxidation by added cytochrome c. Piericidin A did not inhibit the menadione reductase activity even under these conditions.

,,,,+I

FIG. 8. Inhibition of ubiyuinone redllctase by Amytal. The sensitivity to Amytal of Fraction I 126 reductase activity and menadione reductase activity was determined. The assay was carried out, lmder standard assay conditions, i.e., preincubating all reactants together and initiating the react,ioll by adding enzyme. The specific activity of the enzyme in (26 redllctase and menadione reductase was 19.8 and 102, respectively. The Amytal was dissolved in aqlleous sohltion at pII 9.3. Control activities were determined by using the same volumes of a KOII solution at pTI 9.4. There was little effect, of the KOII on 1 he control activity.

with the inability to oxidize KADHY, dist’inguishes these reductase fractions from the menadione reductase (DT-diaphorase) of Martius (19, 20). Piericidin A is an effective inhibitor of respiration in mitochondria and mitochondrial particles (22). The effect of Piericidin A in the submitochondrial particle preparation, which is the routine starting material for the exktct’ion of ubiquinone reductase, is similar to that of hmytal, i.e., t#he menadione reductase activity is inhibited about .50 ‘8 , and the Q6 reductase activity is inhibited 1OO’j:. This occurs at concentrations of 5 X lo-lo mole I’iericidin A per milligram submitochondrial particle protein. Neither of these reductase nctivit,ies is sensitive to Piericidin A in the isolat’ed ubiquinone reductase. Concentrnt,ions as high as 5 X 1OF mole I’iericidin A4 per milligram ubiquinone rcductase protein showed no inhibitory effect. l’iericidin A is t’hought to be a competitive inhibitSor with respect’ t,o ubiquinone

I)ISCUSSION

The two ubiquinone reductase flnvoprotein preparations charact)erized in this report have indistinguish.able catalyt,ic properties. Within the limits of the accuracy of the methods used in the investigation, they could have the same molecular weight. The only reproducible difference bet lyeen them appears t’o be the net electJrical charge on the molecules that allows their separation on ion-exchange columns and by elcctrophoresis. King and Howard (24) have observed that extracts made by snake venom treatment of submitochondrial particles also yield t\vo distinct, N,ZDH dehydrogenase peaks during chromatography on DEAE-cellulose. Consistent with these findings, Biggs et al. (25) have report*ed that the flavoproteins extracted from “pH 5.4 particles” and electron transport particles (ETP) are eluted from hydroxylapatite at different concentrations of phosphate buffer. Thus, the occurrence of two distinct flavoproteins in extracts of mitochondrial particles seems established. Howcvcr, the relat,ionship between the two components is not clear. Since the different methods of extraction (acid-ethanol and phospholipase at 37”) yield two separable flavoproteins with quite similar propert’ics, t)he po~sibilit~y t,hat they arc independentl>v present in mitochondria may be considered. X more likely explanation, however, is that they are derived from th.e same segment, of the resprat)ory chain by cleavage at neighboring sites, as suggcst,cd by King nncl Howard (2-l).

426

PHARO

Many of the experimental observations reported in this series of papers have been confirmed in other laboratories. Singer and co-workers (26) have now successfully prepared active soluble ubiquinone reductase with qualitatively similar rotenone sensitivity from mitochondrial particles. The quantitative differences, e.g., slightly lower magnitude of inhibition by rotenone in the case of Singer’s preparations might be due to either differences in assay conditions or minor modifications in the enzyme protein. Hatefi and Stempel (18) have also isolated a flavoprot’ein with ubiquinone reductase activity and similar biphasic rotenone sensitivity from Complex 1 (NADH-coeneyme Q reductase complex) by dissociation with urea. Relationship of ubiquinone to cytochrome c reductase activity. At all stages of purification, the ubiquinone reductase has activity with menadione, cytochrome c, and ferricyanide as electron acceptors (see also Ref. 5). However, ubiquinone reductase activity is considerably less stable, and prolonged storage of either the particles or the soluble enzyme can lead to complete loss of activity with ubiquinone, whereas the cytochrome c reduction activity is unchanged. The stability difference has been noted also by Biggs et al. (25). The differences in the relative ubiquinone and cytochrome c reductase activities of different extracts is probably related to such preferential loss of the more labile reaction. Thus, acid-ethanol extracts (27), the made from “NADH-oxidase” original ETP (28), and “pH 5.4 particles” from hog heart (29), have negligible ubiquinone reductase activity but high cytochrome c reductase activity (6). However, freshly prepared “pH 5.4 particles” (25) and a modified preparation of ETP (3) also yield extracts which are active in ubiquinone reduction (25). Since ubiquinone and cytochrome c reductase activities behave differently during storage, it would appear that the mechanism of reduction of the two acceptors is different. Hatefi and Stempel (18) have arrived at similar conclusions on the basis of differential sensitivity to inhibitors. An additional point in the comparison is the FMN and Fe content. Both

ET AL.

the ubiquinone reductase and Mahler’s cytochrome c reductase (31) have similar amounts of bound factors. It may be concluded from the comparison that the isolated ubiquinone reductase and Mahler’s cytochrome c reductase are derived from the same component of the respiratory chain. The isolation procedure has preserved the ability to utilize the higher homologs of ubiquinone in the former but has led to it’s lossin the latter preparation. This is not surprising since ubiquinone was unknown at the time of Mahler’s work. Relationship of ubiquinone reduction by the purified jluvoprotein ancl by particles. The sequence of electron transfer steps from NADH to ubiquinone in the respiratory chain is still a matter of dispute. Several views have been expressed, all with only indirect evidence. One hypothesis (1) is that the NADH ubiquinone reductase flavoprot,ein directly reduces the ubiquinone. The supporting evidence is the rotenone sensitivity of the reaction to the extent of 5070% inhibition with 1 mole of rotenone per mole of bound flavin (Fig. 7). Since the affinity of the respiratory chain-linked carrier to rotenone is extremely high (32), it is felt that strong binding between the isolated flavoprotein and the inhibitor may not be fortuitous. The inhibition by Amytal is also characteristic of the response in intact mitochondria, although a 3-fold higher concentration is needed with the soluble enzyme. Several arguments have been advanced against the hypothesis that ubiquinone is the respiratory carrier next to the flavoprotein. First, the biphasic responseto rotenone (Fig. 7) is unlike that seen in mitochondris and the sensitivity to Amytal is lesswith the flavoprotein reaction compared with mitochondrial respiration. However, it may be noted also that the environment of the flavoprotein is different in the particle when it is integrated with lipid, other respiratory carriers, and structural protein into a complex structure. This binding to the other components could conceivably alter the conformational and other properties of the reductase and mask the additional rotenone binding sites, leaving only the primary

UBIQUINONE

inhibition phase of the rotenone effect. Another fact’or for consideration is the state of the substrate. In particulate systems, the ubiquinone may be dissolved in the bound lipid phase and provide a much greater concentration at’ the reaction site of the enzyme, while in the assay of the reductase activity with the purified enzyme, the quinone dissolved in the aqueous phase is at an cxtremely low concent,ration. In fact, lipid seems to be an essential component of t,he particulate system (33). These differences in the state of t,he substrate could modif? the response of the enzyme to both hydroand hydrophobic inhibitors. AIL philic example of such interference is the inhibition of ?\‘ADH oxidation by Q6 with low concent,rations of extraneous protein and det,ergents (1). So inhibit’ion of reactions which may involve the endogenous ubiquinone as a carrier (e.g., NADH oxidat’ion by cytochrome c or oxygen) are observed with t,hese same compounds. It’ must be recognized in comparing inhibition in particles with inhibition in soluble systems that the response could be seriously influenced by factors which are not related t’o the interaction between enzyme and inhibitor per se, but interfere with the availability of t’he inhibitor or substrate. A second and more recent objection is based on the binding of rotenone-14C to particles (34). The bound rotenone remains in the insoluble residue, and only a negligible amount is recovered in the extract containing ubiquinone reductase when the particles are extracted at pH 5.3 and 43” wit,h 10% aqueous ethanol. In this experiment, it is assumed t’hat the rotenone binding to the sensitive site is unaltered during the extraction. The possibility that the rotenone is dissociated and bound to some other lipophilic site or component during the extraction has not been excluded. A third objection to the proposed hypothesis is the requirement for added phospholipid for the restoration of KADH-Q1 reductase activity in particles which had been digested briefly with phospholipase (33). Since t.he soluble flavoprotein-catalyzed ubiquinone reductase reaction shows no requirement for lipid, the two reactions are considered mechanistically different. However, in the

REDUCTASIS.

II

427

particle, a lipid environment around the flavoprotein might be necessary to promote the approach of the lipophilic ubiquinone to the functional site of the enzyme. It has also been argued that the rotenone sensitivity of the Q1 reduction activit’y which is restored by lipid is evidence for hpld requirement for rotenone action (33). Since no activity can be measured with the phospholipase-digested particle wit,hout added phospholipid, the conclusion does not, necessarily follobv from the given experiments. It has not. been sufficiently appreciated that the catalytic properties and inhibitor sensitivity of an enzyme structurally bound to particles could be influenced by the environment and by binding to membraneous structures. If the same enzyme is assayed in similar reactions after it is disengaged from the particles, profound differences in properties may not be unusual. A well-documented example of this phenomenon is the ability of isolated cytochrome c to bind cyanide lvhile endogenous cytochrome c does not bind cyanide (35). A second example is the change in the redox potential of intramitochondrial cytochrome b from around 0.0 mV to a considerably more negative value (-340 mV) after purification (36). On binding to isolated struct’ural protein, the potential again becomes more positive. Another example is the marked alteration in t’he kinetic parameters, K, and V,,,, of purified malic dehydrogenase on binding to the mitochondrial membrane or structural protein (37). A fourth example is the masking of the ATPase activity of the mit’ochondrial coupling factor (FJ on binding to submitochondrial particles (38). The complications expected in a heterogenous assay system, as in the Q6 reduction assay, may be even greater. The above discussion is meant to leave open the question whether ubiquinone is the physiological respiraOory carrier next in sequence to the flavoprotein that catalyzes the oxidation of NADH. ACKNOWLEDGMENT The authors are grateful to Maureen and Roland Lippoldt for expert technical ance.

Maskell assist-

428

PHARO REFERENCES

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