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62 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 46343

SOLUBLE CYTOCHROME b 5 REDUCTASE FROM HUMAN ERYTHROCYTES*

P. G. PASSON** AND D. E. F IULTQUIST ***

The Department of Biological Chemistry, The University of Michigan, Ann Arbor, Mich. 48±o4 (U.S.A.)

(Rece ived F e b r u a r y 2nd, 1972)

SUMMARY

I. An enzyme that catalyzes the reduction of erythrocyte cytochrome b 5 has been isolated from the supernatant fraction of erythrocyte hemolysates by chromatography on DEAE-cellulose, Amberlite CG-5 o, and Bio-Gel P-6o.

2. Erythrocyte cytochrome b 5 reductase has been shown to contain FAD. Incubation of the reductase in the absence of EDTA results in both the appearance of flavin fluorescence and the loss of enzymatic activity with time.

3. The reductase catalyzes the reduction of erythrocyte cytochrome b 5 5o times faster with NADH than with NADPH. The apparent Km of NADH was calculated to be 1.6. IO -~ M and the turnover number is 128o moles of cytochrome b 5 per min per mole of flavin. The reduction of electron acceptors proceeded in the following decreasing order of rate: K~Fe(CN)6, 2,6-dichlorophenolindophenol (DCIP), cytochrome b 5, methylene blue, cytochrome c, 03, oxidized glutathione, and methemoglobin.

4- The FAD prosthetic group, the substrate specificity, and the effect of ionic strength, pH, and EDTA on activity suggest that the reductase is the same enzyme as NADH dehydrogenase I, the enzyme lacking in many cases of congenital methemoglobinemia. The properties of the reductase, including its molecular weight, are very similar to those of the cytochrome b 5 reductases solubilized from microsomes and mitochondria of other tissues.

INTRODUCTION

A number of reductases isolated from red blood cells have been implicated in the reduction of methemoglobinl-lL The various NADPH-dependent "methemoglobin reductases ''1-6, however, catalyze the reduction only in the presence of redox agents such as methylene blue and these enzymes do not appear to have a significant role in the process of methemoglobin reduction under normal conditions~L Two NADH- dependent methemoglobin reductases, NADH dehydrogenase I and II , have been

Abbrev i a t i on : DCIP, 2 ,6-dichlorophenol indophenol . * P a r t s of the d a t a were t a k e n from a doctora l thesis s u b m i t t e d to the D e p a r t m e n t o~

Biological Chemis t ry , R a c k h a m School of G r a d u a t e Studies, The U n i v e r s i t y of Michigan, by P . G . P . in 197 ° .

* * P resen t a d d r e s s : T h e Up john Co., Kalamazoo , Mich. 49 OOl, U.S.A. * '* To whom rep r in t reques t s should be sent.

Biochim. Biophys. Acta, 275 (1972) 62-73

CYTOCHROME b 5 REDUCTASE FROM ERYTHROCYTES 63

partially purified from the supernatant fraction of erythrocytes and studied 7-1°. NADH dehydrogenase I (also referred to as NADH diaphorase) can be distinguished from the other erythrocyte reductases by its substrate specificity, chromatographic behavior, and effect of various ions on its activity~, 1~. This reductase is believed to function in methemoglobin reduction because deficiency of the enzyme results in methemoglobinemia 9,14,1~. A similar reductase has been detected in leucocytes and this enzyme has been reported to be deficient in some cases of congenital methemoglobinemia 16.

An NADH-dependent cytochrome b 5 reductase has been detected in the microsomal fraction front many tissues and species and a solubilized and enzymatically active form of this enzyme has been purified and thoroughly studied 17-~9. The finding that this flavoprotein rapidly catalyzes the reduction of cytochrome bs, which is found with the reductase in microsomes, suggests that these two proteins are a part of the same electron transport system. Recent findings suggest that these proteins are involved in the fa t ty acid desaturation system~°, 2~. A cytochrome b 5 reductase has also been detected in the outer membrane fraction of liver mitochondria22, 2a. This reductase has been solubilized, purified, and shown to be very similar to the microsomal enzyme.

This report describes the isolation and characterization of a soluble, NADH- dependent reductase of human erythrocytes that rapidly catalyzes the reduction of cytochrome b524, isolated from human erythrocytes. The results of this study indicate that the enzyme is NADH dehydrogenase I and demonstrate its similarity to solubilized cytochrome b 5 reductase from liver microsomes. The isolation of this cytochrome b 5 reductase ~5 and a description of its enzymatic properties ~G have been presented in abstract form, and its role in a reconstituted methemoglobin reduction system has been reported previously 27.

EXPERIMENTAL PROCEDURE

Materials KaFe(CN)6 and methylene blue were obtained from Allied Chemicals; NADH,

NADPH, oxidized glutathione, 2,6-dichlorophenolindophenol (DCIP), and horse heart cytochrome c from Sigma; bovine serum albumin (Fraction V, B grade), catalase, Bio-Gel P-2o and P-6o, lOO-2OO mesh, from Calbiochem; Amberlite CG-5o, 2oo-4oo mesh, from Mallinckrodt; and DEAE-cellulose from Fisher. Collodion bags, with porosity less than 5 nm were obtained from Schleicher and Schuell. Dialysis tubing was boiled for I h, washed in I mM EDTA, and then rinsed in water before use. Human hemiglobin, obtained from Calbiochem, was purified by chromatography on Bio-Gel P-2o before it was used.

Naja naja snake venom and FMN were gifts from Dr V. Massey and rat liver microsomal cytochrome b 5 a gift from Dr T. R. Tephly. S-protein ~8 and erythroeu- prein 29 were isolated as described previously. Outdated human blood cells were graciously provided by St. Joseph's Mercy Hospital Blood Bank, Ann Arbor, Mich., The University of Michigan Medical Center Blood Bank, Ann Arbor, Mich., and the Bureau of Laboratories, Michigan Department of Public Health, Lansing, Mich. Cells were stored and, immediately before lysis, washed as described previously2%


6 4 P . G . PASSON, D. E. HULTQUIST

3lethods Prote in concent ra t ions were de te rmined by the me thod of Lowry et al. 3° using

serum a lbumin as s tandard . Fluorescence spec t ra were observed as descr ibed pre- v ious ly 29. Changes of fluorescence with t ime were measured with an A m i n c o - B o w m a n f luorometer . The concent ra t ions of FMN and F A D were de te rmined f luorometr ica l ly using N a j a naja snake venom to l ibera te the FMN from the FAD, and using FMN as s t a n d a r d al.

The ac t i v i t y of the e ry th rocy te reductase wi th cy tochrome b 5 was measured at 25 °C and p H 8.1 on a Gilford recording pho tome te r in a I.O ml react ion mix ture conta in ing: o.78 nmole cy tochrome ba, 6o nmoles N A D H , o.5-1.o /~mole EDTA, 5 o - I o o #moles Tris-HC1, and o.2-o.6 #g of a reductase sample with a specific ac t iv i ty of 2 I . I ~(,dA 6oo nm/min per ml)/A 28o nm~. Increase in absorbance at 424 nm was followed with t ime. A value of lO5 was used for the change in the ext inc t ion coefficient (,:]emM) at this wavelength s2. In most exper iments , the cy tochrome b 5 reducing capac i ty of the reductase was de te rmined in a coupled react ion with cy tochrome c ~7. In these exper iments , the reac t ion mix tu re also conta ined 35 nmoles of horse hear t cy tochrome c and the increase in absorbance at 55 ° n m was followed with t ime. The value of 2I was used for the AemM ~3. The dependence of this assay on each component of the sys tem is shown in Table I. L inea r i ty between ra te of reduct ion and the amount of

T A B L E [

R E Q U I R E M E N T S F O R C Y T O C H R O M E b 5 R E D U C T I O N W I T H C Y T O C H R O M E c AS F I N A L A C C E P T O R

Const i t z~ents ~JA 550 nm / I o nz in

Complete system * o.i2o 3Iinus NADH o.coo Minus reductase o.oo3 Minus cytochrome b5 o.o04 Minus cytochrome c o.ooo M~nus reductase and cytochrmne b5 o.oo2

"The system is that described under Methods with a o.i M Tris-HC1 buffer, pH 8.1, con- t a in ing o. 5 mM EDTA.

reduc tase is shown in Fig. I a for assays carr ied out wi th o.78 nmole of cy tochrome b 5. As can be seen from the plot of ra te of cy tochrome c reduct ion vs the amoun t of cytochrome b 5 (Fig. 2), a t concent ra t ions of cy tochrome b 5 grea ter t han 0.6 #M the ra te of the react ion is independen t of the cy tochrome b 5 concentra t ion. Thus, under these condi t ions the ra te of the overal l react ion is dependen t on the ca ta lys is of the reduc- t ion of cy tochrome b 5 and independen t of the ra te of the react ion between reduced cy tochrome b 5 and oxidized cy tochrome c. The fact t ha t absorbance changes at 550 nm were due to reduct ion of fe r r icy tochrome c was confirmed b y difference spectroscopy. The ra te of cy tochrome b 5 reduct ion was ob ta ined by sub t rac t ing the ra te ob ta ined in the absence of cy tochrome b 5 from the ra te for the complete system.

The enzymat i c reduct ions of ferr icyanide, me thy lene blue, cy tochrome c, g lu ta- thione, methemoglob in and S-prote in were carr ied out as descr ibed for the d i rec t reduct ion of cy tochrome b 5, except t ha t s a tu ra t i ng amounts of these electron acceptors were used in place of the cy tochrome b 5. The reduct ions of ferr icyanide, g lu ta th ione ,

Biochim. Biophys..4cta, 275 (t972) 62-73

CYTOCHROME 65 REDUCTASE FROM ERYTHROCYTES 65

0 28 z

0 ~ 0 2 4

0 02C

016 U z

~ 0 1 2 0 co

< 0 0 8

~ 004 z <I I (_9

O

I i i I

I I

o2 01+ oi+ o8

z

0

020

0 0 w 016

~ 0+12 z

[I?

~ 0.08 m ,<

u_ 0.04 0

u.I (.9 z 0 < I (._)

i J i , 0.2 0.4 0.6 08.

REDUCTASE (jug)

Yig. I. Dependence of rate of reduct ion on the concentrat ion of ery throcyte cy tochrome b~ reductase. Condit ions were those described under Methods except t h a t the buffer was o. i M Tris-HC1 conta in ing o .oo i M EDTA, p H 8.1. a. Reduct ion of h u m a n erythrocyte cy tochrome b5 as assayed by coupl ing the react ion to cy tochrome c. b. Reduct ion of DCIP.

z 0 l - -

0.8

t a uj+~ 0,6

=~ 0 . 4 ~ E

o

w

i i i

0.2

o o12 oi+ oi+ o.8 CYTOCHROME b 5 (n mole)

Fig. 2. Dependence of the reductase-cata lyzed reduct ion of cytochrome c on the concentrat ion of ery throcyte cy tochrome bs. See Methods for details.

methy lene blue, molecular oxygen and S-protein were measured by following with t ime the absorbance of N A D H at 34 ° nm. An 6mM of 6.22 was used in each instance, except for ferricyanide, in which case an CmM of 6.82 was used. This ext inct ion took into consideration the decrease in absorbance at 34 ° nm which accompanies the reduction of ferricyanide. Methemoglobin reduction was measured by following the formation of oxyhemoglob in at 577 nm using a value of 13.7 for the difference between the emM (per heme) of oxyhemoglobin and methemoglobin. When N A D P H was used as the electron donor, saturating amounts were subst i tuted for N A D H in the assays described above.

DCIP reduction was carried out with a DCIP concentration of 25 #M. Reduct ion was measured by fol lowing the decrease in absorbance at 600 nm, assuming an ~mM of - - 2 1 . DCIP reductase act iv i ty was used as a measure of the e n z y m e during the isolation procedure. For this purpose, the assay was carried out at pH 8.1 in a 3 ml


66 P .G . PASSON, D. E. HULTQUIST

solution containing: z5o /~moles Tris-HC1, 1.5 /,moles NADH, z.5 ~moles EI)TA, o.o75/,mole DCIP, and reductase.

Purification of cvtochrome b 5 reductase Washed, outdated, packed human red blood cells (260 lnl) w e r e lysed by force-

fully adding Io4o ml of cold water. The pH of the lysate was adjusted to pH 6.0 with HC1 and centrifuged at 20 ooo X g for 2o rain.

The reductase was removed from the supernatant fraction with DEAE-eellu- lose by modification of published methods for the isolation of erythrocyte reductases~,lL The supernatant fraction was diluted with 1.5 voh of water and adjusted to pH 7.2 with KOH solution. The protein was charged at a rate of 15o ml/h onto a 2.4 cm ~'~" 20 cm DEAE-cellulose column previously equilibrated at 4 °C with o.ooI M buffer of pH 7.2. The column was washed with I 1 of the o.ooi M buffer and then eluted at pH 7.2 with a linear gradient formed with 6oo ml of o.ooI M phosphate in the mixing chamber and 60o ml of 0.2 M phosphate-o.5 M KC1 in the reservoir. Eluent was passed through the column at 75 ml/h. Fractions were collected in tubes that contained concentrated EDTA to give a final concentration of i raM. The DCIP reductase-containing fractions eluted from the column with the linear gradient of buffer (Fig. 3) were pooled, adjusted to pH 6.o with HC1, and dialyzed for 2 h vs 0.05 M phosphate-o.ooI M EDTA, pH 6.o.

The reduetase was further purified by chromatography on Amberlite C(i-5o. The solution was applied to a 3.4 cm < 7 cm column of CG-5o that had been equilibrated with 0.o5 M phosphate o.ooi M EDTA, pH 6.o. The charged column was first washed with 300 ml of o.o5 M phosphate-o.ooI M EDTA buffer and the reductase then eluted with 0.5 M phosphate-o.ooI M EDTA, pH 6.0, at a flow rate of 18o ml/h. Fractions with reduetase activity were pooled and adjusted to pH 7.2 with KOH solution.

In preparation for gel filtration, the reductase was concentrated by (NH4)2SO 4 precipitation. (NH4)2SO ~ was slowly added to 9 ° % saturation (66.2 g/Ioo hal); the pH was maintained at pH 7.2 during the addition. The precipitate was removed by

5.C

4.0

•z30

zc

O.C

, , , , ]

t / ", 08~o~

412 nmA ! k ~ ~ a D 0.4 u

i ",, 0.2

4 0 0 8 0 0 1200 1600 VOLUME OF ELUATE (rnL)

1.4 0.14

.o.21" I.C i / ~&ctivity / ! / o,o

z i

i °" ,,, 100. 0.4 \", / ., I oo4

...... ~ " J ' - ~ - : ~ ....... o o o O'C200 250 300 350 400

VOLUME OF ELUATE (ml)

>_~ B E

4

Fig. 3. Purification of erythrocyte cytochrome b5 reductase on DEAE-cellulose at p H 7.2. Fract ions of 14 rnl were collected. For details see Exper imenta l Procedure.

Fig. 4. Purification of erythrocyte cytochrome b5 reductase on Bio-Gel P-6o. Fractions of 2.46 ml were collected. For details see Exper imenta l Procedure.

t3iochim. Biophys. Acta, 275 (1972) 62-73

CYTOCHROME b5 REDUCTASE FROM ERYTHROCYTES 67

centrifugation, suspended in a minimum volume (28 ml) of o.o5 M phosphate-o.ooI M EDTA, pH 7.2, and dialyzed against this buffer for 6 h. The dialyzed fraction was concentrated to 6 ml by ultrafiltration on a collodion membrane and applied to a 3 4 cm • lO6. 4 cm P-6o column that had previously equilibrated with the phosphate- EDTA buffer. The column was eluted with this buffer at a flow rate of 12 ml/h (Fig. 4).

In order to obtain a more highly purified reductase preparation for enzymatic studies, the most pure fractions of the Bio-Gel P-6o eluates from two reductase preparations were pooled and subjected again to chromatography on Bio-Gel P-6o. Concentration by ultrafiltration and chromatography were carried out as described above,

R E S U L T S

The purification of erythrocyte cytochrome b 5 reductase is summarized in Table II. Rechromatography of the most pure fractions from two preparations gave a sample with a specific activity of 21 [(AA600nm/min per ml enzyme)/A2sonmJ. Because of the presence in red blood cell hemolysates of other reductases which catalyze the reduction of DCIP, the overall yield and recovery can not be calculated. The visible absorbance spectrum of the purified sample indicates that the reductase is contaminated by a small amount of hemoglobin.

TABLE II

P U R I F I C A T I O N O F E R Y T H R O C Y T ] ~ C Y T O C H R O M E b 5 R E D U C T A S E

Fraction Total protein Total activity % of Specific activity (A280 nm )< ml) [(AA/min per ml) original activity (AA/minperml~

× ml] \ A280 nm ]

Hemolysate 113 4oo 792 ioo o.oo7o1 DEAE-cellulose eluate 462 lO9 13.8 o.237 CG-5 o eluate

after concentration 39.6 23.2 2.9 0.587 Bio-Gel P-6o eluate 2.42 16. 7 2.1 6.00

The intact protein showed little fluorescence. However, the supernatant fraction resulting from treatment of the sample with trichloroacetic acid showed fluorescence typical of flavin and the action of phosphodiesterase resulted in a Io-fold increase in fluorescence intensity. Assuming the prosthetic group to be FAD, the concentration of total flavin in the purified sample of erythrocyte cytochrome b 5 reductase was calculated to be 1.42. lo -l° mole/ml. Protein analysis of this sample showed a protein concentration of o.041 mg/ml, which is equivalent to 280000 g protein per mole of flavin. Gel filtration of the reductase indicates a molecular weight of less than 40 000.

Erythrocyte cytochrome b 5 reductase is stable at pH 7.5 or 8.1 when stored at --20 °C in Tris-HCl buffer containing 0.5 mM EDTA and, in this solution, shows no measurable loss of activity over a 6 h period at 4 °C if the protein concentration is no lower than 0.02 mg/ml. However, in more dilute solution a significant loss of activity does occur. Thus, the same sample diluted to 0.002 mg protein/ml with the same


68 P . G . PASSON, D. E. HULTQUIST

>-~ 03 , , , 60

"

O O 0,1 V I I O

'- o| ,° 4 0 8 0 1 2 0

H O U R S

> - o . 3 1 , , 1 6 o

J _ - o . 2 ~ 1 4 o z , j - - o - _ i' "

u~ { / 1.0 mM EDTA

Ooj I - t2° ,~

m ~ o l t I 0 4 0 8 0 1 2 0

H O U R S

Fig. 5. Effect of E D T A on i na c t i va t i on of e r y t h r o c y t e cy toch rome b5 reduc tase and the appea rance of f lavin fluorescence. Samples were i n c u b a t e d a t 4 °C in o.o 4 M p o t a s s i u m p h o s p h a t e buffer, p H 7.2. D C I P reduc tase a c t i v i t y was de t e rmined as descr ibed in E x p e r i m e n t a l Procedure. Fluorescence emiss ion was measured a t 53 ° n m wi th a c t i v a t i o n a t 455 nm. - - - , a c t i v i t y ;

, fluorescence.

180 7- g

,5o c~

~ 12o

~ 9 0

60

3 0 w

c~

0

r ~ J

0 Tr is - HCI Z~.. Po tass i um phosphate

/ Q

I

6!o 7'0 8.0 9)0 pH

c ~. 480

% c E 4 0 0

_ ~ 3 2 0

2 4 0

o_ 160

8O

0

J i i o T r i s -HCt

te"

b

610 710 8 io 910 pH

Fig. 6. p H - a c t i v i t y curve for e r y t h r o c y t e cy toch rome b5 reductase . ©, Tris-HC1 buffers; A, p o t a s s i u m phospha t e buffers. Ra t e s were ad jus t ed to correspond to i .o ml enzyme. For de ta i l s see E x p e r i m e n t a l Procedure. a. Reduc t ion of h u m a n e r y t h r o c y t e cy toch rome bs. Ra t e s were de t e rmined by coupl ing the reac t ion to cy toch rome c. Buffers were o.12 M w i t h o. 5 mM EDTA. b. Reduc t ion of DCIP. Buffers were o. i M w i t h i mM EDTA.

Tr i s -HCI~EDTA buffer lost 32 % ac t i v i t y af ter 6 h at 4 °C. In the absence of EDTA, the loss of ac t i v i t y from even concen t ra ted solutions of the reductase occurs and this is accompan ied b y the appearance of fluorescence (Fig. 5)- Like the fluorescence of free flavin, this fluorescence showed an emission m a x i m u m at 53o nm when ac t i va t ed at 38o or 47 ° n m and showed ac t iva t ion m a x i m a at 378 and 457 nm when emission at 54 ° n m was measured.

The dependence of DCIP reduct ion and cy tochrome b 5 reduct ion on the concen t ra t ion of reductase is shown in Fig. z. The p H - a c t i v i t y curve of the reductase for these two electron acceptors is shown in Fig. 6. Effect of the concent ra t ion of T r i s - HC1 and KC1 on the ca ta lys is of the two reduct ions is shown in Fig. 7. Whereas bo th Tris + and K + inhibi t the reduct ion of cy tochrome bs, only the K + inhib i t s the reduct ion of DCIP. The inhib i t ion b y Tris + and K + appears to be la rge ly responsible for the unusual p H - a c t i v i t y curves shown in Fig. 6. Catalysis of D C I P reduc t ion is una l t e red b y E D T A concent ra t ions up to IO mM, bu t E D T A concent ra t ions of 2, 4 and IO mM inhib i t cy tochrome b 5 reduct ion 6 %, 17 % and 32 %, respect ively.

The subs t ra te specifici ty of the e ry th rocy t e cy tochrome b 5 reductase is shown in Table IH . Of the electron acceptors s tudied, only cy tochrome bs, DCIP , and ferri-

Biochim. Biophys. Acta, 275 (i972) 62-73

CYTOCHROME 35 REDUCTASE FROM ERYTHROCYTES 69

2ool-,-~,>., 0 T r i s - H C I 4 \ \

~ 8 o L ~ a

4o

0.04 0.08 0.12 016 020 024 iONIC STRENGTH

15C g

13(

"- I10

c3 E c 90

o _ v o

,~ 70

5 0

0 T r t s - He!

K CI "e O OS M TFi$- HCt

i i i

0.04 008 01'2 OL=6 0210 0.24 IONIC STRENGTH

Fig. 7- Effect of Tris and K + on the a c t i v i t y of e r y t h r o c y t e cy tochrome ba reduc tase a t p H 8. i . R a t e s were a d j u s t e d to cor respond to i .o ml enzyme. For de ta i l s see E x p e r i m e n t a l Procedure . ©, v a r y i n g concen t ra t ions of Tris-HC1; &, v a r y i n g concen t ra t ions of KC1 in the presence of

0.0 5 M Tris-HC1. a. R e d u c t i o n of h u m a n e r y t h r o c y t e cy tochrome b5 as assayed by the coupled reac t ion wi th cy toch rome c. Buffer con ta ined 0. 5 mM EDTA. b. Reduc t ion of DCIP. Buffer con- t a ined i mM EDTA.

T A B L E I I I

SUBSTRATE SPECIFICITY OF ERYTHROCYTE CYTOCHROME b~ REDUCTASE

Ra te s are compared on a per e lec t ron basis. Condi t ions are those descr ibed in Methods. The buffer was 0.05 M Tris-HC1, p H 8.1, con ta in ing 0. 5 mM EDTA.

Electron acceptor Relative rate Rate with N A D P H with N A D H Rate with N A D H

K3Fe(CN)6 8.1 o.oo2 D C I P 2.45 o.o17 H u m a n e r y t h r o c y t e c y t o c h r o m e b5 * i .oo o.o19 Bovine e r y t h r o c y t e c y t o c h r o m e b5 * 0.35 O.Ol 1 Methy lene blue 0.073 1.5 Cy tochrome e o.o12 1.6 O~ o.oo41 3.7 G lu t a th ione 0.0025 2.5 Methemoglob in o.ooo 5 - -

* Assay was per formed by coupl ing the reac t ion to cy tochrome c.

cyanide were rapidly reduced. With these electron acceptors the reductase is highly specific for NADH. Cytochrome b 5 from rabbit erythrocytes, rabbi t reticulocytes, and rat liver microsomes likewise served well as electron aceeptors for the reductase.

The dependence of reductions on the concentration of NADH is shown in reciprocal plots in Fig. 8, for arb i t rary concentrations of cytochrome b 5 and DCIP. The apparent Km of NADH for the reduction of cytochrome b 5 was calculated to be 1.6. lO -6 M at 25 °C in pH 8.1 buffer. The V of this reaction was 2.06-lO -4 M cyto- cytochrome bJmin per ml enzyme. From the flavin content of this preparation, the turnover number was calculated to be 128o moles of cytochrome b 5 per rain per mole flavin. For the reduction of DCIP the apparent Km of NADH was calculated to be 4.8" io 7 M and, at this concentration of DCIP, the V was 2.3"IO -~ M/rain per ml enzyme. The stoichiometry of the reaction was found to be I.O8 mole NADH oxidized/ mole DCIP reduced.


7 ° P .G. PASSON, D. E. HULTQUIST

2 4

2 0

0 1 6

> L 2

0 8

0 4

i i i i i i

Q

i L i l IO 20 510 20 50 60 710 810

[ S ] - ' x IO - ~

o

140

120

100

80

60 t O 810 i I i I 4 120 160 200 240 280 [S] -f x IO -4

Fig. 8. Double-reciprocal plots of rate of reduction vs NADFI concentrations. IS 1 is the molar concentration of NADH and v is the rate of reduction in moles/1 per rain for the reactions carried out at 2 5 °C in 0.0 5 M Tris-HC1 buffer, pH 8.1, containing 0. 5 mM EDTA. a. Cytochrome b5 reduction, b. DCIP reduction.

0.17

E °' tS :'

LU 0"11 l~

i0.09 l

° % ,;

- - A n a e r o b i c

. . . . . . . Aerobic

2'o ~o 40 TIME(rain)

Fig. 9. Autoxidation of erythrocyte cytochrome b5. Anaerobic and aerobic enzymatic reduction of cytocbrome b5 was carried out at pH 8.1 in the absence of cytochrome c as described in Experi- mental Procedure. The buffer was o.o 5 M Tris-HC1 containing 0. 5 mM EDTA. Air was introduced into the anaerobic cuvette at t ime of 15 rain as indicated.

T h e e n z y m a t i c r e d u c t i o n of c y t o c h r o m e b 5 in a e r o b i c a n d a n a e r o b i c c o n d i t i o n s

is s h o w n in Fig. 9. R e l a t i v e to t h e r e d u c t i o n a c h i e v e d w i t h s o d i u m d i t h i o n i t e , en-

z y m a t i c r e d u c t i o n g a v e 83 % r e d u c t i o n in t h e p r e s e n c e of a i r a n d 96 % r e d u c t i o n

u n d e r a n a e r o b i c c o n d i t i o n s . W h e n a i r w as i n t r o d u c e d i n t o t h e a n a e r o b i c r e a c t i o n

m i x t u r e , p a r t i a l o x i d a t i o n of t h e f e r r o c y t o c h r o m e b 5 occu r r ed . W h e n t h e r e a c t i o n w a s r u n w i t h l i m i t i n g a m o u n t s of N A D H , t h e f e r r o c y t o c h r o m e b~ was c o m p l e t e l y re-

o x i d i z e d a f t e r t h e i n t r o d u c t i o n of air .

T h e r e d u c t i o n of f e r r i c y t o e h r o m e c b y t h e r e d u c t a s e - c y t o c h r o m e b 5 s y s t e m

was i n h i b i t e d b y n e i t h e r c a t a l a s e n o r s u p e r o x i d e d i s m u t a s e .

DISCUSSION

T h e so lub le , N A D H - d e p e n d e n t r e d u c t a s e w h i c h we h a v e i s o l a t e d f r o m h e m o -

l y s a t e s of h u m a n e r y t h r o c y t e s e f f ic ien t ly c a t a l y z e s t h e r e d u c t i o n of so lub le c y t o c h r o m e

b 5 f r o m h u m a n , b o v i n e , a n d r a b b i t r e d cells; so lub i l i z ed e y t o c h r o m e b 5 f r o m r a t

Biochim. Biophys. Acta, 275 (I972) 62-73

CYTOCHROME bs REDUCTASE FROM ERYTHROCYTES 7 1

liver microsomes; DCIP; and ferricyanide. For these electron acceptors, the rate with NADPH is very slow relative to the rate with NADH. The lack of pyridine nucleotide specificity with the less efficient electron acceptors--methylene blue, cytochrome c, molecular oxygen, glutathione, and methemoglobin- -may indicate the presence of a very small amount of a second activity.

There is no indication that the heme-containing component of the reductase preparation is involved in the enzymatic activity. However, the concomitant loss of activity and appearance of flavin fluorescence upon storage of the enzyme in the absence of EDTA indicate that flavin is an essential component of the reductase. The io-fold increase in fluorescence intensity which resulted from treatment of the dissociated flavin with phosphodiesterase is characteristic of the release of FMN from FAD 31, suggesting that the prosthetic group of the reductase is FAD.

The significant electron transfer reactions involving erythrocyte cytochrome b 5 reductase and cytochrome b 5 are summarized in Fig. IO. The reduction of ferri- cytochrome c by the cytochrome bs-cytochrome b 5 reductase system, like the reduction of methemoglobin by this system 2~, is not inhibited by catalase nor superoxide dismutase and thus does not involve unbound H~O 2 or superoxide anion.

NAOH

I / 2 2 / o2 3 D CYTOCHROME b 5 REDUCTASE ~ CYTOCHROME bS---b,~METHEMOGLOBIN

4 "~METHEMOGLOBIN MITOCHONDRIAL CYTOCHROME c

Fig. io. E lec t ron t r ans fe r reac t ions invo lv ing cy t och rome b~ reduc tase and cy toch rome ba f rom e ry th rocy tes . The heav iness of t he arrows indica tes t he re la t ive ra tes of t he electron t ransfers .

The erythrocyte cytochrome b 5 reductase is distinguished from st romal cytochrome c reductase 34, glutathione reductase ~, dihydrofolate reductase ~, the NADPH- dependent "methemoglobin reductases ''1-6, and the other erythrocyte reductases which have been characterized, by its molecular weight, specificity for NADH, specificity toward electron acceptors, and inhibition by KC1.

The results of this s tudy suggest that erythrocyte cytochrome b5 reductase is NADH dehydrogenase I, the enzyme which has been shown to be necessary for normal reduction of methemoglobin in red cells. Erythrocyte cytochrome b 5 reductase pos- sesses an FAD prosthetic group and NADH dehydrogenase I appears to be an FAD- containing protein 7 (although the work of Sugita et al. TM fails to support this view). Erythrocyte cytochrome b 5 reductase shows great similarity to NADH dehydrogenase 17,11,13, 4 in terms of substrate specificity, dependency of activity on pH, inhibition by KC1, and lack of inhibition by o.oi M EDTA. Moreover, we have shown that NADH dehydrogenase I, isolated by the method of Scott et al. 4, rapidly catalyzes the reduction of both liver microsomal and erythrocyte cytochrome ba and that catalysis of methemoglobin reduction by this enzyme preparation is stimulated by erythrocyte cytochrome b~ in a manner similar to that which we reported for erythrocyte cytochrome b 5 reductase 27. The disparity between the K m for NADH (with DCIP as electron acceptor) in the present study (4.8.1o -7 M) as compared to that reported for NADH dehydrogenase I (7.O-lO -6 M) might result from differences in pH, com- position of the buffers, and concentration of electron acceptor.


72 P . G . PASSON, D. E. HULTQUIST

The properties of the erythrocyte cytochrome b 5 reductase are also similar to those reported for cytochrome b 5 reductase from microsomes 1v-19 and from the outer membrane of mitochondria22, 2~ of other tissues. Thus, the erythrocyte and microsomal enzymes are similar in terms of FAD prosthetic group, specificity toward pyridine nucleotides and electron acceptors, pH dependency, inhibition by EDTA, and the apparent Km of NADH. However, the microsomal enzyme does show a considerably greater turnover number for the reaction between NADH and cytochrome b5 than does tile erythrocyte enzyme.

The redox systems of erythrocytes, leucocytes, and the membranes of microsomes, Golgi vesicles, mitochondria (outer membrane), and nuclei appear to have a great deal in common. The erythrocyte cytochrome b 5 reductase is similar to tile NADH-dependent flavoproteins of these other systems. Likewise, erythrocyte cytochrome b524 is similar to cytochrome b a isolated from microsomes, Golgi membranes, mitochondrial outer membranes, nuclear membranes, and leucocytes. In addition, erythrocytes, like most of the other systems, appear to contain either a native or modified form of hemeprotein P-45o. However, the physical state of tile redox proteins of the erythrocyte differs from that of the redox proteins in these other systems. Thus, in microsomes, cytochrome b 5 reductase, cytochrome bs, and hemeprotein P-45o are all an integral part of the membranous fraction and are solubilized only after t reatment with protease, detergent, or lysosomesaL In contrast, very mild methods for the lysis of red blood cells yield cytochrome b 5 reductase and cytochrome b 5 in soluble form, and hemeprotein P-42o loosely associated with the stromal fraction 28.

The similarity of these soluble proteins with the corresponding proteins solubilized from subcellular organelles of other tissues suggests that the soluble erythrocyte proteins are derived from particulate fractions present in precursors of the erythrocyte. Our finding, that greater amounts of the soluble cytochrome b.~ are present in a cell population high in reticulocytes, is in agreement with such a hypo- thesis. Since most of the subeellular organelles of the mammalian red cell disappear prior to the reticulocyte stage, a protein solubilized from the endoplasmic reticulum or other membrane fraction would have had less opportunity to degrade in a reticulocyte than in an erythrocyte.

Whereas microsomal cytochrome b 5 reductase and cytochrome b 5 appear to function in desaturation of fa t ty acids, the soluble reductase and cytochrome of erythrocytes appear to function in the reduction of methemoglobin by the pathway involving reactions I, 2, and 3 of Fig. IO 27. If particulate forms of these proteins are found in immature red blood cells, it will be of great interest to determine whether such forms function in either fa t ty acid desaturation or methemoglobin reduction. If a particulate form of cytochrome b 5 reduetase is present in erythrocyte precursors, it will also be of great interest to determine whether it, like the soluble reductase, is lacking in cells from patients with congenital methemoglobinemia.

ACKNOWLEDGMENTS

We wish to thank Dr Dan Reed for valuable ideas during the conception of this study, Mr Waiter Andrews and Mr Stephen Hansmire for skillful technical assistance, and Mr Michael Flashner for efforts in the preliminary stages of this work. We thank Dr Vincent Massey for recording the fluorescence spectra.

Biochim. Biophys. Acta, 275 (1972) 6"2-73

CYTOCHROME b 5 REDUCTASE FROM ERYTHROCYTES 73

P.G.P. was a predoctoral trainee of U.S. Public Health Service Grant GM-ooI87. This investigation was supported in part by Grant AM-o925o from the U.S.

Public Health Service and by grants 0RA-366o8 and NFR-8 from the University of Michigan.

t ~ E F E R E N C E S

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Biochim. Biophys. Acla, 275 (1972) 62-73

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