Some properties of denatured horse heart cytochrome c

ARCHIVES

OF

BIOCHEMISTRY

AND

Some Properties

BIOPHYSICS

232-238

117,

of Denatured

PAUL BRUMER,

Horse

WALTER Department

(1966)

Heart

G. LEVINE;

Cytochrome

c’

J. PEISACH3

of Pharmacology AND

HAROLD Department

of Biochemistry,

J. STRECKER4

Albert Einstein Bronx, New

College of Medicine, York iO@l

Yeshiva

University,

Received January 25, 1966 A cytochrome c derivative has been prepared by treating horse heart cytochrome c at pH 11 at 65”. This material contains two types of iron. One is associated with the chromophoric center of the protein. The other is nonchromophoric and endows the molecule with an ascorbate oxidase property. Spectral observations indicate protein configurational changes, as well as changes in the accessibility of the chromophoric iron to reducing agents. This derivative no longer can function as part of the electron transport, chain, but does competitively inhibit cytochrome c in the succinoxidase and the succinate cytochrome c reductase systems, but not in the NADH oxidase system.

Cytochrome c has been recognized generally as an unusually thermostable protein (l-3), and this property has been utilized in its isolation from baker’s yeast (3). In 1962, however, Butt and Keilin reported that heating solutions of horse heart cytochrome c at 100” for 3 minutes irreversibly denatured some of the dissolved protein. The altered cytochrome c, although not separated from the native form, was characterized by inactivity with the succinic dehydrogenase system and by the formation of a carbon monoxide complex (4). Johnson and Strecker have noted that heating of dilute solutions of either rat liver or horse heart cytochrome c, at pH near 7, resulted in complete loss of I This work was supported by grants from the United States Public Health Service, GM-10959 and HE -06450. 2 Career Development Awardee of the United States Public Health Service, 5 K3-GM-8102. 3 Career Development Awardee of the United States Public Health Service, 1 K3-GM-31,156. 4 Recipient of a Public Health Service Research Career Program award (1 KG-GM-2487) from the National Institute of General Medical Sciences.

activity for the proline oxidase system (5). Preliminary studies of the conditions required for this type of inactivation of cytochrome c demonstrated that the yield of inactive product was increased by raising the pH at which the heating was conducted. In 1948, Paul (6) incubated a cytochrome c solution at pH 12.7 and observed that the activity of this material with the succinoxidase system decreased with time, Butt and Keilin (4) had also noted that the degree of irreversible heat-denaturation of oxidized cytochrome c increased as the pH was raised from 7.2 to 10.2. These observations led us to a method for preparing larger quantities of the modified product. This report describes a material prepared by denaturing horse heart cytochrome c with heat and under strongly alkaline conditions. Some of the spectral and enzymic properties of this protein are reported. METHODS Preparation

of

heat-denatured

cytochrome

c.

One hundred mg of horse heart cytochrome c (Sigma Type III) was dissolved in water and the 232

DENATUI~ED pH was adjusted to 11 with NaOH. The solution was incubated at 65” for 17 hours, during which time the pH decreased to 10.3, the color became a light brown, and a precipitate formed. This mixture was adjusted t,o pH 7 and lyophilized. The dry material was dissolved in 40 ml of 1 mM ammonium acetate and dialyzed against 4.5 liters of H,O for 6 hours at 5”. Upon centrifugation at 37,OOOg for 5 minutes a brown sediment was obt ained, which was insoluble in ammonium acetate, and was discarded. An Amberlite CG-50 column was prepared by the following modification of the Margoliash procedure (7). Excess concentrated ammonia water was added to 6 gm of Amberlite CC-50 until the mixtllre remained decidedly basic for 10 minutes. The resin was twice washed by decantation with water, eqllilibrated with 0.05 M ammonium acetate, poured into a column 1 cm in diameter, and washed with 500 ml of 0.05 M ammonium acetate. The solution of dialyzed, heat-denatured cytochrome c was passed through the Amberlite col77nul , and the cflluent was collected and dialyzed against 4.5 liters of Hz0 at 5” for 5 hours. IJnchangcd cytochrome c was retained, visible as a red band OII top of the column (7). ?40 cytochrome c tlimer was present in this preparation as indic:tt,ed by the fact that 0.25 hf ammonium acetale ccnnpletely removed the adsorbed red band (7). After dialysis, the preparation was lyophilized, dissolved with difficulty in 7 ml of 1 mM ammonium ac*c:tate, :uld dialyzed oiice more for 3 hours at 5” against 3 liters of 0.05 Y potassium phosphate blll’fer, pH 7.3. The final volume of the material was 10.5 ml containing 2.6G mg of protein per milliliter as cnlculatted from 1 he extinction coefficient (see below). The solut,ion was stored at -20”. Before IIS~, this preparation was thawed and clarified by rentrifugation at) 37,000g for 10 minutes. t’repurcciiov~ of ndochondria. Pig heart mitochondria were prepared by the following revision of 1 he method of Crane et al. (8). All proccdllres wcare condllcted at 5”. Forty gm of pig heart was homogenized in 200 ml of 0.9% KCl, pII 7.2, with a motor-driven Teflon and glass homogenizer. The KC1 treatment removed most, though not all, of the endogenous cytochrome c (9). At the end of the homogenization the pI1 which had decreased to 5.X was :tdjust,ed to 7.2 with NaOH. The suspension was centrifuged al 15,000g for 15 minlltes, and with t,he aid of :I loose-fit,ting all-glass homogenizer (lo), the precipitate was suspended in 200 ml of 0.25 M sucrose containing 1 rnY EDTA, pH 7.2. This suspension was cent’rifrlged for 10 minutes at 65Og, the sediment was discarded, and the supernatant, fraction was recentrifuged for 10 minutes at 10,OOOg. The sediment of the second centrifugation was resuspended as before in 200 ml of 0.25 x

CYTOCIIHOhIt~;

L’:::‘,

c

sucrose containing 1 mu EDTA, pH i.2. Icillally, this mixtllre was centrifrlged again at, 10,OOOg for 10 minutes and the resulting mitochondrial pellrt was resllspended in 10 ml of the sllcrose-El)T:\ mixture. No phosphate was used in the prepar:, tion since it was formd that mitochondria preparcltl or stored with phosphate very rapidly lost, sll(’ cinoxidasr :tctivit)y. Mitochontlri:r WPT~’ IIS(~II within 21 hollrs of prepnrat ion. Eat liver mit ochondria were prepared :iccor~Iing to the met hod of Schllrider :ttltl Ilogohc~c~~~~ (11). Enzymic

assures.

Succiuoxidase

was

tlolerinitif~tl

manometrically with a Warburg apparat IIS ill 2 medium containing 0.075 ~1 potassilun ~~hospl~i~ buffer, pH 7.3, 0.04 hf succinate, 0.15 nil of l)ig heart mi tochondrial preparation iO.296 mg S per milliliter by Kjeldahl procedllrc), a,11d varyit):: concentraliot~s of cytochrome c, as indicatc?d, ill :b total volume of 2.0 ml. Reduction of added cyt,ochrome c by sllccinut,e was measllred spcc~ropho~~~metrically in :L medium contaiiiitlg 0.05 M pot ifssium phosphate, pll 7.3, 0.027 >f sclccirrafc. 1 nrhl KCN, and 0.01 ml of pig heart mitochorttlri:rl preparal ioll (0.431 mg N per millilitrlr) in :L 1 ola1 volume of 3.0 ml (12). Optical density readings were recorded at 550 rnp with a Zeiss PMQ II spec’trophotometer at 37” with cells of l-cm light ~)nt Ii NADH oxidase activity was determinc~tl spect 1’0 photometrically at 340 mp in a medi~un consist ill:: of 0.05 3% potnssilim phosphate, plT 7.3, 0.15 mhl NADH, 0.1 mg of cytochrome c, and 0.02 ml of llip heart mi tochondrial preparation iO.Z%i rng N ]~‘r milliliter) in a total vollime of 3.0 ml. Cytochronic c oxiduse was dctc~rrniiletl ni:1r10 metrically according to thp method of Slat(lr I 13, The mtldillm coll~ainetl 0.075 M pot:cssiltm pl~os phate, 1~11 7.3, 5 m>% El)TA, 0.2 ml 01’ mitcJc*hotldrial preparation (0.130 mg N per millilil(~r). :LIIII varying co]tcent,rat ions of cytochronic, c as itrcli cnted. Akscorb:ttr was added from tllo sitlc:rrm Io M final cnllcerttration of X.8 ml<. 7%~ lot al VO~IIIIII~ was 2.0 ml.

Optical spectra were ol~lainctl wit11 N lS;t~i*c~l~ and Lomb model 505 rrcorditlg sprat rophc~tornelc~r or with :I %riss PMQ I1 spect,rol’l”)tonl(,ter Ibsing cells with B I-cm light path. For dt~termiI1atiorl 01 extinctiofl coeficiotlls, (he protein sollrtioll was dialyzed agdinSt water before mrasliring 1hrx aI)sorption. then Ivophilizr~tl to (IrJ*ncss :it~tl wcighc~tl.

234

BRUMER,

ET AL.

Electrophoresis on paper was conducted with a Beckman model R cell. The strips were developed with bromphenol blue. All chemicals used were the highest quality commercially available. Cyanide solutions were prepared fresh daily. All water used in these experiments was deionized and glass distilled.

pH 7 before and after treatment with sodium dithionite is shown in Fig. 1. At this pH the extinction coefficient of the oxidized protein at 405 rnp is 7.3 mg-l cm-l, and of the reduced protein at 550 rnp is 1.0 mg1 cm-l. The spectral properties and their variation with pH are shown in Table I. Upon reduction with dithionite, there is a shift toward longer wavelength of the Soret band with a concomitant appearance of spectral peaks at

RESULTS

Spectrophotometric studies. The

spectrum of heat-denatured

optica cytochrome c at :’ ! ! ! ’ 1 ! i 1

i !

i

1

0.8

+%O

400

420

440

460

480

WAVELENGTH

500 520

540

560

580 600

(my)

FIG. 1. Optical spectra of heat-denatured cytochrome c and horse heart cytochrome c in 0.1 M Tris-acetate buffer, pH 7.4. (- - -) Heat-denatured cytochrome c; (---) heatdenatured cytochrome c reduced with Na&04; (-- - -) cytochrome c; (-e-.) cytochrome c reduced with NazSz04. All protein concentrations = 0.112 mg/ml. TABLE I SPECTRAL PROPERTIES OF DENATURED CYTOCHROME c PH

Oxidized peak

l.Ob

395 (6.2) 398 (7.5)

3.5b 5.5 7.0

9.0 13.8

403.5 (7.4) 405 (7.3) 405.5 (7.5) 411.5 (5.2)

Reducedpeak9

at 405 (5.5)" 400 (5.3), 414.5 (5.1), 521.5 (0.7), 415.5 (6.0), 521.5 (0.7), 550 (0.8) 417.5 (6.6), 521 (O.S), 550 (1.0) 417 (7.0), 523 (l.O), 549 (1.4) 417 (6.5), 522 (1.2), 551 (1.2)

295, shoulder

550.5

(0.7)

a Reduction with dithionite. b At this pH, the reduction of denatured cytochrome c was accompanied by visible precipitation sulfur. c Numbers in parentheses represent extinction coefficients expressed as mg-km-r.

of

DENATURED

CYTOCIIROMIS

255

c

2.0I.0 ! i

5

;

:

1

I

IO 15 20 3 ‘/IS]* 1o-4 CYTOCHROME

5 IO ‘/IS]’ IT4

I5

C

FIG. 2. Reciprocal plots of inhibition of succinate cytochrome c reduct,asr (Aj and succinoxidase (B). (0) No denatured cytochrome c added; (0) 0.6 mg denatured q-tochrome c added; (0) 0.423 mg denatured cytochrome c added. III A, 1) = 0.001 A/min/ml of mitochondria. In B, ZJ= ~1 O2 consumed/30 min/ml of mitoc~hontlria. Concentrations of cytochrome c are expressed in moles/liter

520 and 550 rnp. At pH 1, no 520 or 550 m/l peaks are observed. The reduced material can be reoxidized either by shaking in air or by adding I<(,Fe(CN)a. When H,S is added there is a shift in t,he Soret peak only, and 110 520 or 550 rnp peaks appear (compare Tables I and II). Ascorbate doesnot alter the spectrum of the denatured cytochrome c in the range from pH 1 to 9, even when added anaerobically. No change is seen in the spectral peaks of denat,ured cytochrome c in the presence of 1 mM azide. However, with cyanide at pH 7 there is an upward shift in spectrum for both the oxidized and the reduced peaks. At pH 13.8, the upward shift with cyanide is even more pronounced. The addition of pyridine, on the other hand, causesa downward shift and sharpening of spectral peaks. Anaerobic addition of cysteine (1 m&r), which reduces native cytochrome c, has no effect of any kind on the denatured cytochrome c. Carbon monoxide does not affect the oxidized spectrum of denatured cytochrome c in the pH range 1-13.8. When denatured cytochrome c is treated with CO at pH 7 and then reduced with dithionite, there is a shift in the Soret peak and the appearance of diffuse peaks around 529 and 561 mp (Fig. 5 and Table II), confirming the work of earlier investigators (4). At pH 7.3

TABLE

II

SPECTRAL PROPERTIES OF DENATUREI) CYTOCHWOME c COMPLEXES~ Condition

HzH HsS IItS NsCN-

(saturated) (saturated) (saturated) (1 rn>l) (5 m>I)

CN- (5 m&l) Pyridine (0.32 ~11 Pyridinc (3.2 M) CO (saturated) CO (sat)urat>ed) EI)T,h

(1 IIlM)

Imidazole

(1 miv)

13.P 7.2 7.2 7.0 13.8h x.5 i.0

417.5 404 401 405 411.5 405 405

a All reductions with dithionitc experiments. h pII adjusted with KOH.

41i, 520, 550 419, 523.5, 552 123. 5‘8, 557 414, 519, 549 414, 518, 549 413, 529, 561 414, 529 562 417, 520: 550 417, *x0, 550 except

II,S

and at 13”, heat-denatured cytochrome c exhibihs no spectral peak at 695 rnp in contrast to native cyt,ochrome c (16) (Fig. 6). The iron content of native cytochrome c is 0.429 %, and the iron content of heatdenatured cytochrome c is 0.8Oti %. Paper electrophoresis experiments were carried out at pH 8.6 in Verona1 buffer (ionic

236

BRUMER:

ET AL.

strength = 0.075). In 15 hours, at a current of 2.5 mA and at 87 V, native cytochrome c migrates 9.5 cm toward the cathode. Heatdenatured cytochrome c, however, under the same conditions, migrates 15.5 cm toward TABLE INHIBITION

OF

Enzymic tally .

III

SUCCINOXIDASE C~T~CHR~ME

activity

BY

DENATURED

c

was measured

manometri-

~102 consumed/ ml mitochondria/ 30 min Contents of Warburg flask inhyb;tor added

(1) Mitochondria (2) Mitochondria + 0.2 mg cytochrome c (3) Mitochondria + 0.3 mg cytochrome c (4) Mitochondria + 0.5 mg cytochrome c (5) Mitochondria + 1.0 mg cytochrome c

0

0.2

0.4

CYTOCHROME

Per cent +0.43 mg inhibition denatured cytochrome c

187 453

133 273

29 40

500

340

32

486

400

18

538

520

3

0.6

0.8

1.0

C (mg)

FIG. 3. Effect of denatured cytochrome c on cytochrome c oxidase activity measured manometrically. (0) No denatured cytochrome c; (0) 0.428 mg denatured cytochrome c added; (0) 0.941 mg denatured cytochrome c added.

0

I

I

0.1 0.2 DENATURED

I 0.3

I 0.4

CYTOCHROME

I 0.5

I 0.6

0.7

C (mg/ml)

FIG. 4. Effect of heat-denatured cytochrome c on the nonenzymic oxidation of ascorbic acid, measured manometrically. The medium contained 0.028 M ascorbate, 5 mM EDTA, 0.075 M potassium phosphate buffer, pH 7.3, in a total volume of 2.0 ml.

the anode. Only a single protein component was observed in both cases. Enzymic activity. The heat-denatured cytochrome c is not active with the succinoxidase system but showscompetitive inhibition with added cytochrome c in concentrations from 0.21 to 0.43 mg per milliliter (Fig. 2 and Table III). Similar competitive inhibition is also found for the reduction of added cytochrome c by succinate. In other manometric experiments, inhibition is observed for the mitochondrial succinoxidase activity without added cytochrome c (line (1) Table III). It is assumed that this endogenous activity is attributable to residual cytochrome c which is not removed by KC1 treatment (9). The addition of exogenous cytochrome c to the assay increasesthe rate of oxygen consumption. Even here, denatured cytochrome c is inhibitory. However, raising the concentration of exogenous cytochrome c overcomes the inhibition due to denatured cytochrome c. In these experiments (Table III) even the lowest concentration of exogenous cytochrome c is nearly saturating. The addition of albumin in a concentration the same as that of the denatured cytochrome c used in these experiments does not inhibit succinoxidase. When

used with a rat liver mitochondria preparation washed three times with 0.9% NaCl, 0.37 mg per milliliter of denatured cytochrome c inhibits the succinoxidase system 53 %. The addition of 0.532 mg denatured cytochrome c to the KADH oxidase system shows little or no inhibition. The denatured cytochrome has essentially no inhibitory effect on the cytochrome oxi-

Ch7

dase system although the rates without added native cytochrome c increase with inhibitor concentration (Fig. 3). This endogenous uptake of oxygen is due t.o ascot’bate oxidation. As seen in Fig. 4, lhe d+ natured cytochrome c catalyzes th(b oxidation of nscorb:tt,e, yet ascorbate has no app:verlt, effect under anaerobic conditiorls on t IIP optical spectrum of &natured c~;vl~ocl~~~~~~ (’ (SW :lt,ovP).

1

c:-4k 4!c4’ 490

530 510 WAVELENGTH

imp)

FIG. 5. Effect, of carbon monoxide on the optical absorbance of dithionite reduced heat-denatured cytochrome c. Protein concentration = 0.44 mg/ ml. (--) Reduced denatured cyt’ochrome C; C---J as above, followed by CO addition.

There arc some major differclncc~s between the optical properties of heat-denatured and native cytochrome c. Although nsrorbatc, H$, or dithionite can be us;cld to rctdu~c~ native cytochromc c, only dithionit,c produces all three spectral hemochronlc: peaks with the denatured prot,ein. This suggesth that the heme iron in denatured cytochrome c is less accessible to ascorbutc aud H,S, either due to conformational ch:~t~gc~ ill t 1~ protein or due to stronger biutlirrg ai, t 11~ x-ligand position of the heme-iron caomplcx. Stabiliza,tioll of iron iri the t rivakll t osidatiorl state is intlic::~tctl by tlrc itl:Mit,y 01 ascorbatc to reduce the heme iron. Schc~jt et antI

GcotJy?

(16)

sl1on-11

11avc

111:11 1 trc> 69.I

1.2 --

0

Ll I II I “630

650

670

WAVELENGTH

I I I I I I I

690

710

730

750

(mp)

FIG. 6. Optical spectrum of native (0) and hea,-denatured 5 mM potassium phosphate buffer, at 13”. The collcentratiorl 10.0 mg/ml, and the pH was 7.8. The con~cnl ration of hwl 1.3 m&ml, and the pH was 7.4.

ferricytochrome c (0) in of native cytochrome c was dtnatllred

cytochnome

c wns

238

BRUMER,

ET

AL.

rnp peak of native ferricytochrome c is re- enous cytochrome c. In the presence of lated to conformational properties of the inhibitor, increasing the amount of added protein but not to the heme iron. The dis- cytochrome c gradually overcomes the appearance of this peak in the denatured inhibitory effect. Thus competition with protein (Fig. 6) suggests that a conformacytochrome c is one possible mechanism of tional change has taken place. The guaniinhibition. In apparent conflict with this dine-induced conformational changes re- interpretation is our finding that denatured ported by Schejter and George were readily cytochrome c exhibits almost no inhibitory reversed by dialysis, while the changes effect on NADH oxidation. However, this produced by heat and alkali treatment, finding does give credence to the suggestion herein described, are irreversible. The dis- of Green and his associates “that the structinct downward shift in the Soret band upon tured electron transport chain is made up of heat-denaturation of ferricytochrome c inditwo intercommunicating but separable cates that a change of bonding to heme iron chains-one for oxidation of succinate and has occurred. A shift downward in wave- the other for oxidation of DPNH” (18). length absorption indicates higher energy ACKNOWLEDGMENTS bonding (17). We wish to thank Miss Patricia Hempstead for We propose that there are two types of iron seen in heat-denatured cytochrome c. help with some of the earlier experiments. One of these is the chromophoric heme iron, REFERENCES and the second is nonchromophoric, nonD., Proc. Roy. Sot. (London), Ser. B. 1. KEILIN, heme iron that is associated with ascorbate 98, 312 (1925). oxidation. This second atom of iron is tightly 2. KEILIN, D., Proc. Roy. Sot. (London), Ser. B. bound. It is not removed by dialysis or the 100, 129 (1926). addition of EDTA. This latter chelating D., Proc. Roy. Sot. (London), Ser. B. 3. KEILIN, agent does not block the oxidation of ascor106, 418 (1930) * bate catalyzed by the heat-denatured proD., Proc. Roy. Sot. 4. BUTT, W. D., AND KEILIN, tein. The source of nonheme iron in the (London), Ser. B. 166, 429 (1962). preparation may be from the base catalyzed 5. JOHNSON, A., AND STRECKER, H. J., J. Biol. Chem. 237, 1876 (1962). oxidation of the heme component of some of 6. PAUL, K. G., Acta Chem. &and. 2, 430 (1948). the native cytochrome c, releasing iron which E., Biochem. J. 66, 535 (1954). 7. MARGOLIASH, binds tightly and specifically to protein. 8. CRANE, F. L., GLENN, J. L., AND GREEN, D. E., Electron paramagnetic resonance (EPR) Biochim. Biophys. Acta 22,475 (1956). studies indicate the presence of two types of 9. SCHNEIDER, W. C., CLAUDE, A., AND HONEFe3+ in the denatured cytochrome c.~ Some BOOM, G. H., J. Biol. Chem. 173, 451 (1948). of the iron resonance peaks are diminished 10. DOUNCE, A. L., WITTER, R. F., MONTY, K. F., with ascorbate while the others are diminPATE, S., AND COTTONE, M. A., J. Biophys. ished with dithionite. Rough comparisons Biochem. Cytol. 1, 139 (1955). 11. SCHNEIDER, W. C., CLAUDE, A., AND HOGEof the components of the EPR spectrum BOOM, G. H., J. Biol. Chem. 183, 123 (1950). indicate that more than half of the bound 12. SINGER, T. P., AND KEARNEY, E. B., in “Methiron in the preparation is nonheme. ods of Biochemical Analysis” (D. Glick, The denatured cytochrome c does not ed.), Vol. 4, p. 331. Wiley (Interscience), inhibit cytochrome oxidase and has only a New York (1957). weak, poorly reproducible inhibitory effect 13. SLATER, E. C., Biochem. J. 44,305 (1949). on NADH oxidation. However, it exhibits a 14. HILL, R., Proc. Roy. Sot. (London), Ser. B. pronounced competition for cytochrome c in 107, 205 (1931). the succinoxidase system whether observed 15. HILL, R. AND KEILIN, D., Proc. Roy. Sot. manometrically or spectrophotometrically. (London), Ser. B. 114, 104 (1933). 3, From Table III it can be seen that the 16. SCHEJTER, A., AND GEORGE, P., Biochemistry 1045 (1964). denatured material inhibits electron transand Metalloporport involving both endogenous and exog- 17. FALK, J. E., “Porphyrins 6 W. E. Blumberg, P. Brumer, W. G. and J. Peisach, unpublished observations.

Levine,

phyrins,” p. 34. Elsevier, Amsterdam D. E., Comp. Biochem. Physiol. 18. GREEN, (1962).

(1964). 4, 81