Studies on the heme environment of oxidized cytochrome b5

Biaehimica et Biophvsica Acta, 336 (1974) 15~-24 [C~Elsevier Scientific Publishing Company. Amsterdam - Pri,~ted in The N~therlands

BBA 30601 STUDIES ON THE HEME E N V I R O N M E N T OF O X I D I Z E D C Y T O C H R O M E b~

MASAO IKEDA', TETSUTARO IIZUKA', HIROSttl TAKAO"," and .rUN J! HA( iHARA ~ "Department o~ BiophyMes, Faculty of EngJneering Science, Osaka University, Toyonaka, O.~aka 560 and bDepartment of Biochemistry, Osaka University Medical Sc,~ool, Osaka 520 (Japan) ( R ~ i v ~ l July 17th, 1973)

SUMMARY

Cytochrome b~, with a molecular weight of a b o u t 1.2" 104 was highiy purified from pig liver. The purified oxidized cytochrome bs was inw-stigated by the following three methods : (|) Absorption spectrophotometry at 23 :C and 77 K. (li) Electron Paramagnetic Resonance (EPR} sp.~ctroscopy at 20 K . ( I l l ) Kinetic measurements of the reaction with C N in the temperature range from 22.25 to 46.75 ~'C. I and ii have demonstrated that: i. In the pH region from pH 5.0 to 11.0, oxidized cytochrome h~ is in a purely low-spin state between 23 °C a n d 20 °K. 2. Below pH 4.0, the spin state reversibly changes to high-spin between 23 °C and 20 "K. This high-spin state is found to b2 due to the heroin released from cytochrome b~. 3. Above pH 12.0, thv spin state reversibly changes to another type of lowspin state between 23 ~C and 20 °K, which is thought to come from a distorted protein structure but not from the ligar.Jing of O H - . 4. Energy for three t,, orbitals c.'~.lculated in one hole formalism shows a high symmetry of ligand c o o r J i n a t i o n for the low-spin state at pH 6.2 and a lowering of the symmetry for anoth~x type of k,~-spin state at pH 12.0. i l l has demonstrated that 5. The reactior with C N - is bi-phasic. The fast reaction is the cytochrome h s m o n o c y a n i d e complex formation, and the slow one is the heroin dicyanide complex formation. 6. The activation energy for fa~t and slow reactions a~e both 25.t kcal ~ole. The values of entropy of activation for fa~t and slow reactions are 12. I and 1 I. 5 , n t copy units, respectively. The protein structure of cytochrome h~ in compariso~ with that of cytochrome c based on the results above as well as those of X-ray studies by Dickerson, R. E., T a k a n o , T., Eisenberg, D.. Kallai, O, B., Samosor.. L.. Cooper. A. * Present Address,: Laboratory of Biochemistry, Osaka College of ~'harrnacy. Matsubara, Japan.

~-)saka,

16 and Margoliash, E. (1")71) J. Biol. Chem. 246, 151 !-1535 and Mathews, F. S., Levine, M. and Argos, P. (1972) J. Mol. Biol. 64, 449--464 are discussed.

INTRODUCTION Cytochrome bs is a hemoprotein which mainly exists in the microsomes o f mammalian liver tissue. Since this cytochrome is tightly bound to the membrane structure of the endoplasmic reticulum, the solubilization procedure is used to purify the cytochrome. Cytochrome bs has been purified from various sources by m a n y investigators [i, 2, 3]. Kajihara and Hagihara [3] and Mathews and Strittmatter [4] have crystallized cytoehromc bs solubiliz~-d with Bacillus subtilis proteinase Nagarse [EC3.4.4,16} and pancreatic lipase (EC3.1.1.3}, respectively. Primary structures were determined by Ozols a,ld Strittmatter [5] and Tsugita et al. [6]. Spectrophotometric studies on cytochrome b5 were reported by Strittmatter and Ozols [7], BoissPehoratsky and Ehrenberg [8, 9] observed light absorption spectra and an EPR signal of cytochrome bs from neutral to alkaline pH regions and reported that oxidized cytochrome bs is present in the mixture of high-spin and tow-spin states. Mathews et al. [10, I I] reported the Zertiary structure of cytochrome h~ by X-ray studies and showed that both the 5th and 6th tigands are histidines. Keller and Wiithrich [12] studied the electronic g tensors by the measurements of the proton NMR spectra of cytochrome b~ and discussed the ligand coordination of this cytochrome. In order to determine the electronic state of oxidized cytochrome hs, we measured the optical and EPR spectra in a wide range fo pH values. Based on these results as ,~,elt as tha~ ~,f X-ray analysis [10, I I], we quantitatively elucidated the mechanism of the kinetic process of oxidized ~ytochrome bs with C N - using thermodynamic equations. MAT[:~RIALS AND METHODS Materials

Cytochrome hs was purified from pig liver according to Kajihara and Hagihara [31 with considerable modificatior~s for a large scale preparation. Pig liver {30 •gt has minceC and homogenized in 0.25 M sucrose t120 I), and tissue debris, nuclei and 3art of the mitchondria were removed by a Sharpres centrifuge. Microsomes was precipitated at pH 5.0 (with acetic acid) together with some proteins and some mitothor dna, and collected by the Sharpres centrifuge. The precipitate was suspended in 0.1 M phosphate buffer at pH 7 4 containing I mM EDTA at a concentration of about 20 mg/ml. For the solubilization of the cytochrome, crystalline Nagarse (1 nag of protein) was added to the sus F nsion, and the mixture was brought to pH 7.8, and then kept at about 15 ~C for about 48 h. The suspension was brought to pH 5.0 and filtered :rough a Buchner funnel with ~.he aid of cellite. The filtraLe was adjusted to pH 7 4 (with Tris) and to t;ais solution a sul~ci¢td, amount ~r DEAE-Sephadex l equii,brated with 0.05 M Trisacetate at pH 7.6'~ v, as added to absorb the cytochrome b s, and the Sephadex was col-

17 lected on a glass funnel. The Sephadex was washed with 0.05 M Tris-ace't3tte at p H 7.5, then transferred to a column. C y t o c h r o m e bs was eluted with 0.5 M TlJsacetate at p H 7.6. The elute was fractionated with (NH4)2SO 4 an -~. the fract on prec i p i t a t e d bet~'een 70 and 90,% s a t u r a t i o n was collected. The precipitate was dissolved with 0.1 M T r i s - a c e t a t e buffer at p H 7.6 and charged on a Sephadex G-70 c,~lumn, then developed by passing the same buffer. The fraction containing cytod~ dine bs was passed t h r o u g h a D E A E - S e p h a d e x column ~equilibrated with 0.05 M T r i s acetate at p H 7.6) and the column was washed with 0.1 M T r i s - a c e t a t e at p H 7.6, The c y t o c h r o m e on the c l o u m n v a s eluted with 0.5 M Tris-acetate at p H 7.6. T h e elute was dialyzed against 0.35 M T r i s - a c e t a t e at pH 6.2 and charged on a D E A E Sephadex column equilibrated with the same buffer, and then developed with the same buffer. C y t o e h r o m e bs separated into three bands on the column and the effluent c o r r e s p o n d i n g to each band was collected as separate fractions. The Ist, 2nd and 3rd band fractions contained about 50, 40 and 10'~,, of total c y t o c h r o m e h~, respectively. The !st band fraction was used in the present investigation. As discussed earlier [3], the above preparation may not represent the natural c y t o c h r o m e b5 molecule but a fragment e f hemoprotein having the same physiological and spectral properties as those o f the c y t e c h r o m e m microsomes. Primary structures [5, 6] were found to be quite similar between the cytochrome solubilized with pancreatic Lipase [!] and N a g a r s e [3}, and the space group o f crystalline cytochrome b5 is P2~2,2~ [4, 13] in both preparation.,,. So we can discuss the environment a r o u n d the heine o f c y t o c h r o m e h S solubilized with Nagarse based on the results o f X-ray studies on c y t o c h r o m e hs with pancreatic lipase [10, I I],

Methods Light a b s o r p t i o n spectra at 23 C and 77 " K were recorded by a Hitachi Model 124 s p e c t r o p h o t o m e t e r and a Shimadzu D-40 D F S split-beam spectrophotometer [I 4], respectively. The cell used for the cry:~genic temperature measurements was ~ specially constructed twin cuvette [14] with the apparent light path of 3 ram, to which :~ telfon diffuser was attach,:d. The aqu-'ous solution of cytochrome he was frozen without the a d d i t i o n o f glycerol. A n identical solution without the protein was used as the reference. EPR spectra were recorded by Varian Model E-12 at X-band frequency with a 100 k Hz field m o d u l a t i o n at hquid H_, temperature {20 K). The magnetic field strcr g~h was determined by the nuclear magnetic resonance o f pro.~on in water. Reactions with cyanide io~ were recorded by a Shimadzu D-40 D F S sFtitbeam s p e c t r o p h o t o m e t e r [t4] at 412 nm with an a p p a r a t u s to keep the temperature of the cell c o m p a r t m e n t constant within ~ 0.05 ' C by ~ a t e r circulation with Komatsu Y a m a t o Coolnics Model C'FE 120. The magne:ic stir~:er was equipped under the cell. The temperature was measured exactly by an A u - C o alloy vs Cu thermocouple. Detailed features of this a p p a r a t u s will be reported elsewhere. RESU LTS Fig. 1 s h o ~ s the a b s o r p t i o n spectra o f oxidized cytochrome b~ in the region from p H 3.5 to 12.0 at 23 C. Spectra a r o u n d neutral pH, having a g-band at 530 nm

18

SSJ~V

cyf b, (OXidiZed) ~'-

~30C

o~o.I

5

a #H3S

t s

' "" 400

I 500

"~'~-,o i 600

7O0

LtNGrH (rim) l-ig. I. Absorption spectra of oxidized cytochrorr_e bs a. 23 ~C. Cytochrome bs ~as dissolved in 0.5 M H3PO,-NaH2PO4 at pH 3.5. in 0.5 M Tris--aeetat¢ at pFl 4.0. 5.0 and 8.0, and in 0.5 M NaHCO3Na2COa-NaOH at pH 11.0 and 12.0. *Avt

and an a-band shoulder at 560 nm, are typical o f the low-spin type o f ferric hemoproteins [15]. Above p H I 1.0, the a - b a n d is slightly b r o a d e n e d , suggesting the appearance o f another type <~:"low-spin state, and the spectrum at p H 13.0 is stable and similar to that at p H 12.0. Below p H 4.0, the spectrum o f the low-spin type disappears, and new peaks appear at 637 nrn a n d 510 nm. Since these p e a k s are characteristic o f ferric high spin [15], oxidized c y t o c h r o m e hs seems to transit from low-spin to highspin ~itates on lowering the p H value. Together with the a b o v e change, the shar~ soret peak disappears on lowering the p H value below p H 4.0. These transitions in acidic and a3kaline p H regions are b o t h observed reversibly by s p e c t r o p h o t o m e t r y . Ab~;orption spectra at 77 °K exhibit the similar features to those in Fig. I. Fig. 2 illustrates the p H dependence o f the ratio o f the a b s o r b a n c e s Aa~o ,,1/ A , ~ , m for 23 °C and A~7,,m/Asso.,~ for 77 °K, where the m i d p o i n t o f this transition (pH 3.7) at 23 ~C is slightly sh.ifted to a lower p H value (pH 3.5) at 77 K . This p H dependence o f the spin state :~ simpler than that o f o x i t , z e d c y t o c h r o m e c having three forms below neutral p H [o f~]. Fig. 3 illustrates the E P R spectrum o f oxidized cyt.~chrome bs at p H ~.2. This E P R spectrum has a low-spin signal (el ~ 3.05, g2 := ".22 and g3 ~ 1.41) ar~J a

I,)

I-IsPO.-NaH~PO,

23"e

1.4

<[ !

•~ HIGH SPIN

..............

1.2 1.0 0.8 (16 0.4 0.2

LOW SPIN

. . . . . . . . . . . . . . . . .

0 "/'7'~K H , p O 4 - N o H ~ P Q ,

0.8

h ...............

L

~

. . . . . . . . . . .

2.0

30

40

5r"

i ~ HIGH SPIN

2 LOW SPtN

pH

Fig. 2. The pH dependence of the ratios ofthe absorbances A~0 n~/A4,~ ,,, for 23 C and A~2,7 nra!ASS0 nrn for 77 K . We adopted the ratios of the soret region at 23 C and the visible region at 77 ~K for several experimental reasons and since the pH dependence of this transition observed in the visible region is the same as that in the sorer region at 23 'C and 77 K . small a m o u n t o f h i g h - s p i n signal at g - 4.3. This high-spih signal m a y be due to a little c o n t a m i n a t i o n o f free iron a t o m w h i c h might c o m e f r o m the distorted h e m e o f the d e n a t u r a t e d f 6 r m o f the c y t o c h r o m e . Ehrenberg and B o i s s - P o l t o r a t s k y [8, 9] reported the s a m e lo',.-spin and h i g h - s p i n signals, together with the high-spin signal at g = 6.2 w h i c h w e c o u l d n o t observe at neutral p H . A t p H 12.0, tile E P R s p e c t r u m has a smaPl a m o u n t o f t w o kinds o f high-spin s i ~ a l s ( g -- 5 . 8 4 a n d s -- 4 . 3 ) a n d the o t h e r low-spin s i g n a l ( g 1 :~ 2.76, g, 2.26and g3 -~ 1.67) than the s p e c t r u m in the neutral p H region. In the alkaline p H region, these features are similar to t h o s e o f E h r e n b e r g and B o i s s - P o l t o r a t s k y [8, 9] except

bs

cyt

20*K

pH6?

.i,o "7i,, . . . . . . .

Qz,2 22

/ '\_ k/

0

I

2

3

4

5

= GAUSS I iS. 3. E P R s p e c t r u m o f oxidized c y t o c h r o m e t,s. C y t t x ' h r o r n e at pH 6.2, Measurements were carriea out dt 20 "K.

h s w a s dissolved in 0,5 M T r i s - , a . etate

20 TABLE I THE ~, VALUES OF OXIDIZED CYTOCHROME b~ AT N E U T R A L A N D A L K A L I N E pH REGIONS Measurements at pH 6.2 a~d ! 2.0 were carried out at 20 °K. The data for the pH values 7.0 and 12.14 at 88 ~K were taken from Ehrenberg and Boiss-PoRoratsky [8] and Boiss-Po[toratsky and Ehrenberg [9}. The high-spin absorption at g =- 2,0 could not be identified at pH 12.0 in the present investigation. pH

Low spin

High spin

6.2

gl = 3 . 0 5 g~ = 2.22 g~ : 1.41

7.0

gt :- 3.03 g2 - 2.23 g~ 1,43 gt ~, 2.76 ,¢2 2.26 g3 ~ 1,67 ~'~ ~ 2.76 g : : - 2.28 g 3 1,6g

12.0

12.14

ref. this paper

~8, 9 g - 6.2 (g 21 this paper g" ~: 5.98 L¢ - 2) 8, 9 g , 6.2 (g 2)

t h a t t h e r e is a large a m o u n t o f h i g h spin a t g :. 6.2 in t h e i r s p e c t r a . T h e s e g v a l u e s are listed in T a b l e 1. W e c a l c u l a t e d t h e e n e r g y f o r t h r e e t2~ o r b i t a l s in ot~e-hole f o r m a l i s m [17] u s i n g the l o w - s p i n g v a l u e s b o t h at p H 6.2 a n d 12.0. F r o m t h e s e v a l u e s o f e n e r g y , we o b t a i n e d t h e axial a n d r h o m b i c d i s tt o r t i o n s a n d listed t h e m in T a b l e I1. TABLE !I THE ENERGIES FOR THREE t~, ORBITALS 3. is t~ae spin-orbit coupling parameterlapprox. 400 c m - 4 , 1a and R are the .~,Jal and rhombic distortions, respectively. pH

p

6.2 12.0

3.2212 3.833).

R 1.6213. 2.356a

Fig. 4 i l l u s t r a t e s t h e r e a c t i o n o f o x i d i z e d c y t o c h r o m e b s ( 1 7 / ~ M ) o c c u r r i n g w i t h the a d d i t i o n o f K C N (20 r a M ) in 0.1 M s o d i u m p h o s p h a t e b u f f e r a t p H 7.6 b e t w e e n 22.25 a n d 46.75 cC. As d e s c r i b e d q u a l i t a t i v e l y b y H a g i h a r a a n d l i z u k a [14], t h i s rea c , i o n is very slow at r o o m t e m p e r a t u r e in s p i t e o f t h e a d d i t i o n o f excess K C N , a n d see ms t o c o n s i s t o f t w o f i r s t - o r d e r r e a c t i o n s . T h e a b s o r p t i o n s p e c t r a o f o x i d i z e d c y t o c h r o m e bs Lt 30 °C a f t e r t h e a d d i t i o n o f K C N h a v e i s o s b e s t i c p o i n t s a t 380 a n d 4 1 9 n r l w i t h i n the first | 0 r a i n ancl 386.5 a n d 422 n m a f t e r 10 rain in t h e s o r e t r e g i o n , c o r r e s p o n d i n g t o t h e i n i t a l p a r t t0--10 m i n ) a n d s u b s e q u e n t :~att ( 1 0 - 4 0 rain) o n C u r v e 3 in Fig. 4, respectively. A c c o r d i n g to G e o r g e a n d T s o u [18], we performe~ ~ a t h e r m o d y n a m i c a n a l y s i s

c-

t

cJ c,'

..

®

,;o

qc

C~_

---J-I0

I 2'0

I 30 l!me

32 ? G ZS~ 3~ 36 59 4 3 4~

I 40

ZS*C 5L)'C 75°C 90"C OO'C 75°C 75°C 75~C I 5,0

L~____ 6o

(mln~

Fig. 4. Reaction of oxidized cytochrome b~ with C N - between 22.25 and 46.75 ~C at pH 7.6. Plot of logarithm of the ratio of absorbance (A, -.4oo)/(A0 - A~:~) xs time, showing that the reaction consists of two first-order reactions. The concentrations of cytochrorne bs and KCN were 17 t~M and 20 raM, respectively. o f the a b o v e bi-phasic r e a c t i o n as f o l l o w s : (i) F r o m the results o f H a r m a n and W o r l e y [I 9] on the h y d r o l y s i s o f K C N , we h a v e calculated the c o n c e n t r a t i o n o f C N at each t e m p e r a t u r e d e s c r i b e d in Fig. 4. (ii) T h e values o f the a c t i v a t i o n energy for fast a n d slow r e a c t i o n s are d e t e r m i n e d to be b o t h 25.1 k c a l / m o t e f r o m the slopes o f A r r h e n i u s plots A a n d B in Fig. 5, respectively. (iii) T h e vzdues o f the e n t h a l p y o f a c t i v a t i o n A H * are given by E q n t :IH"

=

(1)

E~ - - R T

w h e r e E~ is the a c t i v a t i o n e n e r g y and T is the a b s o l u t e t e m p e r a t u r e a n d R is the gas c o n s t a n t T h e values o f the e n t r o p y o f a c t i v a t i o n zlS* are calculated f r o m the d a t a at 26.50 '~C using E q n 2

3 I

32

33

3,4

I/T x tO I

Fig. 5. Plots to dcternline the activation energy for fast and slow reactions, the so,ailed Arrhenius plot. as lines A and B, respectively, k was defined as ( M - ' - s e c - ' ) .

22 TABLE II1 THERMODYNAMIC DATA FOR THE REACTION OF CYTOCHROME bs WITH CNIFor the definition of symbols, see text. Kind of

k at 26.50 °C

E~

reaction

(M-Lsec -~)

(kcal/mole) (kt:al/mole) (¢. u.)

AH °

AS*

Fast Slow

! .93

25. I

24.5

12. I

1.02

25. I

24.5

l 1.5

where k is the rate constant and ks is the Boltzmann constant and h is Plank's constant. The thermodynamic data thus obtained are listed in Table II!. DISCUSS,ON Since there appears to be no non-conservative mutation near the heme between the eytochrome bs of calf and pig [6, 20], it seems reasonable to discuss the home environment of cytochrome bs o f pig with reference to the results o f X-ray studies on calf [lO, 11]. H o m e release in the acidic region

Since not only the light absorption spectrum (Fig. !) but also the E P R signal t,f cytochrome hs at pH 3.0 (adjusted with H3PO4) were found to be very similar to those of hemin at pH 3.0 (unpublished result), the high-spin state observed below pH 4.0 is considered to be due to the heroin released from the protein. The release o f home was also confirmed by the fact that 12 h dialysis of cytochrome bs against a buffer solution at pH 3.0 indicated the loss o f 73 % home through the dialysis tube. Because of the easy release o f home in the acidic region, cytochrome bs did not show such a complicated, .~pin change as observed in cytochrome c [16]. Electronic state o f the h o m e iron

Oxidi2ed cytochrome bs in the neutral pH region was found to be in a lowspin state belween 20 K and 23 "C, indicating the absence o f thermal mixing of the low-spin and high-spin states. Since the results o f X-ray studies on cytochrome ;'5 [10, 1 I] ~aggest the covalent binding of the 5th and 6th ligands to home iron, the t,dstence of the purely low-spin state at neutral pH is quite reasonable. So it might be r ~ther difficult to think that the high-spin E P R signal at g ---- 6.2, which was observed Ly Ehrenberg and Boiss-Poltoratsky [8, 9], is due to native cytocbrome b~ at neutral I'H region. Although the EPR spectrum at pH 12.0 has two kinds o f high-spin signal, the major part of the sample see,.,~ to be a low-spin comr~,und, because of the higher sensitivity of the high-spin signal than the low-spin one. " H~lisE P R spectrum is thou--ht to correspond to the absorption spectrum at pH 12.0 in ~-~g. I suggesting the exi.,tence of another type of low-spin than that in the neutral pH ~e~ion. The low-spin signal at pH 12.0 (gt = 2.75, g2 -~ 2.26 and ga -- 1.67) is very similar to the low-spin signal of acid Hb + (dihistbdine-type low-spin ; gl - 2.75, gz ~ 2.25 and g3 = 1.69) rep~,:ted by

23 Yonetani et al. [2t] but not to that of the alkali Hb + ( O H - - t y p e low-spin; g~ 2.56, g, -----2.17 and g3 : 1.83) [21]. The values o f the axial and rbombic distortions in the low-spin state in Table !I show that the ligand coordination at pH 6.2 has a higher symmetry than that at p H 12.0. This lowering o f symmetry will come from a distorted protein str~ :ture but not from the coordination o f O H - . In the alkaline p H region, the high-spin signal near g -- 6 appears and an increase of the high-spin signal at g ~ 6.2 was reported by Ehrenberg and BoissPoltoratsky [8, 9]. As reported b~ Boiss-Labbe [22], the high-spin signal at g ~ 6.2 might be due to hemin released fr< m cytochrome b s above pH I2.0. Smith and Williams [23] mentioned the import, r ce of the high-spin (g ~ 6.2) in the neutral pH region. But the spin state of c y t o c h : ,,e bs in the neutral p H region is essentially lowspin as discussed above, and this Iow-spie state is considered to be important in the functioning o f cytochrome b~ as the electron-transferring protein. Reaction with C N As shown in Fig. 4, the reaction of cytochrome bs with C N - is considered to consist of two first-order reactions. The absorption spectrum o f cytochrome b s (! 7/~M) 60 min after the addition of K C N (20 raM) at 30 ~C exhibits the same features as that of the heroin--cyanide complex. After 24 h of dialysis of cytochrome b~ at 23 °C against a buffer s o h t i o n at pH 7.6 containing 30 m M K C N , 76.9 j~ heme were removed through a dkdysis tube. Considering the rate constant at 25 C, the second phase o f this reaction is thought to indicate a reaction process such that the coordination position o f the home iron in cytochrome bs i~ attached to the C N - ion, thus breaking the strong interaction between the home and apoprotein. It might be reasonable to describe this reaction schematically as follows: cytochrome bs + 2 C N - ~-~,t .~ cytochrome hs CN '- C N - ~-,t,~,, ~ a p c c y t o c h r o m e h 5 : hemin(CNL,

13)

Thermodynamic prop," "ties o f the reaction The values o f the activation energy were 25.1 kcal/mole for both the fast and slow reactions, indicating the similarity in the environment of the 5th and 6th ligands. From X-ray studies on cytochrome b5 [I 1], the histidine residues 39 and 63, both the 5th and 6th ligands, bind the h e r e to the main chains firmly as bridges via a coordination bond and both residues are hydrogen-bonded to the main chain. Substitution o f the C N - ion f o r an internal ligand breaks the bridge, which will induce a major disruption o f the protein structure. In the case o f cytochrome c [24], 6th ligand is methionine which does not have such a hydrogen bond with the main chain as the histidine residues ofcyt'~chrome hs. It is. therefore, reasonable that the value of the entropy o f activation (12. t entropy units) for the monocyanide complex tbrmation in cytoehrome bs is remarkably ,arger than that in cytochrome c (0.5 entropy units) [18]. The difference in the value of the entropy o f activation shows that the protein structure of cytochrome bs is more disturbed by the coordination of the external ligand that, that of cytochrome c.

24 ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k Professor M. K o t a n i for his kind discussion, and they also thank D r M. A. Rashid for his technical assistance, T h e y t h a n k Professor T. Yonetani for his e n c o u r a g e m e n t s a n d gift o f m a g n e t i c stirrer m o t o r s . T h i s investigation has been supported in part by a Research G r a n t f r o m the M i n i s t r y o f E d u cation, Japan, and it has been u n d e r the auspice o f the J a p a n - U n i t e d Stateg Cooperative Science P r o g r a m , 6R005. REFERENCES 1 2 3 4

Strittmatter, P. and Vetic, S. F. (1956) J. Biol. Chem. 221,253-264 Boiss-Pohcratsky, R. and Chaix, P. (1964) Bull. Soc. China. Biol. 46, 867-880 Kajihara, T. and Hagihara, B. (1968) J. Biochem. Tokyo 63, 453--461 Mathews, S. F. and Strittmatter, P. (19691 J. Mol. Biol. 41,295-297 Ozols, J. and Strittmatter, P. (1969) J. Biol. Chem. 244, 6617-6618 6 Tsugita, A., Kobayashi, M., Tani, M., Kyo, S., Rashid, M. A., Yoshida, Y., Kajihara, T. a:td Hagihara, B. (1970) Proc. Natl. Acad. Sci. U.S. 67, 442-447 7 Strinmatter, P. and Ozols, J. (19661 in Heroes and Hemoproteins (Chance, B., Estabrook, R. and Yonetani. T., eds), pp. 447-461, Academic Press, New York 8 Ehrenberg, A. and Boiss-Poltoratsky, R. (1968) in Structure and Function of Cytochromes (Okunuki, K., Kamen, M. D. and Sekuzu, l., eds), pp. 59~598, Univ, of Tokyo Press, Tokyo 9 Boiss-Poltoratsky, R. and Ehrenberg. A. (1967) Eur. J. Bioehem, 2, 361-365 10 Mathews, F. S., Levine, M. and Argos, P. (1972) J. Mo|. Biol. 64, 449-464 t I Mathews, F. S.. Le,,ine, M. and Argos, P. (1971) Cold Spring Harbor Syrup. Quant. Biol. 36, 387-395 12 Keller, R. M. and Wiithrich, K. (1972) Biochim. Biophys. Acta 285, 326-336 13 Kretsinger, R. H., Hagihara, B. and Tsugita, A. (i9701 Biochim, Biophys. Acta 200, 421-422 14 Hagihara, B. and lizuka, T. (1971) J. Biochem. TokTo 69, 355-362 15 lizuka, T. and Yonetani, T. (1970) Adv. Biophys. 1, 157-182 16 Theorell, H. and Akeson, ~. (1941) J. Am, Chem. Scat. 20, 502-508 17 Kotani, M. (1961) Prog. Theor. Phys. (Kyoto) Suppl. No 17, 4--13 18 George, P. and Tsou, C. L. (1952) Biochem. J. 50, 440--448 19 Harman, R. W. and Worley, F. P. (1924) Trans. Farady Soc. 20, 502-508 20 Nobrega, F. G. and Ozols, J. (1971) J. Biol. Chem. 246, 1706-1717 21 Yonet~ni, T., lizuka, T. and Waterman, M. R.. (19711 J. Biol. Chem. 246, 7683-7689 22 Boisg-Labee, P. (1972) in Structure anti Function of Oxidation-Reduction Enzymes (Akeson, A.. and Ehrenberg. A., edsL pp. 303-308, Pergamon Pre~, London 23 Smith, D W~ and Williams, R. J. P, (1969) Struct. Bonding 7, 1~15 24 Dickerson, R. E.. Takano, T., Eisenberg, D., Kallai, O. B., Samoson, L., Co,per, A. and Margoliash, E. (1971) 3. Biol. Chem, 246, 1511-1535