Biochimica et Biophysica Acta, 495 (1977) 287-298
© Elsevier/North-HollandBiomedicalPress BBA 37797 NUCLEAR MAGNETIC RESONANCE STUDIES OF HEMOPROTEINS IX. pH DEPENDENT FEATURES OF HORSE HEART FERRIC CYTOCHROME c
ISAO MORISHIMA",SATOSHI OGAWA", TEIJIRO YONEZAWA"and TETSUTARO IIZUKAb " Department of Hydrocarbon Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606, and b Department of Biochemistry, School of Medicine, Keio University, Tokyo 160 (Japan)
(Received May 23nd, 1977)
SUMMARY The pH-dependent structural change of horse heart ferric cytochrome c was studied by high resolution proton nuclear magnetic resonance (PMR) spectroscopy at 220 MHz by monitoring the heine peripheral methyl peaks of the native protein, its cyanide complex, and chemically modified (carboxymethylated) ferric cytochrome c. The spectral changes reversible with respect to raising or lowering of the pH value were noted at pHs ~ 2.5, 4, 9, and 11, and at higher pH region above 12 for the native protein. However, in the case of the cyanide complex and the carboxymethylated protein the spectral changes were found to occur in alkaline pH region only above pH 11.5. These results were interpreted as follows: (1) The sixth ligand (methionine sulfur) is bound more weakly to the heme iron than the fifth one (histidine imidazole). The isomerization at pH 9 monitored by the disappearance of the absorption band at 695 nm was confirmed to associate with the displacement of the methionine ligand (Met 80) by the lysyl residue (Lys 79) by referring to the hyperfine shifted resonance of the amine complex and the carboxymethylated form of ferric cytochrome c. (2) Coordination mode of the Lys 79 residue to the heme iron is perturbed at pH 11 probably due to the breaking of the hydrogen bond between Tyr 67 and Thr 78 residues. The ligand displacement of the Lys 79 residue also occurs again at a higher pH region above 12, and hydroxide form of ferric cytochrome c is formed. (3) Acidification of the protein to pH 4.0-2.5 results in the low spin acidic form characterized by weakning of methionine-iron coordination which allows displacement of the methionine by external fluoride ligand. Further acidification below pH 2.5 changes the ferric conformation of cytochrome c drastically from the low spin form to high spin form by disruption of the methionine-iron coordination bond. In this very low pH region there are no well resolved hyperfine shifted resonances of the proton NMR, indicating the unfoldings of the protein from globular conformation into a random coil.
288 INTRODUCTION Cytochrome c is an important hemoprotein in mitochondrial electron transfer having a molecular weight of 12 400 and an iron porphyrin covalently attached to two cysteine sulfhydryls of the polypeptide chain. The tertiary structure of ferric cytochrome c has recently been visualized by the X-ray study to have a compact globular structure with the heme group buried in a heme crevice [1]. Because of its unique spectral property of pH dependence, the acid-base transition behavior ofcytochrome c, especially the pH-dependent profile of the linkage of the axial ligand, has been the subject of recent extensive studies. In the early studies, Theorell and Akesson demonstrated that ferric cytochrome c shows different absorption spectra in acidic, neutral, and alkaline solutions [2]. They distinguished five different spectra (types I-V) depending on the pH value of the protein solution: type I below pH 0.42, type II between pH 0.42 and 2.5, type III between pH 2.5 and 9.35, type IV between pH 9.35 and 12.76, and type V above pH 12.76. Although numerous studies using hydrodynamic, spectrophotometric, and potentiometric techniques have been made to elucidate the nature of the pH dependence of ferric cytochrome c after this work [3-9, 11, 27], detailed discussion on the intrinsic nature of the conformational transition of the protein has been open to further studies. This is probably due to the unfeasibility of the spectrophotometric method in the identification of the different low spin and high spin hemoproteins since the optical spectrum in the visible region only reflects the electronic structure of the heme and have no advantages to differenciate several forms of each low spin or high spin hemoprotein. For example, conformational isomerization of ferric cytochrome c in the alkaline solution monitored by the disappearance of the absorption band at 695 nm has usually been attributed to the displacement of the sixth methionine ligand by lysyl residue of the protein chain. However, the evidence that is incompatible with the lysine hypothesis has recently been presented on the basis of the pH titration of the chemically modified cytochrome c such as guanidinated and amidinated derivatives [12, 13]. These authors attributed this isomerization reaction either to the concentration of hydroxide anion required to displace the methionine ligand or to the ionization of the imino group of the fifth axial iron ligand of histidine. In the present study we have used high resolution proton nuclear magnetic resonance spectroscopy (NMR) at 220 MHz as an additional means of probing the pH dependent features of conformational change of ferric cytochrome c. The proton NMR studies of ferric and ferrous cytochrome c have been reported, including its pH dependences, by many investigators [14-19]. We have made here a comprehensive study of the pH dependence of ferric cytochrome c at acidic and alkaline region as well as its derivatives such as cyanide complex and carboxymethylated ferric cytochrome c. The hyperfine shifted resonances of the heme peripheral groups were monitored here as a sensitive probe of the chemical and physical environments of the heine prosthetic group of ferric cytochrome c. MATERIALS AND METHODS Horse heart ferric cytochrome c, type VI, purchased from Sigma was dissolved in 0.1 M potassium phosphate buffer, pH 6.0, and then it was quantitatively oxidized
289 by adding potassium ferricyanide to the protein solution. After dialysing the protein solution against the buffer, cytochrome c was purified by passing through the CM-52 cellulose column equilibrated with the same phosphate buffer. The elution of the protein from the column was done with 0.5 M potassium phosphate buffer, pH 7.0. The eluate was dialysed against water to obtain the salt-free sample and then lyophilized. The lyophilized sample was dissolved in ZH20 and was used for the NMR measurement after removing the insoluble portion of the sample by centrifugation. The protein concentration was about 3 mM. The complex of ferric cytochrome c with cyanide was prepared by adding the ligand to the 2H20 solution of the protein until the NMR spectral change of it was saturated. Carboxymethylation of the methionine residues at positions 65 and 80 was performed as the method previously described [20]. The pert value in alkaline and acidic regions were adjusted by successive additions of dilute NaO2H or dilute zHC1 to the protein solution in NMR sample tube, respectively. The p2H values are direct readings of the pH meter (Radiometer) using a micro-combination glass electrode (Ingold) inserted into the protein solution in the NMR sample tube. No correction was made for the deuterium isotope effect on the direct reading of the p2H values. There were no visible signs of protein precipitation in any pH region examined throughout the entire course of the titration. Proton NMR spectra were recorded in a pulsed Fourier transform mode (PFT) with a Varian Associates HR-220 spectrometer operating at 220 MHz equipped with a Varian variable temperature control unit and Nicolet TT-100 PFT operating instrument. The quadrature phase detection (QPD) method was used with pulse width 37.5 ps (90 degree pulse). The spectra were obtained by 4 K points transform of 40 kHz spectral width after 1024-8192 pulses with a repetition time of 0.05 s. The temperature of the ambient probe was kept at 22 °C unless otherwise noted. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internal standard and chemical shifts to low field at constant radio frequency were assigned as negative values. RESULTS The high resolution NMR spectra of ferric cytochrome c in the downfield hyperfine shifted region showed markedly the three step transitions as the pH of the protein solution is increased stepwise from neutral pH (Fig. 1). The peaks at --35, --32, and +25 ppm of the two heme peripheral methyl and methionine (Met 80) methyl resonances at pH 7.5 lose their intensity and new peaks, presumably, the heme ring methyl signals appeared at --21.5 and --24 ppm (al, a2) above pH 9. The spectral changes at pH ~ 11 and pH ~12 of ferric cytochrome c were also as drastic as those at pH ~ 9. The signals al and az expend progressively their intensity and concurrently the new peaks of the heme methyl resonances appear at --22 and --22.5 ppm (b I and b2) above pH 10.5. By further raising the pH above 12 these peaks again disappeared from their original position and finally ferric cytochrome c gave broadened resonances at -- 11.5, -- 15.1, --20.2, and --25.2 ppm (signals 1-4). It is also possible to obtain information about the alkaline induced structural transition of ferric cytochrome c by studying the proton NMR spectra of cyano- and carboxymethylated cytochrome c in the alkaline pH region since the 6th coordination site is blocked by the external ligand in the cyanide complex and the methionine residue could no more be responsible for the coordination to the heme iron in the
290 Cyt ¢ (Fe 3")
4
3
2
p21.e
1
I
-3S
-30
-25
-20
"15
PPM
"40
S
I0
15
2(]
25
30
Fig. 1. pH dependence of the proton NMR spectra of ferric cytochrome c at 22 °C in alkaline pH region. High and lowfield parts of the hyperfine shifted spectrum are shown. Spectral changes are noted at pH 9, 11, and at higher pH region above 12. carboxymethylated protein. In Fig. 2 is shown the pH dependence of the N M R spectra in the downfield region of the carboxymethylated cytochrome c. This modified protein gives their heme peripheral methyl peaks at --13, --21.5, and --24 ppm at neutral pH and shows no such spectral change below pH 11 as seen in the native protein. Above pH 11, however, the hyperfine shifted resonances of this protein decrease their intensity with a slight shift to the lower field side, and concomitantly broad peaks appear at --11.5, --15.1, --20.2, and --25.5 ppm. Fig. 3 shows the proton N M R spectra in the hyperfine shifted region of the amine complex, carboxymethylated, and alkaline (at pH 9.9) cytochromes c, and these are compared with that of the native protein. In the spectra of the cytochrome c at pH 9.9, and of the amine complex at pH 7.5 the signals coming from the residual native form remain at their original position. It should be noted here that the spectra of the carboxymethylated and the alkaline cytochrome c are almost similar, indicating that the structure of the heme environment, especially the iron axial ligand in these cytochromes c are identical. Moreover amine derivatives of cytochrome c such as methylamine and ethylamine complexes afford their heme methyl peaks at the spectral region between --20 and --27 ppm [30]. As depicted for the case of amine complex, two heme peripheral methyl pe~ks are seen at --23 and --24.5 ppm, suggesting that the iron axial ligand of ferric cytochrome c at pH 9.9 and of the carboxymethylated protein is primary amine, probably supplied by the lysyl residue at position 79. Ferric cytochrome c shows pH-dependent spectra in the alkaline p H region
291 CM Cyt ~(Fe 3°)
p2H 12.5
i
-30
i
-~
-20
-15
~m
i
i
-10
10
Fig. 2. pH dependence of the proton N M R spectra of carboxymethylated ferric cytochrome c at 22 °C. The hyperfine shifted region of the spectra is shown. The spectral change reversible with raising or lowering of pH is noted at pH 12.
more explicitly than its cyanide complex, suggesting that the spectral changes of the native protein at pH 9 and 11 are attributable to the conformational transition of the sixth coordination side of the heme (Fig. 4). The cyanide complex of the protein gives their heme methyl proton N M R peaks at --11.5, --15.1, --20.2, and --25.2 ppm at the pH region between pH 7 and 10.5, and shows no spectral change in thi6 pH region. Above pH 11, however, these peaks decrease their intensity with small progressive shift to the upper or lower field side and finally new signals appears concomitantly at --11.5, --15.3, --20.2, and --25.3 ppm above pH 11.5. The small spectral shifts of the cyanide complex may result from the heme environmental perturbation caused by pH variation. It is quite interesting to note that the spectra of the native protein, carboxymethylated protein and the cyano-proteins quite resemble each other above pH 12. It is likely that the internal and external ligands in these cytochromes c are replaced by the hydroxide anion and hence they gave the similar spectra at higher pH region above pH 12. The shift of this transition to the lower pH region in the cyanide complex and the slight difference in these spectra, particularly the broader peaks for the native and carboxymethylated cytochromes c than the cyanide complex, may be indicative of the slight difference in the mode of unfolding of the protein moiety caused by the pH variation.
-25
i
-20
3*)
-15 PPM
I
-10
t
5
I
10
15
i
20
25
I
30
Fig. 3. Comparison of the proton N M R spectra of the hyperfine shifted signals of amine complex of cytochrome c at pH 7.0, carboxymethylated cytochrome c at pH 7.0, cytochrome c at pH 9.9, and native cytochrome c at pH 7.0. The spectrum of the native protein at pH 9.9 is almost similar to that of the carboxymethylated one at pH 7.0.
i
-30
-35
I
-40
Cyt-c(Fe
293 Cyt¢ (Fe3*)'CN" pin
3
-10
-IS
2
-~
I
-IS mM
Fig. 4. Proton NMR spectra of the cyanide complex of ferric cytochrome c at several pHs. Lowfield region of the hyperfine shifted signals is shown. The pH-dependent spectral change is seen at pH 11.5. Fig. 5 illustrates the pH dependence of the proton N M R spectra of ferric cytochrome c in acidic pH region, indicating the presence o f three different forms. The low field peaks at --35, and --34 ppm and the broad high field absorption at + 2 4 ppm have been previously assigned to the resonances of two of the heine ring methyl groups and of axial methionine methyl, respectively [16, 19]. These signals lose their intensity with progressive line broadening below pH 4, and concomitantly a new peak comes out at + 2 0 ppm. This new peak grew gradually with decreasing pH at the expense of the methionine methyl signal at + 2 4 ppm. The peak at + 2 4 ppm was finally replaced by this new signal at about pH 2.6. The sum of the intensity of two resonances at + 2 0 and + 2 4 ppm was half of the total intensity o f the two heme peripheral methyl signals at --34 and --35 ppm, indicating that the new peak at + 2 0 ppm is attributable to the methyl signal o f the methionine ligand dislocated slightly from the original coordination position. The two heme side methyl signals which correspond to the methionine resonance at + 2 0 ppm appear to be located at --32 to --37 ppm with substantial line broadenings. To further clarify the acid induced conformational change of ferric cytochrome c, the binding of the external fluoride to the heme iron was monitored by the proton NMR. It was then noted that the fluoride ligand binds to the heme iron to produce a ferric high spin complex in a narrow pH region in which the new peak at + 2 0 ppm was observable in the upfield spectrum region for the native protein (see Fig. 5). This was confirmed by the observation of broad signals
294 I--'
,
I
I
,
I
p=H
Cyt £ (Fe ~'+)
2.6
Jk_j,/
I
-40
i
-30
I
-20
3.1
I
-lO
I
*
i
t
0
10
20
30
ppm
Fig. 5. pH dependence of the proton NMR spectra of ferric cytochrome c at acidic pH region. The hyperfine shifted signals are illustrated. Three spectra are distinguished depending on pH of the protein solution, between pH 7 and 4, between pH 4 and 2.5, and below pH 2.5. Below pH 2.5 all the hyperfine resonances are broadened out beyond detection.
of the heme side methyl groups at the spectral region between --40 and --70 ppm, which was similar to the spectrum of metmyoglobin fluoride. In this p H re~ion between 4 and 2.5, the addition of fluoride afforded the spectrum of ferric high spin cytochrome c fluoride contaminated with that of the native form. It appears safe to say that in this p H region there exist two forms of ferric cytochrome c, one of which can replace the methionine ligand by the external fluoride. This may allow us to expect that the coordination bond between the iron-methionine is weak enough to be replaced by the weak external ligand. With further lowering the p H of the protein solution below p H 2.5, all the hyperfine shifted signals disappeared and no peaks were noted down to --100 p p m from DSS, indicating that the resonances of the heme peripheral groups were broadened out beyond detection. These results are obviously associated with the well known conformational transition of ferric cytochrome c from the low spin form to the high spin form.
295 DISCUSSION The hyperfine shifted proton NMR spectra of ferric cytochrome c at pH 9.9 as well as the carboxymethylated one at pH 7 showed that the environment of the sixth coordination site in these cytochromes c are closely similar. In addition, the spectra of these cytochromes c are also similar to those of the complexes of ferric cytochrome c with primary amines. These findings suggest that the new ligand to the heme iron of cytochrome c above pH 9 is the lysine residue at position 79. It has previously been suggested that the conformational transition of ferric cytochrome c at pH 9 monitored by the disappearance of the absorption band at 695 nm is attributable either to the displacement of the methionine ligand by the hydroxide anion or to the ionization of the imino group of the fifth histidine ligand [12, 13]. Neither the former nor the latter hypothesis can be affirmed by our present NMR study since the cyanide complex and the carboxymethylated cytochrome c show no such a change at corresponding pH region. Moreover, the spectra of carboxymethylated one at pH 7 is almost identical with that of the native protein at pH 9.9, indicating clearly that the change of ferric cytochrome c at pH 9 is not attributable to the concentration of hydroxide anion required to displace the Met 80 ligand. It has been pointed out from the examination of the Kendrew skeletal model of cytochrome c that Met 80 can be swung away from the original position by rotating the polypeptide chain and then the Lys 79 residue can be swung up until its amino nitrogen is exactly at the sixth coordination position [21]. Beside this suggestion, a variety of spectral evidences support the lysine hypothesis including ESR and NMR evidences by Peisach et al. and Gupta and Koenig [9, 19, 22]. The present study confirmed that the isomerization of ferric cytochrome c at pH 9 is resulted from the coordination of the lysyl residue to the heme iron by displacing the methionine residue. The proton NMR spectra in the hyperfine shifted region at pH above 12 suggest that the heme axial group of ferric cytochrome c, carboxymethylated cytochrome c, and the cyanide complex are the same in these pH region. It is tempting for us to exclude the possibility of the ionization of the His 18 imino group in the interpretation of the spectral change occurring above pH 12. If the change of ferric cytochrome c at pH 12 is attributed to the ionization of the His 18, a spectrum different from that of the native protein should be obtained for the cyanide complex in the corresponding pH region. The above results and discussion suggest that the new sixth iron ligand of ferric cytochrome c above pH 12 is the external hydroxide anion. Although the appearance of the hydroxide form of ferric cytochrome c has been suggested by ESR and spectrophotometric studies [2, 22], no conclusive evidence has been presented presumably because of the difficulty in the interpretation of the spectra due to the unfoldings of the protein molecule. The broadend proton NMR resonances in the hyperfine shifted region at high pH indicates that the compact globular structure of ferric cytochrome c is partly broken above pH 12. The hydroxide form of ferric hemoproteins has been known to appear by the alkaline ionization of the iron bound water molecule as evidenced for metmyoglobin and methemoglobin. In these hemoproteins the alkaline derivatives are thermal spin mixture between high spin and low spin states, and the heme methyl proton NMR signals of these were observed in the spectral region between --30 and --50 ppm [23, 24]. In ferric cytochrome c, the hyperfine shifted spectrum of the hydroxide form is
296 characteristic of low spin hemoprotein since the heme methyl peaks are in the spectral region between --10 and --25 ppm. This difference between the hydroxide forms of these hemoproteins is probably due to the covalent bond of the heme to the apoprotein of ferric cytochrome c. According to Otsuka. thermal mixing between the low and high spin states of hemoprotein is deeply related to the rigid fixation of the heme group to the apoprotein [29]. The absence of thermal mixing of spin state in ferric cytochrome c complex has been briefly discussed for its azide and imidazole complexes on the bases of the van der Waals contact interaction between the heme group and the apoprotein by Saito and Iizuka [25]. Having made the assignment of the spectral change of cytochrome c occurring at pH 9 and 12, let us now consider why the change of the spectra was noted at pH 11 only for the native cytochrome c. The pH jump and potentiometric studies of cytochrome c indicated that the ionization of the tyrosine residue at position 67 occures at about pH 11 [19, 26, 27]. The hydrogen bond between this tyrosine residue and the threonine residue at position 78 has been visualized by X-ray crystallographic studies [1]. These results allow us to suggest that the change at pH 11 is associated with the change of the coordination mode of the lysyl residue at position 79 to the heme iron due to the breaking of the hydrogen bond between Thr 78 and Tyr 67. Accordingly, we designate this cytochrome c formed at pH 11 as "strained lysine form". This assignment is compatible with the observation that the cyanide complex shows no substantial change in the spectra at corresponding pH. However, the effect of the breaking of the hydrogen bond between Thr 78 and Tyr 67 is hardly manifested in the NMR spectrum of the carboxymethylated cytochrome probably due to the effect of the chemical modification since this modified cytochrome c did not exhibit a substantial spectral change in these pH region. A variety of the spectral evidence suggested that acidification of ferric cytochrome c down to pH 2 results in the displacement of the methionine ligand by a weak field ligand supplied by a solvent molecule [2-4, 8-10, 14]. For example, Peisach et al. have indicated that at below pH 4 the protein is converted from low spin form to high spin form, evidenced by low field splitting (g = 6.3, 5.8) in the ESR signal and by an optical spectrum similar to that of metmyoglobin [22]. Although this drastic acid induced transition of ferric cytochrome c has been studied by many investigators, confirmative evidence of the displacement of the Met 80 ligand from its coordination position is quite limited. Gupta and Koenig showed that the methionyl methyl proton NMR signal of native cytochrome c at +24 ppm disappears at low pH, indicating a displacement of the methionine ligand [19]. We pay much attention here to the peak at ~-20 ppm which is presumably assigned to the methionyl methyl signal dislocated from the native one by acid-induced perturbation of the heme environment. Our observation that this methionine is replaced by a weak high spin ligand F- and that its signal at +20 ppm disappeared below pH 2.5 may lead to conclude that the methionine-iron bond is weaken with the lowering of the pH of the protein solution to produce an acidic low spin form, so that it is replaced by external fluoride. This acidic form of ferric cytochrome c in a low spin state has not been identified in other studies. To confirm further this methionine replacement, cross-saturation experiments on the two forms below pH 2.5 appear to be desirable and are now being planned in our laboratory. It is likely that the protonation of some amino acid residue except Met 80 is the first step to cause the acid conformational
297 transition of cytochrome c because the methionine residue has no ionizable group in aqueous solution. Although several assumption were presented for this group such as the His 26 and the heme propionate [10, 17], we have no evidence to assign this group. It is also probable that further acidification of cytochrome c below pH 2.5 results in the complete disruption of the protein conformation and thus no hyperfine shifted peaks were observed due to line broadening. In summary, the present N M R study of the hyperfine shift distinguished six different forms of ferric cytochrome c depending on the pH of the protein solution. They are acid high spin form (below pH 2.5), acidic low spin form (between p H 2.5 and 4), native form (between pH 4 and 9), lysine form (between pH 9 and 11), the so-termed "strained lysine form" (between pH 11 and 12.5) and hydroxide form (above pH 12.5). These results also suggest that the conformation of ferric cytochrome c is maintained in a compact and functional form by specific interaction of the ionizable residue of the polypeptide chain. The breaking of these interaction effected by pH and chemical modification results in a conformational change in the heme vicinity of the protein. ACKNOWLEDGEMENTS The authors are grateful to Dr. M. Ikeda-Saito for preparation of carboxymethylated cytochrome c. They also wish to thank Dr. T. Hosoya and Dr. T. Inubushi for their encouragement and discussion. This work is supported by Toray Research Grant for Science and Technology and by research grant in aid from Ministry of Education, Japan.
REFERENCES 1 Dickerson, R. E., Takano, T., Eisenberg, D., Kalliai, O. B., Samson, L., Cooper, A. and Margoliashi, E. (1971) J. Biol. Chem. 246, 1511-1534 2 Theorell, H. and Akesson, A. (1941) J. Am. Chem. Soc. 63, 1812-1818 3 Babul, J. and Stellwagen, E. (1972) Biochemistry 11, 1195-1200 4 Aviram, I. (1973) J. Biol. Chem. 248, 1894-1896 5 Sutin, N. and Yandell, J. K. (1972) J. Biol. Chem. 247, 6932-6936 6 Eakin, R. T., Morgan, L. O. and Matwiyoff, N. A. (1975) Biochemistry 14, 4538-4543 7 Hartzell, C. R. and Shaw, P. W. (1976) Biochemistry 15, 1909-1914 8 CzeLinski, G. and Bracokova, V. (1971) Arch. Biochem. Biophys. 147, 707-716 9 Schejter, A., Aviram, I., Margalit, R. and Goldkorn, T. (1975) Annal. N.Y. Acad. Sci. U.S. 244, 51-59 10 Stellwagen, E. and Babul, J. (1975) Biochemistry 14, 5135-5140 11 Greenwood, C. and Wilson, M. T. (1971) Eur. J. Biochem. 22, 5-10 12 Stellwagen, E., Babul, J. and Wilgus, H. (1975) Biochim. Biophys. Acta 405, 115-121 13 Pettigrew, G. W., Aviram, I. and Schejter, A. (1976) Biochem. Biophys. Res. Commun. 68,807813 14 Lanir, A. and Aviram, I. (1975) Arch. Biochem. Biophys. 166, 439-455 15 Wuthrich, K., Aviram, I. and Schejter, A. (1971) Biochim. Biophys. Acta 253, 98-103 16 McDonald, C. C. and Phillips, W. D. (1973) Biochemistry 12, 3170--3186 17 Cohen, J. S., Fisher, W. R. and Schechter, A. N. (1974) J. Biol. Chem. 249, 1113-1118 18 Wuthrich, K. (1970) Struct. Bonding. 8, 53-121 19 Gupta, R. K. and Koenig, S. H. (1971) Biochem. Biophys. Res. Commun. 45, 1134-1143 20 Schejter, A. and Aviram, I. (1970) J. Biol. Chem. 245, 1552-1557
298 21 Dickerson, R. E., Takano, T., Kallai, O. B. and Samson, L. (1970) "Structure and Function of Oxidation Reduction Enzymes" (Akesson, A. and Ehrenberg, A., eds.), pp. 69-83, Pergamon Press, Oxford 22 Peisach, J., Blumberg, W. E., Ogawa, S., Rachmilewitz, F. A. and Oltzik, R. (1971) J. Biol. Chem. 246, 3342-3355 23 Morishima, I. and Iizuka, T. (1974) J. Am. Chem. Soc. 96, 5279-5283 24 Iizuka, T. and Morishima, I. (1975) Biochim. Biophys. Acta 400, 143-153 25 Saito, M. and lizuka, T. (1975) Biochim. Biophys. Acta 393, 335-342 26 Rupley, J. A. (1964) Biochemistry 3, 1648-1650 27 Czerlinski, G. H. and Dar, K. (1971) Biochim. Biophys. Acta 234, 57-61 28 Wada, K. and Okunuki, K. (1969) J. Biochem. 66, 263-272 29 Otsuka, T. (1970) Biochim. Biophys. Acta 214, 233-235 30 Morishima, I., Ogawa, S., Yonezawa, T. and Iizuka, T. (1977) Biochim. Biophys. Acta, in the press
© Elsevier/North-HollandBiomedicalPress BBA 37797 NUCLEAR MAGNETIC RESONANCE STUDIES OF HEMOPROTEINS IX. pH DEPENDENT FEATURES OF HORSE HEART FERRIC CYTOCHROME c
ISAO MORISHIMA",SATOSHI OGAWA", TEIJIRO YONEZAWA"and TETSUTARO IIZUKAb " Department of Hydrocarbon Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606, and b Department of Biochemistry, School of Medicine, Keio University, Tokyo 160 (Japan)
(Received May 23nd, 1977)
SUMMARY The pH-dependent structural change of horse heart ferric cytochrome c was studied by high resolution proton nuclear magnetic resonance (PMR) spectroscopy at 220 MHz by monitoring the heine peripheral methyl peaks of the native protein, its cyanide complex, and chemically modified (carboxymethylated) ferric cytochrome c. The spectral changes reversible with respect to raising or lowering of the pH value were noted at pHs ~ 2.5, 4, 9, and 11, and at higher pH region above 12 for the native protein. However, in the case of the cyanide complex and the carboxymethylated protein the spectral changes were found to occur in alkaline pH region only above pH 11.5. These results were interpreted as follows: (1) The sixth ligand (methionine sulfur) is bound more weakly to the heme iron than the fifth one (histidine imidazole). The isomerization at pH 9 monitored by the disappearance of the absorption band at 695 nm was confirmed to associate with the displacement of the methionine ligand (Met 80) by the lysyl residue (Lys 79) by referring to the hyperfine shifted resonance of the amine complex and the carboxymethylated form of ferric cytochrome c. (2) Coordination mode of the Lys 79 residue to the heme iron is perturbed at pH 11 probably due to the breaking of the hydrogen bond between Tyr 67 and Thr 78 residues. The ligand displacement of the Lys 79 residue also occurs again at a higher pH region above 12, and hydroxide form of ferric cytochrome c is formed. (3) Acidification of the protein to pH 4.0-2.5 results in the low spin acidic form characterized by weakning of methionine-iron coordination which allows displacement of the methionine by external fluoride ligand. Further acidification below pH 2.5 changes the ferric conformation of cytochrome c drastically from the low spin form to high spin form by disruption of the methionine-iron coordination bond. In this very low pH region there are no well resolved hyperfine shifted resonances of the proton NMR, indicating the unfoldings of the protein from globular conformation into a random coil.
288 INTRODUCTION Cytochrome c is an important hemoprotein in mitochondrial electron transfer having a molecular weight of 12 400 and an iron porphyrin covalently attached to two cysteine sulfhydryls of the polypeptide chain. The tertiary structure of ferric cytochrome c has recently been visualized by the X-ray study to have a compact globular structure with the heme group buried in a heme crevice [1]. Because of its unique spectral property of pH dependence, the acid-base transition behavior ofcytochrome c, especially the pH-dependent profile of the linkage of the axial ligand, has been the subject of recent extensive studies. In the early studies, Theorell and Akesson demonstrated that ferric cytochrome c shows different absorption spectra in acidic, neutral, and alkaline solutions [2]. They distinguished five different spectra (types I-V) depending on the pH value of the protein solution: type I below pH 0.42, type II between pH 0.42 and 2.5, type III between pH 2.5 and 9.35, type IV between pH 9.35 and 12.76, and type V above pH 12.76. Although numerous studies using hydrodynamic, spectrophotometric, and potentiometric techniques have been made to elucidate the nature of the pH dependence of ferric cytochrome c after this work [3-9, 11, 27], detailed discussion on the intrinsic nature of the conformational transition of the protein has been open to further studies. This is probably due to the unfeasibility of the spectrophotometric method in the identification of the different low spin and high spin hemoproteins since the optical spectrum in the visible region only reflects the electronic structure of the heme and have no advantages to differenciate several forms of each low spin or high spin hemoprotein. For example, conformational isomerization of ferric cytochrome c in the alkaline solution monitored by the disappearance of the absorption band at 695 nm has usually been attributed to the displacement of the sixth methionine ligand by lysyl residue of the protein chain. However, the evidence that is incompatible with the lysine hypothesis has recently been presented on the basis of the pH titration of the chemically modified cytochrome c such as guanidinated and amidinated derivatives [12, 13]. These authors attributed this isomerization reaction either to the concentration of hydroxide anion required to displace the methionine ligand or to the ionization of the imino group of the fifth axial iron ligand of histidine. In the present study we have used high resolution proton nuclear magnetic resonance spectroscopy (NMR) at 220 MHz as an additional means of probing the pH dependent features of conformational change of ferric cytochrome c. The proton NMR studies of ferric and ferrous cytochrome c have been reported, including its pH dependences, by many investigators [14-19]. We have made here a comprehensive study of the pH dependence of ferric cytochrome c at acidic and alkaline region as well as its derivatives such as cyanide complex and carboxymethylated ferric cytochrome c. The hyperfine shifted resonances of the heme peripheral groups were monitored here as a sensitive probe of the chemical and physical environments of the heine prosthetic group of ferric cytochrome c. MATERIALS AND METHODS Horse heart ferric cytochrome c, type VI, purchased from Sigma was dissolved in 0.1 M potassium phosphate buffer, pH 6.0, and then it was quantitatively oxidized
289 by adding potassium ferricyanide to the protein solution. After dialysing the protein solution against the buffer, cytochrome c was purified by passing through the CM-52 cellulose column equilibrated with the same phosphate buffer. The elution of the protein from the column was done with 0.5 M potassium phosphate buffer, pH 7.0. The eluate was dialysed against water to obtain the salt-free sample and then lyophilized. The lyophilized sample was dissolved in ZH20 and was used for the NMR measurement after removing the insoluble portion of the sample by centrifugation. The protein concentration was about 3 mM. The complex of ferric cytochrome c with cyanide was prepared by adding the ligand to the 2H20 solution of the protein until the NMR spectral change of it was saturated. Carboxymethylation of the methionine residues at positions 65 and 80 was performed as the method previously described [20]. The pert value in alkaline and acidic regions were adjusted by successive additions of dilute NaO2H or dilute zHC1 to the protein solution in NMR sample tube, respectively. The p2H values are direct readings of the pH meter (Radiometer) using a micro-combination glass electrode (Ingold) inserted into the protein solution in the NMR sample tube. No correction was made for the deuterium isotope effect on the direct reading of the p2H values. There were no visible signs of protein precipitation in any pH region examined throughout the entire course of the titration. Proton NMR spectra were recorded in a pulsed Fourier transform mode (PFT) with a Varian Associates HR-220 spectrometer operating at 220 MHz equipped with a Varian variable temperature control unit and Nicolet TT-100 PFT operating instrument. The quadrature phase detection (QPD) method was used with pulse width 37.5 ps (90 degree pulse). The spectra were obtained by 4 K points transform of 40 kHz spectral width after 1024-8192 pulses with a repetition time of 0.05 s. The temperature of the ambient probe was kept at 22 °C unless otherwise noted. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internal standard and chemical shifts to low field at constant radio frequency were assigned as negative values. RESULTS The high resolution NMR spectra of ferric cytochrome c in the downfield hyperfine shifted region showed markedly the three step transitions as the pH of the protein solution is increased stepwise from neutral pH (Fig. 1). The peaks at --35, --32, and +25 ppm of the two heme peripheral methyl and methionine (Met 80) methyl resonances at pH 7.5 lose their intensity and new peaks, presumably, the heme ring methyl signals appeared at --21.5 and --24 ppm (al, a2) above pH 9. The spectral changes at pH ~ 11 and pH ~12 of ferric cytochrome c were also as drastic as those at pH ~ 9. The signals al and az expend progressively their intensity and concurrently the new peaks of the heme methyl resonances appear at --22 and --22.5 ppm (b I and b2) above pH 10.5. By further raising the pH above 12 these peaks again disappeared from their original position and finally ferric cytochrome c gave broadened resonances at -- 11.5, -- 15.1, --20.2, and --25.2 ppm (signals 1-4). It is also possible to obtain information about the alkaline induced structural transition of ferric cytochrome c by studying the proton NMR spectra of cyano- and carboxymethylated cytochrome c in the alkaline pH region since the 6th coordination site is blocked by the external ligand in the cyanide complex and the methionine residue could no more be responsible for the coordination to the heme iron in the
290 Cyt ¢ (Fe 3")
4
3
2
p21.e
1
I
-3S
-30
-25
-20
"15
PPM
"40
S
I0
15
2(]
25
30
Fig. 1. pH dependence of the proton NMR spectra of ferric cytochrome c at 22 °C in alkaline pH region. High and lowfield parts of the hyperfine shifted spectrum are shown. Spectral changes are noted at pH 9, 11, and at higher pH region above 12. carboxymethylated protein. In Fig. 2 is shown the pH dependence of the N M R spectra in the downfield region of the carboxymethylated cytochrome c. This modified protein gives their heme peripheral methyl peaks at --13, --21.5, and --24 ppm at neutral pH and shows no such spectral change below pH 11 as seen in the native protein. Above pH 11, however, the hyperfine shifted resonances of this protein decrease their intensity with a slight shift to the lower field side, and concomitantly broad peaks appear at --11.5, --15.1, --20.2, and --25.5 ppm. Fig. 3 shows the proton N M R spectra in the hyperfine shifted region of the amine complex, carboxymethylated, and alkaline (at pH 9.9) cytochromes c, and these are compared with that of the native protein. In the spectra of the cytochrome c at pH 9.9, and of the amine complex at pH 7.5 the signals coming from the residual native form remain at their original position. It should be noted here that the spectra of the carboxymethylated and the alkaline cytochrome c are almost similar, indicating that the structure of the heme environment, especially the iron axial ligand in these cytochromes c are identical. Moreover amine derivatives of cytochrome c such as methylamine and ethylamine complexes afford their heme methyl peaks at the spectral region between --20 and --27 ppm [30]. As depicted for the case of amine complex, two heme peripheral methyl pe~ks are seen at --23 and --24.5 ppm, suggesting that the iron axial ligand of ferric cytochrome c at pH 9.9 and of the carboxymethylated protein is primary amine, probably supplied by the lysyl residue at position 79. Ferric cytochrome c shows pH-dependent spectra in the alkaline p H region
291 CM Cyt ~(Fe 3°)
p2H 12.5
i
-30
i
-~
-20
-15
~m
i
i
-10
10
Fig. 2. pH dependence of the proton N M R spectra of carboxymethylated ferric cytochrome c at 22 °C. The hyperfine shifted region of the spectra is shown. The spectral change reversible with raising or lowering of pH is noted at pH 12.
more explicitly than its cyanide complex, suggesting that the spectral changes of the native protein at pH 9 and 11 are attributable to the conformational transition of the sixth coordination side of the heme (Fig. 4). The cyanide complex of the protein gives their heme methyl proton N M R peaks at --11.5, --15.1, --20.2, and --25.2 ppm at the pH region between pH 7 and 10.5, and shows no spectral change in thi6 pH region. Above pH 11, however, these peaks decrease their intensity with small progressive shift to the upper or lower field side and finally new signals appears concomitantly at --11.5, --15.3, --20.2, and --25.3 ppm above pH 11.5. The small spectral shifts of the cyanide complex may result from the heme environmental perturbation caused by pH variation. It is quite interesting to note that the spectra of the native protein, carboxymethylated protein and the cyano-proteins quite resemble each other above pH 12. It is likely that the internal and external ligands in these cytochromes c are replaced by the hydroxide anion and hence they gave the similar spectra at higher pH region above pH 12. The shift of this transition to the lower pH region in the cyanide complex and the slight difference in these spectra, particularly the broader peaks for the native and carboxymethylated cytochromes c than the cyanide complex, may be indicative of the slight difference in the mode of unfolding of the protein moiety caused by the pH variation.
-25
i
-20
3*)
-15 PPM
I
-10
t
5
I
10
15
i
20
25
I
30
Fig. 3. Comparison of the proton N M R spectra of the hyperfine shifted signals of amine complex of cytochrome c at pH 7.0, carboxymethylated cytochrome c at pH 7.0, cytochrome c at pH 9.9, and native cytochrome c at pH 7.0. The spectrum of the native protein at pH 9.9 is almost similar to that of the carboxymethylated one at pH 7.0.
i
-30
-35
I
-40
Cyt-c(Fe
293 Cyt¢ (Fe3*)'CN" pin
3
-10
-IS
2
-~
I
-IS mM
Fig. 4. Proton NMR spectra of the cyanide complex of ferric cytochrome c at several pHs. Lowfield region of the hyperfine shifted signals is shown. The pH-dependent spectral change is seen at pH 11.5. Fig. 5 illustrates the pH dependence of the proton N M R spectra of ferric cytochrome c in acidic pH region, indicating the presence o f three different forms. The low field peaks at --35, and --34 ppm and the broad high field absorption at + 2 4 ppm have been previously assigned to the resonances of two of the heine ring methyl groups and of axial methionine methyl, respectively [16, 19]. These signals lose their intensity with progressive line broadening below pH 4, and concomitantly a new peak comes out at + 2 0 ppm. This new peak grew gradually with decreasing pH at the expense of the methionine methyl signal at + 2 4 ppm. The peak at + 2 4 ppm was finally replaced by this new signal at about pH 2.6. The sum of the intensity of two resonances at + 2 0 and + 2 4 ppm was half of the total intensity o f the two heme peripheral methyl signals at --34 and --35 ppm, indicating that the new peak at + 2 0 ppm is attributable to the methyl signal o f the methionine ligand dislocated slightly from the original coordination position. The two heme side methyl signals which correspond to the methionine resonance at + 2 0 ppm appear to be located at --32 to --37 ppm with substantial line broadenings. To further clarify the acid induced conformational change of ferric cytochrome c, the binding of the external fluoride to the heme iron was monitored by the proton NMR. It was then noted that the fluoride ligand binds to the heme iron to produce a ferric high spin complex in a narrow pH region in which the new peak at + 2 0 ppm was observable in the upfield spectrum region for the native protein (see Fig. 5). This was confirmed by the observation of broad signals
294 I--'
,
I
I
,
I
p=H
Cyt £ (Fe ~'+)
2.6
Jk_j,/
I
-40
i
-30
I
-20
3.1
I
-lO
I
*
i
t
0
10
20
30
ppm
Fig. 5. pH dependence of the proton NMR spectra of ferric cytochrome c at acidic pH region. The hyperfine shifted signals are illustrated. Three spectra are distinguished depending on pH of the protein solution, between pH 7 and 4, between pH 4 and 2.5, and below pH 2.5. Below pH 2.5 all the hyperfine resonances are broadened out beyond detection.
of the heme side methyl groups at the spectral region between --40 and --70 ppm, which was similar to the spectrum of metmyoglobin fluoride. In this p H re~ion between 4 and 2.5, the addition of fluoride afforded the spectrum of ferric high spin cytochrome c fluoride contaminated with that of the native form. It appears safe to say that in this p H region there exist two forms of ferric cytochrome c, one of which can replace the methionine ligand by the external fluoride. This may allow us to expect that the coordination bond between the iron-methionine is weak enough to be replaced by the weak external ligand. With further lowering the p H of the protein solution below p H 2.5, all the hyperfine shifted signals disappeared and no peaks were noted down to --100 p p m from DSS, indicating that the resonances of the heme peripheral groups were broadened out beyond detection. These results are obviously associated with the well known conformational transition of ferric cytochrome c from the low spin form to the high spin form.
295 DISCUSSION The hyperfine shifted proton NMR spectra of ferric cytochrome c at pH 9.9 as well as the carboxymethylated one at pH 7 showed that the environment of the sixth coordination site in these cytochromes c are closely similar. In addition, the spectra of these cytochromes c are also similar to those of the complexes of ferric cytochrome c with primary amines. These findings suggest that the new ligand to the heme iron of cytochrome c above pH 9 is the lysine residue at position 79. It has previously been suggested that the conformational transition of ferric cytochrome c at pH 9 monitored by the disappearance of the absorption band at 695 nm is attributable either to the displacement of the methionine ligand by the hydroxide anion or to the ionization of the imino group of the fifth histidine ligand [12, 13]. Neither the former nor the latter hypothesis can be affirmed by our present NMR study since the cyanide complex and the carboxymethylated cytochrome c show no such a change at corresponding pH region. Moreover, the spectra of carboxymethylated one at pH 7 is almost identical with that of the native protein at pH 9.9, indicating clearly that the change of ferric cytochrome c at pH 9 is not attributable to the concentration of hydroxide anion required to displace the Met 80 ligand. It has been pointed out from the examination of the Kendrew skeletal model of cytochrome c that Met 80 can be swung away from the original position by rotating the polypeptide chain and then the Lys 79 residue can be swung up until its amino nitrogen is exactly at the sixth coordination position [21]. Beside this suggestion, a variety of spectral evidences support the lysine hypothesis including ESR and NMR evidences by Peisach et al. and Gupta and Koenig [9, 19, 22]. The present study confirmed that the isomerization of ferric cytochrome c at pH 9 is resulted from the coordination of the lysyl residue to the heme iron by displacing the methionine residue. The proton NMR spectra in the hyperfine shifted region at pH above 12 suggest that the heme axial group of ferric cytochrome c, carboxymethylated cytochrome c, and the cyanide complex are the same in these pH region. It is tempting for us to exclude the possibility of the ionization of the His 18 imino group in the interpretation of the spectral change occurring above pH 12. If the change of ferric cytochrome c at pH 12 is attributed to the ionization of the His 18, a spectrum different from that of the native protein should be obtained for the cyanide complex in the corresponding pH region. The above results and discussion suggest that the new sixth iron ligand of ferric cytochrome c above pH 12 is the external hydroxide anion. Although the appearance of the hydroxide form of ferric cytochrome c has been suggested by ESR and spectrophotometric studies [2, 22], no conclusive evidence has been presented presumably because of the difficulty in the interpretation of the spectra due to the unfoldings of the protein molecule. The broadend proton NMR resonances in the hyperfine shifted region at high pH indicates that the compact globular structure of ferric cytochrome c is partly broken above pH 12. The hydroxide form of ferric hemoproteins has been known to appear by the alkaline ionization of the iron bound water molecule as evidenced for metmyoglobin and methemoglobin. In these hemoproteins the alkaline derivatives are thermal spin mixture between high spin and low spin states, and the heme methyl proton NMR signals of these were observed in the spectral region between --30 and --50 ppm [23, 24]. In ferric cytochrome c, the hyperfine shifted spectrum of the hydroxide form is
296 characteristic of low spin hemoprotein since the heme methyl peaks are in the spectral region between --10 and --25 ppm. This difference between the hydroxide forms of these hemoproteins is probably due to the covalent bond of the heme to the apoprotein of ferric cytochrome c. According to Otsuka. thermal mixing between the low and high spin states of hemoprotein is deeply related to the rigid fixation of the heme group to the apoprotein [29]. The absence of thermal mixing of spin state in ferric cytochrome c complex has been briefly discussed for its azide and imidazole complexes on the bases of the van der Waals contact interaction between the heme group and the apoprotein by Saito and Iizuka [25]. Having made the assignment of the spectral change of cytochrome c occurring at pH 9 and 12, let us now consider why the change of the spectra was noted at pH 11 only for the native cytochrome c. The pH jump and potentiometric studies of cytochrome c indicated that the ionization of the tyrosine residue at position 67 occures at about pH 11 [19, 26, 27]. The hydrogen bond between this tyrosine residue and the threonine residue at position 78 has been visualized by X-ray crystallographic studies [1]. These results allow us to suggest that the change at pH 11 is associated with the change of the coordination mode of the lysyl residue at position 79 to the heme iron due to the breaking of the hydrogen bond between Thr 78 and Tyr 67. Accordingly, we designate this cytochrome c formed at pH 11 as "strained lysine form". This assignment is compatible with the observation that the cyanide complex shows no substantial change in the spectra at corresponding pH. However, the effect of the breaking of the hydrogen bond between Thr 78 and Tyr 67 is hardly manifested in the NMR spectrum of the carboxymethylated cytochrome probably due to the effect of the chemical modification since this modified cytochrome c did not exhibit a substantial spectral change in these pH region. A variety of the spectral evidence suggested that acidification of ferric cytochrome c down to pH 2 results in the displacement of the methionine ligand by a weak field ligand supplied by a solvent molecule [2-4, 8-10, 14]. For example, Peisach et al. have indicated that at below pH 4 the protein is converted from low spin form to high spin form, evidenced by low field splitting (g = 6.3, 5.8) in the ESR signal and by an optical spectrum similar to that of metmyoglobin [22]. Although this drastic acid induced transition of ferric cytochrome c has been studied by many investigators, confirmative evidence of the displacement of the Met 80 ligand from its coordination position is quite limited. Gupta and Koenig showed that the methionyl methyl proton NMR signal of native cytochrome c at +24 ppm disappears at low pH, indicating a displacement of the methionine ligand [19]. We pay much attention here to the peak at ~-20 ppm which is presumably assigned to the methionyl methyl signal dislocated from the native one by acid-induced perturbation of the heme environment. Our observation that this methionine is replaced by a weak high spin ligand F- and that its signal at +20 ppm disappeared below pH 2.5 may lead to conclude that the methionine-iron bond is weaken with the lowering of the pH of the protein solution to produce an acidic low spin form, so that it is replaced by external fluoride. This acidic form of ferric cytochrome c in a low spin state has not been identified in other studies. To confirm further this methionine replacement, cross-saturation experiments on the two forms below pH 2.5 appear to be desirable and are now being planned in our laboratory. It is likely that the protonation of some amino acid residue except Met 80 is the first step to cause the acid conformational
297 transition of cytochrome c because the methionine residue has no ionizable group in aqueous solution. Although several assumption were presented for this group such as the His 26 and the heme propionate [10, 17], we have no evidence to assign this group. It is also probable that further acidification of cytochrome c below pH 2.5 results in the complete disruption of the protein conformation and thus no hyperfine shifted peaks were observed due to line broadening. In summary, the present N M R study of the hyperfine shift distinguished six different forms of ferric cytochrome c depending on the pH of the protein solution. They are acid high spin form (below pH 2.5), acidic low spin form (between p H 2.5 and 4), native form (between pH 4 and 9), lysine form (between pH 9 and 11), the so-termed "strained lysine form" (between pH 11 and 12.5) and hydroxide form (above pH 12.5). These results also suggest that the conformation of ferric cytochrome c is maintained in a compact and functional form by specific interaction of the ionizable residue of the polypeptide chain. The breaking of these interaction effected by pH and chemical modification results in a conformational change in the heme vicinity of the protein. ACKNOWLEDGEMENTS The authors are grateful to Dr. M. Ikeda-Saito for preparation of carboxymethylated cytochrome c. They also wish to thank Dr. T. Hosoya and Dr. T. Inubushi for their encouragement and discussion. This work is supported by Toray Research Grant for Science and Technology and by research grant in aid from Ministry of Education, Japan.
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298 21 Dickerson, R. E., Takano, T., Kallai, O. B. and Samson, L. (1970) "Structure and Function of Oxidation Reduction Enzymes" (Akesson, A. and Ehrenberg, A., eds.), pp. 69-83, Pergamon Press, Oxford 22 Peisach, J., Blumberg, W. E., Ogawa, S., Rachmilewitz, F. A. and Oltzik, R. (1971) J. Biol. Chem. 246, 3342-3355 23 Morishima, I. and Iizuka, T. (1974) J. Am. Chem. Soc. 96, 5279-5283 24 Iizuka, T. and Morishima, I. (1975) Biochim. Biophys. Acta 400, 143-153 25 Saito, M. and lizuka, T. (1975) Biochim. Biophys. Acta 393, 335-342 26 Rupley, J. A. (1964) Biochemistry 3, 1648-1650 27 Czerlinski, G. H. and Dar, K. (1971) Biochim. Biophys. Acta 234, 57-61 28 Wada, K. and Okunuki, K. (1969) J. Biochem. 66, 263-272 29 Otsuka, T. (1970) Biochim. Biophys. Acta 214, 233-235 30 Morishima, I., Ogawa, S., Yonezawa, T. and Iizuka, T. (1977) Biochim. Biophys. Acta, in the press
