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Nuclear magnetic resonance studies of metal substituted horse cytochrome c
Nuclear Magnetic Resonance Studies of Metal Substituted Horse Cytochrome c Geoffrey R. Moore and Robert J. P. Williams Inorganic
Chemisrq
Laborarov.
Univesiry
of O_@ord
James C. W. Chien and L- Charles Dickson Department
of Chemisny.
University
of Massachusetts
ABSTRACT* The proton nuclear magnetic resonance spectra of various metal substituted derivatives of horse cytochrome c have been studied and compared to the spectra of native cyto-
chrome c. The proteins studied were the cobalt(W), copper(H), iron(II), iron(III), manganese(lII), nickel(R), and zinc(U) derivatives. Spectxa of the diamagnetic cobalt(W), iron( and zinc(II) proteins were we&resolved and specific resonance assignments were made. All three proteins possessed a methionine ligand to the metal. The spectrum of cobalt(IIl) cytochrome c was Investigated in some detail as this protein was used as a diamagnetic control for iron(II1) cytochrome c. Comparison of the spectra of cobalt(II1) and iron cytochromes c revealed that their conformations were very similar but the foIlowing conclusion couid be made; the oxidation of cytochrome c is accompanied by a smah conformation change.
INTRODWXION Utilization
of the different
probe properties
of metal ions by replacement
of the native
metal ion of a metaUoprotein with another metaf ion is widely exploited in physical studies of proteins [l] _ Until recently application of this principle to haemoproteins
had been restricted to haemoglobin [2] and myoglobin [3], proteins in which the haem group is not covalently bound to the protein. With the development of suitable preparative techniques for cytochrome c, in which the haem group is covalently bound to the protein, a range of metal-substituted cytochromes c have recently been prepared [4-S] . Many of the metal-substituted cytochrome c derivatives have been used in biological studies whose aims are directed towards further understanding of the function of native cytochrome c. For example, the oxidase activity of cobalt cytochrome c [4] and Address reprint requests to: R. J. P. Williams, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K.
* The abbreviations used in this paper, Co(III)c, respectively, the cobaIt(III), cobaIt(II), copper( horse cytochrome c.
Joumat of Inorganic Biochemisny
12,1-15
@ EIsevier North Hohand, Inc., 1980
Co(II)c, Cu(II)c, Mn(III)<:, Ni(II)-c, and Zn(II)c are, manganese(III), nickel(II), and zinc(I1) derivatives of
1
(1980) 0162-0134/80/010001-15$02.25
2
G. FL Moore et aL
the interactions of Sn(IV) and Zn(Il) cytochromes c with cytochrome c oxidase and rnitochondria [9] have been investigated. The relevance of such studies to native cytochrome c cannot yet be judged. One criterion which has to be established is the nature of any structural modifications accompanying the metal replacement. With this in mind we have studied the nmr spectra of the Co(III), Zn(II), Cu(II), Ni(II), and Mn(II1) derivatives of horse cytochrome c. -4dditional impetus for these investigations has come from the success of nmr in probing the solution structures of a wide range of eukaryotic and prokaryotic cytochromes c [lo-151 . In these previous investigations a major problem has been the comparison of nrnr data for the diamagnetic ferrocytochrome c and the paramagnetic ferricytochrome c. Differences in chemical shifts of corresponding resonances of the two- forms could arise from changes in conformation accompanying oxidation state change and from additional shifts caused by the pararnagnetism of ferricytochrome c. As cobalticytochrome c is six coordinate with Met 80 and His 18 providing the axial ligands [16] , and as this protein is active in an electron transfer capacity with cytochrome c oxidase [4] , it was hoped that cobalticytochrome c would serve as a diarnagnetic control for ferricytochrome c. In this paper we report a detailed analysis of the nrnr spectra of the Co(II1) and Zn(I1) derivatives of horse cytochrome c and a more limited analysis of the spectra of the Cu(II), Ni(II), and Mn(II1) derivatives.
MATERIALS
AND METHODS
The metal-substituted proteins were prepared as previously described [4-s] . Native horse cytochrome c was obtained from Sigma Chemical Co.;Type VI grade was used. Samples for nmr were prepared by dissolution of the metal substituted proteins in 0.05 M deuterated phosphate buffer at pH 7.0 Samples of native cytochrome c were dissolved in DaO. Protein concentrations were usually 3 X 10-a M. Ferrocytochrome c was obtained by reduction of ferricytochrome c with ascorbic acid under argon. The operating temperature was varied depending on the stability of the protein and on the nature of the nmr experiment. Temperatures are recorded in the legends to the figures and tables. Spectra were obtained using a Bruker 270 MHz spectrometer operating in the Fourier Transform mode. Convolution difference [17] , cross-saturation [ IS], spindecoupling [19] , andspinecho double resonance [20] were carried out as previously described. Acetone and dioxan were used as internal standards but all chemical shifts are quoted in parts per million @pm) downfield from 2,2&xnethyl-2silapentane-Ssulphonate-
RESULTS
Co(m) Cytochrome
c
The aromatic and aliphatic regions of the convolution difference spectra of Co(HI)< and ferrocytochrome c are shown in figures 1 and 2. To a cursory inspection, the spectra of ferrocytochrome c and Co(III)+z have many areas of close sim&rity, for example, the resonances between 0 and -S ppm, many of which arise from the axial
Studies of Horse Cytochrome
c
3
a.
b.
10 8 6 ppm FIGURE I_ The aromatic regions of the convolution difference spectra of (a) 3-mM cobalticytochrome-c in 0.05-Mdeuterated phosphate buffer at pH 7.0 and 57°C; and (b) S-mM horse ferrocytochromec in D20 at pH 5.0 and 57°C.
ligand Met 80. Where there are overlapping resonances the similarity is less striking as the general appearance of the spectra are greatly altered by small (ca. 0.02 to 0.03 ppm) differences in chemical shifts. However, detailed assignment allows exact comparison of corresponding resonances. Resonances of native cytochrome c have been assigned [14, 15]_ Resonances of Co(I.II)c were assigned first to amino acid type, based on identification of coupling patterns with spin-decoupling and spin-echo double-resonance, and then to a specific amino acid in the protein sequence by comparison with the corresponding, previously assigned resonances of ferrocytochrome c [14, 15]_ This procedure was only possible because of the close similarity of the resonance chemical shifts between Co(III)c and ferrocytochrome c. Assignments are summarized in Table 1 and 2. The dependence of the spectrum of Co(II& upon temperature, below 65OC, and pH is also similar to that of ferrocytochrome c. The C-2 and C4 resonances of His 33 shift with variation in pH with a pK, of 6.4 f 0 .l at 27OC. The methyl resonance at -0.24 ppm (57°C) from Ile 57 is also temperaturedependent shifting downfield with increasing temperature; between 27OC and 57°C it shifts by -0.24 ppm. Again, this parallels the behavior of the corresponding resonance of ferrocytochrome c [21] _ Additionally, this methyl resonance is pH dependent_ Between pH 4.2 and pH 9.6 it is pH independent, but outside this range the resonance shifts downfield. This shift is not a result of denaturation since the remainder of the spectrum is only little affected. Between pH 9.6 and pH 109 the methyl resonance shifts by -0.07 ppm and
G. EL Moore et al.
4
a.
1
2
I
I
0
-2
I
-r;
ppm
FIGURE 2. The aliphatic regionsof the convolutiondifferencespectraof (a) 3-mM cobalticytochrome-c in 0.05-M deuterated phosphatebuffer at pH 7.0 and STC!; and (b) 5-mM horse ferrocytochromw in D@ at pH 5.0 and S?C_
between pH 42 and pH 3.4 it shifts by > -0.2 ppm. A final illustration of the temperature dependent resonances is afforded by resonances of Tyr 48_ At temperatures <35OC, this residue is in slow exchange between equivalent orientations for ferrocytochrome c 1221. With increasing temperature its resonances broaden, finalIy disappearing from ‘;he spectrum at 3YC. In the spectrum of Co(III)i: at 37OC there are anaIogus resonances, related by a cross-saturation effect, at 5.47 and 6.72 ppm. These exhibit a similar temperature dependence to the corresponding resonances of ferrocytochrome c, and they broaden and disappear above 42OC There are differences in stability between the two proteins; ferrocytochrome c is stable up to Q?C with its spectrum little changed from the low-temperature form 121, 231, whereas Co(1IQ-c begins to denature at ca. 65°C. This is analogous to the behavior of ferricytochrome c. &r(n) Cytochrome c In order to study the protein by nmr, soiutions of Zn(II) cytochrome c were made immediateIy prior to use from mate&d prepared and stored in the dark, and the sohrtions
Studies of Horse Cytochrome
TABLE
c
1. Metal Ligand Resonances for Diamagnetic Cytochromes c
Assignment Haem meso Haem me50 Haem meso Haem meso Thioether CH Thioether CH3 His 18 Met 80 CH3 Met 80 7 CH Met 80 p CH Met 80 7 CH Met 80 @ CH
Fe(II)-c 9.62 9.59
9.32 9.04
6.36 2.57 0.13 -3.28 -3.73 -2.58 -1.87 -0.19
Co(iII)-c
Zn(ii)-c
10.71 10.43 10.26 9.77 6.53 2.76 -0.07 -3.77 -4.93 -2.98 -2.21 -0.40
10.11 9.94 9.49 nd nd nd 30.05 -3.07 -3.60 -2.36 -1.62 nd
a AU chemical shifts measured in ppm at pH 7 and, for Fe(II)-c and Co(III)s at 57°C but for ZnQI)-c at 2?C. The resonances of the metal ligands of Fe(II)c and Co(III)-c are virtually temperature independent but for Zn(II)-c they are strongly temperature dependent. b The meso CH assignments for Co(III)-c corrects previous work [ 161 where they had been inconectly assigned.
placed into smoked glass nmr tubes previously flushed with argon. Even so, protein denaturation, evident from spectral quality and gradual precipitation of the sample, began to occur within ca. 2 hr. As with Co(III)c, comparison of the spectrum of Zn(II) cytochrome c with that of ferrocytochrome c reveals many similarities between them (compare Figure 3 with Figures 1 and 2). In particular, the ringcurrent-shifted methyl regions, between 0 and 4 ppm, contain analogous resonances indicating that Met 80 provides an axiaI Iigand to the Z&II) ion. Spectra of Zn(I1) cytochrome c are less well-resolved than spectra of Co(II1) cytochrome c or ferrocytochrome c and resolution was not improved with increasing temperature because of the relative instability of Zn(I1) cytochrome C. Thus, assignment methods based on multiplet structure could not be applied. However, as the spectra of Zn(I1) cytochrome c and ferrocytochrome c at 27O are similar it proved possible to compile a list of tentative assignments based on comparison of resonance intensities and chemical shifts (Tables 1 and 3). It can be seen from figure 3 that the spectrum, and therefore the structure, of Zn(I1) cytochrome c is strongly temperature dependent with many resonances shifting with variation in temperature and some, such as the methyl resonance of Met 80 at -3.07 ppm (27OC) decreasing in intensity with increasing temperature. Indeed at 27°C the Met 80 resonance is only of ca. two proton intensity and at 57°C it has decreased to < one proton intensity compared to the remaining methyl resonances between 0 and -1 .O ppm (57°C). The chemical shift variations between 27°C and 47°C are not completely reversible_ Cu(II), Ni(IK), and Mn@I) Derivativesof Cytochrome E
z
C0(11)-, CU(II)-, Ni(II)-, and Mn(III)-cytochromes c are considered together as they are paramagnetic. Electron paramagnetic resonance measurements show that the electron-
G. R. Moore et al.
spin relaxation times of Co(II)c and Cu(II)-c are long, indicating that their paramagnetic centers are powerful relaxation probes, whereas those for Mn(III)c and Ni(II)-c are short. Thus, spectra of Mn(III)c and Ni(II)c were predicted to exhibit relatively sharp hype&me shifted resonances of the haem group [6, 71. However, in none of the above cases were resonances observed outside the spectral region 12 to -2 ppmSpectra of Fe(III)-c, Cu(II)c, and Ni(I1) c are shown in figure 4 over the region 12 to -2 ppm. The spectrum of Cu(II)-c appears to be considerably broadened (compared, for example with the spectrum of ferricytochrome c), confiig the earlier predictions. The quality of the spectra of the paramagnetic metal-substituted proteins is low and assignments have not been obtained. DISCUSSION
Co(III)-Cytochrome
c
Clearly, from the data of Tables 1 and 2 it can be concluded that the structures of ferrocytochrome c and Co(III)c are essentiaZZy similar with both proteins possessing 6 co-ordinate metal ions, the axial ligands of which are His 18 and Met 80. However, there are some variations in chemical shifts for corresponding resonances. Many df these variations are small (2 0.03 ppm) but some are significantly larger. Chemical shift differences could arise by a number of mechanisms: I_ Changes in metal-ligand bond lengths and/or haem plane-metal-l&and bond angles, giving rise to different haem ring-current shifts for resonances of ligand nuclei_ 2. Variation in electron delocalisation in the porphyrin leading to a variation in o-bond electron density for nuclei of the porphyrin and a variation in the magnitude of the haem ring-current. 3 _ Temperature independent pammagnetism of the metal. 4_ Electric field effects caused by change in charge of the metal ion. 5. A change induced in the protein structure. This could be a metal linked conformation change, an oxidation-state linked conformation change or a structural change brought about by protein modification during metal replacement. We will consider resonances in turn, in the following order: haem resonances, axial ligand resonances, and other amino acid resonances. Many studies of metal substituted porphyrins have established that, for porphyrins with in-plane metal ions the chemical shift of the porphyrin resonances increase as charge on the metal ion increases; for dipositive metal ions a,,,, = 9.75 to 10.08 ppm and for tripositive metal ions 6,,,, = 10.13 to 10.39 [24]_ The results for ferrocytochrome c and Co(III)c are in general accordance with this scheme (Table 1) but note that the chemical shifts for the meso resonances are spread over a wider range than for the ‘model’ compounds. There are two conflicting mechanisms contributing to the different chemical shifts for meso resonances of ferrocytochrome c and Co(III)c: the cr withdrawing power of Co(III) is greater than for Fe(D) while Co(III) is a poorer srdonor than Fe(I1). The former leads to a deshielding of the meso protons, and the latter to a decrease in the haem ringcurrent for Co(III)-c relative to ferrocytochrome c. As the nett shifts of the meso resonances for Co(III)-c are downfield from those of ferrocytochrome, the o-withdrawing property of the metal ion is more im-
Studies
of Horse Cytocbrome c
TABLE
2. Compadson
Assignment Leu 32 Leu 32 lie 57 Met 65 N-acetate
of Chemical
Shifts for Fe(II)-c
Fe(I0-c
Co(IiI)c
-0.76 -0.60 -0.43 2.09 2.06
-0.75 -0.68 -0.24 2.12 2.09
I
His 33 His33 His 26 His 26 Trp 59
7.79 7.29 7.52 7.06 6.99
7.77 7.27 7.51 7.06 7.11
Trp 59 Trp 59 Trp 59 Trp 59 Phe 82 F’he 36 Phe 36 Phe 10 Phe 10 Tyr 97 Tyr 97 Tyr 48 Tyr 48
7.60 6.70 5.74 7.09 6.34 7.40 6.89 6.71 7.40 7.22 6.64 6.78 5.59
7.60 6.62 5.77 7.07 6.19 7.36 6.88 6.73 7.42 7.25 6.62 6.72 5.47
and Co(III)-@
Shift Fe(II)-Co(III)
% Differenceb
-0.01 +0.08 +0.19
+4.8 -14.5
+-O-O8 -0.03 +0.02 +-o-15 +0.04 +0.01 -0.02 -0.02 -0.03 +-o-o2 +0.06 +0.12
16-W -0.7 6.8 14.2
14.3d 7.9
o All chemical shifts measured at 570 and pH 7 e.ucept those of Tyr 48, measured at 27”. b % difference is Fe(II)-Co(III) difference expressed as percentage difference from secondary shift for resonance of fenocytochrome c. C Assuming ass@ment scheiner the resonance at 6.70 ppm arises from the C-6 proton. d Assuming these resonances are meta proton resonances.
portant. systems
The difference between the meso resonance shifts for the ‘model’ porphyrin and the cytochromes may be due to the donor/acceptor properties of the
axial ligands and to secondary structure shifts, such as amino acid ring-current shifts. Whereas the chemical shifts of the Met 80 resonances (Table 4) are different for the two proteins, those of the singlet resonances of His 18 are very similar; the shift from primary position is only ca. 3% greater for Co(III)c. As the His 18 C-2 and C3 protons are closer to the metal than the Met 80 protons a significant contribution to the chemical shift differences from temperature independent paramagnetism can be discounted_ The electronic effects caused by metal substitution would cause the Met 80 resonances of Co(III)c to shift downfield if the distances between the haem plane and the Met 80 protons were unaffected by the metal replacement. Therefore, the large upfield shifts of the Met 80 resonances for Co(III)x can be attributed to a decrease in the haem plane-Met 80 proton distances. This could arise by a simple shortening of the metalsulphur bond length or by a decrease in the metal-sulphur bond length concomitant with tilting of the Co(III)-S bond. The geometry of the Fe(II)-S-Met 80 bond system
G.
I
>-t3
2
I
0
I
R. Moore et al.
I
-4
ppm (a) ;b: JTIGURE 3. The aromaticregion(a) and aliphatic region (b) of the spectrum of 3-1&f zinc-cytochrome-c in 0.05-M deuterated phosphate buffer at pH 7.0 and various temperatures_ The vertid scale in (a) is 4 times that in @). of cytocbromes c has recently been correlated with redox potentials [25]_ In these studies it was shown that as the Fe(lI)-S bond length decreased the chemical shift of the resonances of the methionine ligands CHa and yCH protons decreased whilst that of the r’CH proton increased. A change in Fe(II)-S bond angles was not indicated by these studies. Now consider the differences in chemical shifts for the Met 80 resonances of Co(III)-c compared to ferrocytochrome c (Table 1); all the resonances are shifted further upfield for Co(III)-c and that of ‘tbe $H proton by a considerably greater amount than the others. This is a very different situation than for the studies with different ferrocytochromes c and indicates that the Co(III)-S bond length is not only shorter than the Fe(ll)-S bond length but also that the Co(III)-S bond is nonaxial. From the decreased haem ring-current of Co(IU)-c it would be predicted that the secondary shifts for resonances of groups close to the haem should decrease_However, for a number
Studies of Horse Cytochrome
c
TABLE 3. Comparison
of Chemical Shifts for Fe(II)-c and Zn(II>-c”
Assignment
l.eu 32 Leu 32 lle57 Met 65 N-acetate
Fe(H)*
-0.76 -0.60 -0.60 2.06 2.06
1
-0.90 -0.70 -0.24 2.12 2.05
Trp 59 Trp 59
5.74 6.99
5.65
7.40 6.88
7.01 7.37 6.89
Phe 10 Phe 10 Tyr 97 Tyr 97
6.72 7.40 7.22 6.61
6.59 7.37 7.22 6.59
Phe 36 Phe 36
0 Chemical shifts measured at 27Oand pH 7-k b Assignments for Zn(II)-c have not been contirmed with double-resonancetechniques.
inspection of the data in Table 2 shows that those resonances which are significantly shifted @ 10%) from ferrocytochrome c to Co(III)c experience a larger secondary shift; note the resonances of Trp 59, Phe 82, and Tyr 48. In the case of Try 48, much of the secondary shift experienced by its resonances is derived from Phe 46 while the haem group is responsible for the secondary shifts of resonances of Trp 59 and Phe 82_ The increased shift of the Phe 82 para proton resonance may be associated with the change in metal-S bond length for Co(III)c which, transmitted along the peptide chain, ‘pulls’ Phe 82 closer to the haem. The shift of the Trp 59 resonance at 6.62 ppm (Co(III)c), the only significantly shifted resonance of Trp 59, is not readily explicable_ Any change in Trp 59-haem orientation would affect most of the tryptophan resonances and probably strongly perturb the resonance at 5.77 ppm (Co(III)-c). The shifts of the Tyr 48 resonances are also not readily explicable although a small change in Phe 46-Tyr 48 orientation could account for them. It is relevant to note that both Trp 59 and Tyr 48 are H-bonded to the haem propionate side chains [26] and therefore a change in the haem o-bond electron distribution could affect the strength of the H-bonds. The resonance of lle 57 at -024 ppm (Co(III)c) is an exception to the aromatic resonances discussed above in that it experiences a significantly smaller secondary shift. Ile 57 is far from the haem and the methyl resonance at -0.24 ppm derives its secondary shift mainly from Tyr 74. The corresponding resonance of ferrocytochrome c is temperature and pH sensitive [27] , and this sensitivity has been interpreted in terms of conformational mobility in the region of the protein about Ile 57 [21] _ Similarly this Ile 57 resonance of Co(III)-c is temperature and pH dependent and exhibits analogous behavior to the ferrocytochrome c resonance. Thus, the difference in secondary shift for the methyl resonances of Ile 57 probably reflects a small difference in structure between the two proteins. Movement of the aromatic ring of Tyr 74 away from the WH, of IIe 57 has been suggested as the cause for the pH and temperature
10
G. R. Moore et al.
I 0
I 6
I
I
I
4
2
0
I -2
pm
FIGURE 4. Spectra of horse cytochromesc at pH 7.0 and 37OC. (a)Ferricytochrome-c in D20; @I) copper0-cytochrome-c in 0.05-M deuterated phosphate buffer; (c) manganese(III)-cytochrome-c in 0.05-M deuterated phosphate buffer; (d) nickel(Ii)sytocbrome c in 0.05-M deuterated phosphate btlffer.
dependent shifts of this methyl resonance [21] _It is attractive to speculate that transmission of the decrease in the metal-S bond length for Co(III)-c along the peptide chain may be responsible for the difference in shifts between the corresponding resonances of Ile 57. The Tyr 48 resonances of ferrocytochrome c at 5.59 and 6.78 ppm broaden with increasing temperature as the aromatic ring of Tyr 48 undergoes more rapid motion placing its resonances in intermediate exchange 1221. The corresponding resonances of Co@)) possess a similar temperature dependence and this, along with the temperature dependence of the Ile 57 6CHa resonance, reveals that not only is Co(III)-c similar to ferrocytochrome c in its static structural aspeck but also in its dynamic aspects. It proved impossible to obtain the ortho and meta resonance of Tyr 48, Phe 46, and
Studies of Horse Cytochrome
4. Resonances
TABLE
Secondary Shift for Assignment Met 80 SCH3 E
11
c
WIGc +5.40 +6.43 i-4.85 +4.57 +2.46
of Met 80 Fe(II)-Co(III) difference % H-49 +1.20 +0.40 +0.34 +0.21
9.1 18.7 8.2 7.4 8.5
Fe(H)-Zn(II) difference -0.21 -0.13 -0.22 -0.25 nd
% -3.9 -2.0 -4.5 -5.5 nd
Primary Position for Met Resonvlces -SCH3
2.12 ppm 2.70ppm 2.27 ppm
Phe 82 in fast exchange, as has been done for ferrocytochrome c at 97O [13,14,22], because of denaturation of Co(III)-c above 65°C. In respect of its thermal stability Co(III)c resembles ferricytochrome c rather than ferrocytochrome c and thus the overall charge is the determining factor. However, in respect of its pH stability Co(I11)1: is like ferrocytochrome c rather than ferricytochrome c. The methionine ligand of ferricytochrome c is replaced at ca pH 9.2 by a nitrogen ligand but the methionine ligands of Co(III)c and ferrocytochrome c remain intact even at pH 11 (27°C). Thus in this case the charge is not the determining factor. The previous discussion has centred about chemical shift differences arising from small structural rearrangements, for the data tell us there are no large-scale rearrangements_ However, the additional charge on the metal ion for Co(III)c may induce small electric-field effects in resonances of groups close to the haem which would lead to upfield chemical shifts. This would be predominantly an isotropic effect and thus, resonances of Leu 32 should experience larger shifts due to this mechanism than the para proton resonance of Phe 82. Similarly on this model the Trp 59 resonance at 5.77 ppm (Co(III)c) and the His 18 singlet resonance should experience large shifts compared to their corresponding resonances of ferrocytochrome c. This is not the case and we conclude that electric-field effects are not important in this conqarison. Additionally, the expected decrease in haem ring-current was not observed in the chemical shifts of assigned resonances. Resonance shifts due to modification of the protein other than those associated with the metal replacement have not been observed_ This is not surprising as the ion-exchange and electrophoretic mob&ties of Co(III)c and fenicytochrome c are the same [4] _ Zn(II)-cytochrome
c
The problems concerning the assignrnent of resonances of Zn(II)c have been discussed in the results section. There a list of tentative assignments was constructed (Table 3) based on comparison of the spectrum of Zn(II)c with that of ferrocytochrome c_ Differences in structure are apparent between Zn(II)< and ferrocytochrome c, however; note, for example, the additional two two-proton intensity peaks at 6.23 and
G. R Moore et al.
12
5.48 ppm in the spectrum of Zn(II)-c and the different secondary shifts for the upfield ringcurrent-shifted methyl resonances. These uncertainties prohibit detailed discussion of the structure of Zn(II)-c in terms of the structure of ferrocytochrome c other than to say that immediately on dissolution of Zn(II)-c, the protein conformation closely resembles that of ferrocytochrome c. The spectra of Zn(II)-c given in figure 3 show that, in contrast to ferrocytochrome c and Co(III)-c, there are many, large temperature-dependent effects not all of which se reversible_ In the absence of firm assignments we will confme ourselves to a discussion of the Met 80 resonances-but note that the resonances, in figure 3, disappearing with increasing temperature from the region 8-10 ppm, probably arise from exchangeable NH protons. Shifts of the resonances of Met 80 with temperature are represented in figure 5. These shifts are in the direction of decreasing secondary shift with increasing temperature and they extrapolate at T = m to: for the CH3 and @CH resonances 2.0 f 0.2 ppm and for the flH resonances 2.8 + 02 ppm. Additionally, the intensity of the methyl resonance decreases with increasing temperature. The intensity variation for this resonance implies that ca. 66% of the Zn(II)c molecules possess Met 80 ligation at 27” while ca. 34% do not. At 57O more than ca. 34% of the Zn(II)-c molecules possess Met 80 ligation_ These data are consistent with schemes such as
where bond length b >a_ Kis required to be>103 set-l in order that the resonance condition for fast exchange is met. The extrapolated values for the Met 80 chemical shifts at T = m accord well witk the primary positions for methionine resonances; 2 -12 ppm for the CH, and PCH resonances, and 2.64 ppm for the 7CH resonances_ In fact the spectrum of Zn(II)-c after exposure of a solution to light at 27°C for ca. 3 hr is that of a random coil protein. Crystal structure determinations for Zn(Ii)-porphyrin complexes have shown that the Zn(I1) ions are displaced from the center of the porphyrin plane; for (HaO)Zn(tetraphenylporphyrin) this displacement is 0.35 A [28] _ A similar disphcement for Zn(II)-c, presumably on the side of the His 18 ligand, would lead to a wrtakening of the metal-S bond. This is probably one cause of the relative instability of Zn(II)-c, but note also that Zn(II)-porphyrins are light sensitive [29] _
Co@),
Cu(II), Ni(II), and Mn(II1) Derivatives of Cytochrome
c
We can add nothing new to our previous knowledge concerning the structures of Co(II)c, Cu(II)-c, Mn(III)-c, and Ni(1Q-c as the quality of the spectra was insufficient to obtain assignments. The lack of contact shifted resonances for Mn(III)c and Ni(II)-c presents a problem as for paramagnetic ions with short electron spin-lattice relaxation tirres the resonances of the ligand nuclei should be observable [30]. Thus, nmr studies [3 l] of Mn(III> and NiII) acetylacetonates revealed sharp, strongly shifted resonances of the acetylacetonate ligands. It is feasible that exchange processes such as that occurring for Zn(II)c may operate in these proteins; with K = lo3 to lo4 set-l and a con-
13
Studies of Horse Cytochrome c
6 ppm
-3-5
\
“
-3-O
-2-5
-1-S
’
;7
37
-‘I
47
57
Temperature
“C
FIGURE 5. Temperaturedependenceof the resonancesof Met80 of zinc-cytochromec.
siderably greater chemical shift difference the resonances of the &and nuclei would then be in intermediate exchange and therefore not observable_
General Diiussion Our studies have shown that the structures of Co(III)c and Zn(II)c are similar to that of ferrocytochrome c, although in the case of Zn(1Q-c there are some structural modifications in addition to the exchange processes at the coordination site. Co(II)-c has been shown to be 6 coordinate with Met 80 and His 18 providing the axial ligands [16] and this protein possesses similar pH transitions to ferrocytochrome c; pKa’s at ca. 3 and ca. 12 [32] _ In view of the data presented in this paper it must be the case that only a small conformation change accompanies oxidation-state change for cobalt
G. R. Moore et al.
14
.
cytochrome c. Therefore, these proteins may serve as viable probes for the biological action of native cytochrome c. As discussed in an earlier report [4], there is also a thermodynamic element involved where cobait-cytochrome c undergoes oxidation state change as its Em = -140 mV whereas that of native cytochrome c is + 260 mV. Nevertheless, the lack of activity for cobalt cytochrome c with cytochrome reductase preparations (it is active with cytochrome oxidase) has been deduced as arising from structural modification involving the surface of the protein [4] . This is ofgreat interest as the chemical properties of native cytochrome c also suggest that there is a difference between the structure of the protein surface in its two oxidation states [33] _ An important conclusion from the comparison of Co(III)< and ferrocytochrome c is that, as the additional charge for Co(IIIb has no major effect upon the protein conformation the same should be true for ferricytochrome c, i.e., the oxidation of native cytochrome c is not accompanied by a Zarge conformation change. It has been necessary to establish this by a technique independent of x-ray crystallography because although the most recent x-ray structure determinations have shown that there is no oxidation state linked conformation change, [26,34,35], previous x-ray studies [36, 371 have been interpreted in the light of massive conformation changes. However, the present results show that there are small differences in conformation between the Co(II1) and Fe(I1) proteins. There must be a small change in metal-S bond length on oxidation state change of iron analogous to the difference between ferrocytochrome c and Co(IIl)-c, but as the bond length for Fe(III)-S is likely to be intermediate between that of Fe(II)-S and Co(III)-S the repercussions of this change will not be any greater than those observed for Co(III)c_ Even so we know that this change affects the surface of the protein around IIe 57. We can now proceed in our investigation 1381 of the solution structure of cytochrome c confident that the difference in chemical shifts for corresponding resonances of ferricytochrome c and ferrocytochrome c arise predominantly from contact and pseudocontact shifts, except in one or two regions of the protein. The central role of the metal ion in dete r-mining the stability of the protein is demonstrated by these studies_ It has been known for a long time that ferrocytochrome c is more stable to extremes of temperature than ferricytochrome c [23,33]. This is mirrored by the behaviour of cobalt cytochrome c where Co(III)-c is less stable than ferrocytochrome c. Yet the protein conformations of Co(III)-c and ferrocytochrome c are very similar. Clearly the additional charge for the +3 oxidation state leads to a destabillsation of the protein. We thank the Science Research Council. rhe Medical Research Council and the Royal Society for jkancti support_ R.PW k a member of the Oxford Enzyme Group_
REFERENCES 1: 2. 3. 4. 5.
6.
B. L. Vallee and R. J. P. WiUiams,Proc_iVatL Acad_ Sci. USA 59,498 (1968). F. W. Snyder Jr. and J. C. W. Chien, Eur. J. Biochem. 91,83 (1978); and references therein. LV IkedsSaito, T. Inubushi, G. G. McDonald, and T_Yonetmi, J. BioL Chem_ 253, (1978). L. C. Dickinsonand J. C. W. Chien,Biochemistry 14,3526 (1975). M. C. Findlay, L. C. Dickinson, and J. C. W. Chien,J. Am. 0zem Sot. 99,5168 (1977). L. C. Dickinsonand J. C. W. Chien, J. BtiL C7zem. 252,6156 (1977).
7134
Studies of Horse Cytochrome
7. 8. 9_ 10. 11. 12. 13. 14. 1.5. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29_ 30. 31. 32. 33. 34. 35. 36. 37. 38.
15
c
M_ C. Findlay and J. C. W. Chien, Eur. J. Biochem 76,79 (1977). J. M. Vanderkooi, F. Adar, and M. Ericinska, Eur. J. Biochem 64,381 (1976). J. M_ Vanderkooi, R_ Lam&berg, G_ W_ Hayden, and C- S- Owen, Ew. J. B&hem
81,339
(1977). A. G. Redfield and R. K. Gupta, CWdSpringHarbow Symp. Quant. Biol. 36,405 (1971). C. C. McDonald and W. D. Phillips, Biochemkny 12,317O (1973). R. M. Keller and K_ Wuthrich, Biochem Biophys Acta. 533,195 (1978). D. 3. Cookson, G. R. Moore, R. C. Pitt, R. 3. P. Williams, I. D. Campbell, R. P. Ambler, M. Bruschi, and J. L&all, Eur. J. B&hem. 83,261(1978). G. R. Moore and R. J. P. Williams, submitted to Eur. J. Biochem G. R. Moore and R. J. P. Williams, submitted to Ew. J. Biochem. L. C_ Dickinson and J_ C_ W_ Chien, Biochemistry 14,3534 (1975). I. D. Campbell, C. M. Dobson, R. J. P. Williams, and A. V. Xavier, J. Mag. Res. 11, 172 (1973). R. A. Hoffman and S. Forsen, in Progress in NMR Spectroscopy, 3. W. Emsley, J. Feeney, and L. H. Sutcliffe, Eds., Pergamon Press, Oxford, 1966, p. 15. I. D. Campbell, C. M. Dobson, and R. J. P. Williams,Proc. R. Sot. B189,503 (1975). I. D. CaxnpbeB and C. M. Dobson,J. Chem. Sot. Chem Commun. 1975,750. G. R. Moore, D-Phil. Thesis, University of Oxford (1978). I. D. Campbell, C. M. Dobson, G. R. Moore, S. J. Perkins, and R. J. P. Wiiliams, FEBS Left. 70,96 (1976). W. D. Butt and D. Keilin. Proc. R. Sot. B156,429_ H. Scheer and J. J. Katz, in Porphyrins andfiferalloporphyrins, K. M. Smith, Ed., Elsevier, Amsterdam, p_ 399. G. R. Moore and R. J_ P. Williams, FEBS Left_ 79,229 (1977). N. Mandel, G. Mandel, B. L. Trus, J. Rosenberg, G. Carlson, and R. E. Dickerson, J. Biol. Chem. 252,4619 (1977). R. K. Gupta and S. H. Koenig, Biochem. Biophys. Res. Commun. 45,1134 (1971). D. M. Collins and J_ L. Hoard,1 Am. Chem. Sot. 92,3761(1970)_ P. K. W. Wasser and J. -H. Fuhrhop, Ann. NY Acad_ Sci 206,533 (1973). T_ J. Swift, in NMR of Paramagnetic Molecules. G. N. La Mar, W. de W. Horrocks Jr., and R. H. Hohn, Eds., Academic Press, New York, 1973. D. R. Eaton,J. Am. Chem. Sot. 87,3097 (1965). R. E. Dickerson and R. Timkovich, in The Enzymes. 3rd ed., P. Boyer, Ed., Academic Press, New York, Vol XI, p. 397. E. Margoliash and A_ Schejter, Adv. Prot. Chem. 21,113 (1966). R. Swanson, B. L. Trus, N. Mandel, G. Mandel, 0. B. KaBai, and R. E. Dickerson, J. BioL Chem. 252 759 (1977). T. Takano, B. L. Trus, N. hfandel, G. Mandel, 0. B. KaBai, R. Swanson, and R. E. Dickerson, J. Biol. Chem. 252,776 (1977). T. Takano, R. Swanson, 0. B. Kallai, and R. E. Dickerson, Cold SpringHarbour Symp. Quant. BtiZ. 36,397 (1971). T. Takano, 0. B_ Kallai. R. Swanson, and R. E. Dickerson,1 BioL Chem 248,5234 (1973). G. R. Moore and R. J. P. Williams, manuscript in preparation_
Received June 6.1979; accepted June II.
1979
Chemisrq
Laborarov.
Univesiry
of O_@ord
James C. W. Chien and L- Charles Dickson Department
of Chemisny.
University
of Massachusetts
ABSTRACT* The proton nuclear magnetic resonance spectra of various metal substituted derivatives of horse cytochrome c have been studied and compared to the spectra of native cyto-
chrome c. The proteins studied were the cobalt(W), copper(H), iron(II), iron(III), manganese(lII), nickel(R), and zinc(U) derivatives. Spectxa of the diamagnetic cobalt(W), iron( and zinc(II) proteins were we&resolved and specific resonance assignments were made. All three proteins possessed a methionine ligand to the metal. The spectrum of cobalt(IIl) cytochrome c was Investigated in some detail as this protein was used as a diamagnetic control for iron(II1) cytochrome c. Comparison of the spectra of cobalt(II1) and iron cytochromes c revealed that their conformations were very similar but the foIlowing conclusion couid be made; the oxidation of cytochrome c is accompanied by a smah conformation change.
INTRODWXION Utilization
of the different
probe properties
of metal ions by replacement
of the native
metal ion of a metaUoprotein with another metaf ion is widely exploited in physical studies of proteins [l] _ Until recently application of this principle to haemoproteins
had been restricted to haemoglobin [2] and myoglobin [3], proteins in which the haem group is not covalently bound to the protein. With the development of suitable preparative techniques for cytochrome c, in which the haem group is covalently bound to the protein, a range of metal-substituted cytochromes c have recently been prepared [4-S] . Many of the metal-substituted cytochrome c derivatives have been used in biological studies whose aims are directed towards further understanding of the function of native cytochrome c. For example, the oxidase activity of cobalt cytochrome c [4] and Address reprint requests to: R. J. P. Williams, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K.
* The abbreviations used in this paper, Co(III)c, respectively, the cobaIt(III), cobaIt(II), copper( horse cytochrome c.
Joumat of Inorganic Biochemisny
12,1-15
@ EIsevier North Hohand, Inc., 1980
Co(II)c, Cu(II)c, Mn(III)<:, Ni(II)-c, and Zn(II)c are, manganese(III), nickel(II), and zinc(I1) derivatives of
1
(1980) 0162-0134/80/010001-15$02.25
2
G. FL Moore et aL
the interactions of Sn(IV) and Zn(Il) cytochromes c with cytochrome c oxidase and rnitochondria [9] have been investigated. The relevance of such studies to native cytochrome c cannot yet be judged. One criterion which has to be established is the nature of any structural modifications accompanying the metal replacement. With this in mind we have studied the nmr spectra of the Co(III), Zn(II), Cu(II), Ni(II), and Mn(II1) derivatives of horse cytochrome c. -4dditional impetus for these investigations has come from the success of nmr in probing the solution structures of a wide range of eukaryotic and prokaryotic cytochromes c [lo-151 . In these previous investigations a major problem has been the comparison of nrnr data for the diamagnetic ferrocytochrome c and the paramagnetic ferricytochrome c. Differences in chemical shifts of corresponding resonances of the two- forms could arise from changes in conformation accompanying oxidation state change and from additional shifts caused by the pararnagnetism of ferricytochrome c. As cobalticytochrome c is six coordinate with Met 80 and His 18 providing the axial ligands [16] , and as this protein is active in an electron transfer capacity with cytochrome c oxidase [4] , it was hoped that cobalticytochrome c would serve as a diarnagnetic control for ferricytochrome c. In this paper we report a detailed analysis of the nrnr spectra of the Co(II1) and Zn(I1) derivatives of horse cytochrome c and a more limited analysis of the spectra of the Cu(II), Ni(II), and Mn(II1) derivatives.
MATERIALS
AND METHODS
The metal-substituted proteins were prepared as previously described [4-s] . Native horse cytochrome c was obtained from Sigma Chemical Co.;Type VI grade was used. Samples for nmr were prepared by dissolution of the metal substituted proteins in 0.05 M deuterated phosphate buffer at pH 7.0 Samples of native cytochrome c were dissolved in DaO. Protein concentrations were usually 3 X 10-a M. Ferrocytochrome c was obtained by reduction of ferricytochrome c with ascorbic acid under argon. The operating temperature was varied depending on the stability of the protein and on the nature of the nmr experiment. Temperatures are recorded in the legends to the figures and tables. Spectra were obtained using a Bruker 270 MHz spectrometer operating in the Fourier Transform mode. Convolution difference [17] , cross-saturation [ IS], spindecoupling [19] , andspinecho double resonance [20] were carried out as previously described. Acetone and dioxan were used as internal standards but all chemical shifts are quoted in parts per million @pm) downfield from 2,2&xnethyl-2silapentane-Ssulphonate-
RESULTS
Co(m) Cytochrome
c
The aromatic and aliphatic regions of the convolution difference spectra of Co(HI)< and ferrocytochrome c are shown in figures 1 and 2. To a cursory inspection, the spectra of ferrocytochrome c and Co(III)+z have many areas of close sim&rity, for example, the resonances between 0 and -S ppm, many of which arise from the axial
Studies of Horse Cytochrome
c
3
a.
b.
10 8 6 ppm FIGURE I_ The aromatic regions of the convolution difference spectra of (a) 3-mM cobalticytochrome-c in 0.05-Mdeuterated phosphate buffer at pH 7.0 and 57°C; and (b) S-mM horse ferrocytochromec in D20 at pH 5.0 and 57°C.
ligand Met 80. Where there are overlapping resonances the similarity is less striking as the general appearance of the spectra are greatly altered by small (ca. 0.02 to 0.03 ppm) differences in chemical shifts. However, detailed assignment allows exact comparison of corresponding resonances. Resonances of native cytochrome c have been assigned [14, 15]_ Resonances of Co(I.II)c were assigned first to amino acid type, based on identification of coupling patterns with spin-decoupling and spin-echo double-resonance, and then to a specific amino acid in the protein sequence by comparison with the corresponding, previously assigned resonances of ferrocytochrome c [14, 15]_ This procedure was only possible because of the close similarity of the resonance chemical shifts between Co(III)c and ferrocytochrome c. Assignments are summarized in Table 1 and 2. The dependence of the spectrum of Co(II& upon temperature, below 65OC, and pH is also similar to that of ferrocytochrome c. The C-2 and C4 resonances of His 33 shift with variation in pH with a pK, of 6.4 f 0 .l at 27OC. The methyl resonance at -0.24 ppm (57°C) from Ile 57 is also temperaturedependent shifting downfield with increasing temperature; between 27OC and 57°C it shifts by -0.24 ppm. Again, this parallels the behavior of the corresponding resonance of ferrocytochrome c [21] _ Additionally, this methyl resonance is pH dependent_ Between pH 4.2 and pH 9.6 it is pH independent, but outside this range the resonance shifts downfield. This shift is not a result of denaturation since the remainder of the spectrum is only little affected. Between pH 9.6 and pH 109 the methyl resonance shifts by -0.07 ppm and
G. EL Moore et al.
4
a.
1
2
I
I
0
-2
I
-r;
ppm
FIGURE 2. The aliphatic regionsof the convolutiondifferencespectraof (a) 3-mM cobalticytochrome-c in 0.05-M deuterated phosphatebuffer at pH 7.0 and STC!; and (b) 5-mM horse ferrocytochromw in D@ at pH 5.0 and S?C_
between pH 42 and pH 3.4 it shifts by > -0.2 ppm. A final illustration of the temperature dependent resonances is afforded by resonances of Tyr 48_ At temperatures <35OC, this residue is in slow exchange between equivalent orientations for ferrocytochrome c 1221. With increasing temperature its resonances broaden, finalIy disappearing from ‘;he spectrum at 3YC. In the spectrum of Co(III)i: at 37OC there are anaIogus resonances, related by a cross-saturation effect, at 5.47 and 6.72 ppm. These exhibit a similar temperature dependence to the corresponding resonances of ferrocytochrome c, and they broaden and disappear above 42OC There are differences in stability between the two proteins; ferrocytochrome c is stable up to Q?C with its spectrum little changed from the low-temperature form 121, 231, whereas Co(1IQ-c begins to denature at ca. 65°C. This is analogous to the behavior of ferricytochrome c. &r(n) Cytochrome c In order to study the protein by nmr, soiutions of Zn(II) cytochrome c were made immediateIy prior to use from mate&d prepared and stored in the dark, and the sohrtions
Studies of Horse Cytochrome
TABLE
c
1. Metal Ligand Resonances for Diamagnetic Cytochromes c
Assignment Haem meso Haem me50 Haem meso Haem meso Thioether CH Thioether CH3 His 18 Met 80 CH3 Met 80 7 CH Met 80 p CH Met 80 7 CH Met 80 @ CH
Fe(II)-c 9.62 9.59
9.32 9.04
6.36 2.57 0.13 -3.28 -3.73 -2.58 -1.87 -0.19
Co(iII)-c
Zn(ii)-c
10.71 10.43 10.26 9.77 6.53 2.76 -0.07 -3.77 -4.93 -2.98 -2.21 -0.40
10.11 9.94 9.49 nd nd nd 30.05 -3.07 -3.60 -2.36 -1.62 nd
a AU chemical shifts measured in ppm at pH 7 and, for Fe(II)-c and Co(III)s at 57°C but for ZnQI)-c at 2?C. The resonances of the metal ligands of Fe(II)c and Co(III)-c are virtually temperature independent but for Zn(II)-c they are strongly temperature dependent. b The meso CH assignments for Co(III)-c corrects previous work [ 161 where they had been inconectly assigned.
placed into smoked glass nmr tubes previously flushed with argon. Even so, protein denaturation, evident from spectral quality and gradual precipitation of the sample, began to occur within ca. 2 hr. As with Co(III)c, comparison of the spectrum of Zn(II) cytochrome c with that of ferrocytochrome c reveals many similarities between them (compare Figure 3 with Figures 1 and 2). In particular, the ringcurrent-shifted methyl regions, between 0 and 4 ppm, contain analogous resonances indicating that Met 80 provides an axiaI Iigand to the Z&II) ion. Spectra of Zn(I1) cytochrome c are less well-resolved than spectra of Co(II1) cytochrome c or ferrocytochrome c and resolution was not improved with increasing temperature because of the relative instability of Zn(I1) cytochrome C. Thus, assignment methods based on multiplet structure could not be applied. However, as the spectra of Zn(I1) cytochrome c and ferrocytochrome c at 27O are similar it proved possible to compile a list of tentative assignments based on comparison of resonance intensities and chemical shifts (Tables 1 and 3). It can be seen from figure 3 that the spectrum, and therefore the structure, of Zn(I1) cytochrome c is strongly temperature dependent with many resonances shifting with variation in temperature and some, such as the methyl resonance of Met 80 at -3.07 ppm (27OC) decreasing in intensity with increasing temperature. Indeed at 27°C the Met 80 resonance is only of ca. two proton intensity and at 57°C it has decreased to < one proton intensity compared to the remaining methyl resonances between 0 and -1 .O ppm (57°C). The chemical shift variations between 27°C and 47°C are not completely reversible_ Cu(II), Ni(IK), and Mn@I) Derivativesof Cytochrome E
z
C0(11)-, CU(II)-, Ni(II)-, and Mn(III)-cytochromes c are considered together as they are paramagnetic. Electron paramagnetic resonance measurements show that the electron-
G. R. Moore et al.
spin relaxation times of Co(II)c and Cu(II)-c are long, indicating that their paramagnetic centers are powerful relaxation probes, whereas those for Mn(III)c and Ni(II)-c are short. Thus, spectra of Mn(III)c and Ni(II)c were predicted to exhibit relatively sharp hype&me shifted resonances of the haem group [6, 71. However, in none of the above cases were resonances observed outside the spectral region 12 to -2 ppmSpectra of Fe(III)-c, Cu(II)c, and Ni(I1) c are shown in figure 4 over the region 12 to -2 ppm. The spectrum of Cu(II)-c appears to be considerably broadened (compared, for example with the spectrum of ferricytochrome c), confiig the earlier predictions. The quality of the spectra of the paramagnetic metal-substituted proteins is low and assignments have not been obtained. DISCUSSION
Co(III)-Cytochrome
c
Clearly, from the data of Tables 1 and 2 it can be concluded that the structures of ferrocytochrome c and Co(III)c are essentiaZZy similar with both proteins possessing 6 co-ordinate metal ions, the axial ligands of which are His 18 and Met 80. However, there are some variations in chemical shifts for corresponding resonances. Many df these variations are small (2 0.03 ppm) but some are significantly larger. Chemical shift differences could arise by a number of mechanisms: I_ Changes in metal-ligand bond lengths and/or haem plane-metal-l&and bond angles, giving rise to different haem ring-current shifts for resonances of ligand nuclei_ 2. Variation in electron delocalisation in the porphyrin leading to a variation in o-bond electron density for nuclei of the porphyrin and a variation in the magnitude of the haem ring-current. 3 _ Temperature independent pammagnetism of the metal. 4_ Electric field effects caused by change in charge of the metal ion. 5. A change induced in the protein structure. This could be a metal linked conformation change, an oxidation-state linked conformation change or a structural change brought about by protein modification during metal replacement. We will consider resonances in turn, in the following order: haem resonances, axial ligand resonances, and other amino acid resonances. Many studies of metal substituted porphyrins have established that, for porphyrins with in-plane metal ions the chemical shift of the porphyrin resonances increase as charge on the metal ion increases; for dipositive metal ions a,,,, = 9.75 to 10.08 ppm and for tripositive metal ions 6,,,, = 10.13 to 10.39 [24]_ The results for ferrocytochrome c and Co(III)c are in general accordance with this scheme (Table 1) but note that the chemical shifts for the meso resonances are spread over a wider range than for the ‘model’ compounds. There are two conflicting mechanisms contributing to the different chemical shifts for meso resonances of ferrocytochrome c and Co(III)c: the cr withdrawing power of Co(III) is greater than for Fe(D) while Co(III) is a poorer srdonor than Fe(I1). The former leads to a deshielding of the meso protons, and the latter to a decrease in the haem ringcurrent for Co(III)-c relative to ferrocytochrome c. As the nett shifts of the meso resonances for Co(III)-c are downfield from those of ferrocytochrome, the o-withdrawing property of the metal ion is more im-
Studies
of Horse Cytocbrome c
TABLE
2. Compadson
Assignment Leu 32 Leu 32 lie 57 Met 65 N-acetate
of Chemical
Shifts for Fe(II)-c
Fe(I0-c
Co(IiI)c
-0.76 -0.60 -0.43 2.09 2.06
-0.75 -0.68 -0.24 2.12 2.09
I
His 33 His33 His 26 His 26 Trp 59
7.79 7.29 7.52 7.06 6.99
7.77 7.27 7.51 7.06 7.11
Trp 59 Trp 59 Trp 59 Trp 59 Phe 82 F’he 36 Phe 36 Phe 10 Phe 10 Tyr 97 Tyr 97 Tyr 48 Tyr 48
7.60 6.70 5.74 7.09 6.34 7.40 6.89 6.71 7.40 7.22 6.64 6.78 5.59
7.60 6.62 5.77 7.07 6.19 7.36 6.88 6.73 7.42 7.25 6.62 6.72 5.47
and Co(III)-@
Shift Fe(II)-Co(III)
% Differenceb
-0.01 +0.08 +0.19
+4.8 -14.5
+-O-O8 -0.03 +0.02 +-o-15 +0.04 +0.01 -0.02 -0.02 -0.03 +-o-o2 +0.06 +0.12
16-W -0.7 6.8 14.2
14.3d 7.9
o All chemical shifts measured at 570 and pH 7 e.ucept those of Tyr 48, measured at 27”. b % difference is Fe(II)-Co(III) difference expressed as percentage difference from secondary shift for resonance of fenocytochrome c. C Assuming ass@ment scheiner the resonance at 6.70 ppm arises from the C-6 proton. d Assuming these resonances are meta proton resonances.
portant. systems
The difference between the meso resonance shifts for the ‘model’ porphyrin and the cytochromes may be due to the donor/acceptor properties of the
axial ligands and to secondary structure shifts, such as amino acid ring-current shifts. Whereas the chemical shifts of the Met 80 resonances (Table 4) are different for the two proteins, those of the singlet resonances of His 18 are very similar; the shift from primary position is only ca. 3% greater for Co(III)c. As the His 18 C-2 and C3 protons are closer to the metal than the Met 80 protons a significant contribution to the chemical shift differences from temperature independent paramagnetism can be discounted_ The electronic effects caused by metal substitution would cause the Met 80 resonances of Co(III)c to shift downfield if the distances between the haem plane and the Met 80 protons were unaffected by the metal replacement. Therefore, the large upfield shifts of the Met 80 resonances for Co(III)x can be attributed to a decrease in the haem plane-Met 80 proton distances. This could arise by a simple shortening of the metalsulphur bond length or by a decrease in the metal-sulphur bond length concomitant with tilting of the Co(III)-S bond. The geometry of the Fe(II)-S-Met 80 bond system
G.
I
>-t3
2
I
0
I
R. Moore et al.
I
-4
ppm (a) ;b: JTIGURE 3. The aromaticregion(a) and aliphatic region (b) of the spectrum of 3-1&f zinc-cytochrome-c in 0.05-M deuterated phosphate buffer at pH 7.0 and various temperatures_ The vertid scale in (a) is 4 times that in @). of cytocbromes c has recently been correlated with redox potentials [25]_ In these studies it was shown that as the Fe(lI)-S bond length decreased the chemical shift of the resonances of the methionine ligands CHa and yCH protons decreased whilst that of the r’CH proton increased. A change in Fe(II)-S bond angles was not indicated by these studies. Now consider the differences in chemical shifts for the Met 80 resonances of Co(III)-c compared to ferrocytochrome c (Table 1); all the resonances are shifted further upfield for Co(III)-c and that of ‘tbe $H proton by a considerably greater amount than the others. This is a very different situation than for the studies with different ferrocytochromes c and indicates that the Co(III)-S bond length is not only shorter than the Fe(ll)-S bond length but also that the Co(III)-S bond is nonaxial. From the decreased haem ring-current of Co(IU)-c it would be predicted that the secondary shifts for resonances of groups close to the haem should decrease_However, for a number
Studies of Horse Cytochrome
c
TABLE 3. Comparison
of Chemical Shifts for Fe(II)-c and Zn(II>-c”
Assignment
l.eu 32 Leu 32 lle57 Met 65 N-acetate
Fe(H)*
-0.76 -0.60 -0.60 2.06 2.06
1
-0.90 -0.70 -0.24 2.12 2.05
Trp 59 Trp 59
5.74 6.99
5.65
7.40 6.88
7.01 7.37 6.89
Phe 10 Phe 10 Tyr 97 Tyr 97
6.72 7.40 7.22 6.61
6.59 7.37 7.22 6.59
Phe 36 Phe 36
0 Chemical shifts measured at 27Oand pH 7-k b Assignments for Zn(II)-c have not been contirmed with double-resonancetechniques.
inspection of the data in Table 2 shows that those resonances which are significantly shifted @ 10%) from ferrocytochrome c to Co(III)c experience a larger secondary shift; note the resonances of Trp 59, Phe 82, and Tyr 48. In the case of Try 48, much of the secondary shift experienced by its resonances is derived from Phe 46 while the haem group is responsible for the secondary shifts of resonances of Trp 59 and Phe 82_ The increased shift of the Phe 82 para proton resonance may be associated with the change in metal-S bond length for Co(III)c which, transmitted along the peptide chain, ‘pulls’ Phe 82 closer to the haem. The shift of the Trp 59 resonance at 6.62 ppm (Co(III)c), the only significantly shifted resonance of Trp 59, is not readily explicable_ Any change in Trp 59-haem orientation would affect most of the tryptophan resonances and probably strongly perturb the resonance at 5.77 ppm (Co(III)-c). The shifts of the Tyr 48 resonances are also not readily explicable although a small change in Phe 46-Tyr 48 orientation could account for them. It is relevant to note that both Trp 59 and Tyr 48 are H-bonded to the haem propionate side chains [26] and therefore a change in the haem o-bond electron distribution could affect the strength of the H-bonds. The resonance of lle 57 at -024 ppm (Co(III)c) is an exception to the aromatic resonances discussed above in that it experiences a significantly smaller secondary shift. Ile 57 is far from the haem and the methyl resonance at -0.24 ppm derives its secondary shift mainly from Tyr 74. The corresponding resonance of ferrocytochrome c is temperature and pH sensitive [27] , and this sensitivity has been interpreted in terms of conformational mobility in the region of the protein about Ile 57 [21] _ Similarly this Ile 57 resonance of Co(III)-c is temperature and pH dependent and exhibits analogous behavior to the ferrocytochrome c resonance. Thus, the difference in secondary shift for the methyl resonances of Ile 57 probably reflects a small difference in structure between the two proteins. Movement of the aromatic ring of Tyr 74 away from the WH, of IIe 57 has been suggested as the cause for the pH and temperature
10
G. R. Moore et al.
I 0
I 6
I
I
I
4
2
0
I -2
pm
FIGURE 4. Spectra of horse cytochromesc at pH 7.0 and 37OC. (a)Ferricytochrome-c in D20; @I) copper0-cytochrome-c in 0.05-M deuterated phosphate buffer; (c) manganese(III)-cytochrome-c in 0.05-M deuterated phosphate buffer; (d) nickel(Ii)sytocbrome c in 0.05-M deuterated phosphate btlffer.
dependent shifts of this methyl resonance [21] _It is attractive to speculate that transmission of the decrease in the metal-S bond length for Co(III)-c along the peptide chain may be responsible for the difference in shifts between the corresponding resonances of Ile 57. The Tyr 48 resonances of ferrocytochrome c at 5.59 and 6.78 ppm broaden with increasing temperature as the aromatic ring of Tyr 48 undergoes more rapid motion placing its resonances in intermediate exchange 1221. The corresponding resonances of Co@)) possess a similar temperature dependence and this, along with the temperature dependence of the Ile 57 6CHa resonance, reveals that not only is Co(III)-c similar to ferrocytochrome c in its static structural aspeck but also in its dynamic aspects. It proved impossible to obtain the ortho and meta resonance of Tyr 48, Phe 46, and
Studies of Horse Cytochrome
4. Resonances
TABLE
Secondary Shift for Assignment Met 80 SCH3 E
11
c
WIGc +5.40 +6.43 i-4.85 +4.57 +2.46
of Met 80 Fe(II)-Co(III) difference % H-49 +1.20 +0.40 +0.34 +0.21
9.1 18.7 8.2 7.4 8.5
Fe(H)-Zn(II) difference -0.21 -0.13 -0.22 -0.25 nd
% -3.9 -2.0 -4.5 -5.5 nd
Primary Position for Met Resonvlces -SCH3
2.12 ppm 2.70ppm 2.27 ppm
Phe 82 in fast exchange, as has been done for ferrocytochrome c at 97O [13,14,22], because of denaturation of Co(III)-c above 65°C. In respect of its thermal stability Co(III)c resembles ferricytochrome c rather than ferrocytochrome c and thus the overall charge is the determining factor. However, in respect of its pH stability Co(I11)1: is like ferrocytochrome c rather than ferricytochrome c. The methionine ligand of ferricytochrome c is replaced at ca pH 9.2 by a nitrogen ligand but the methionine ligands of Co(III)c and ferrocytochrome c remain intact even at pH 11 (27°C). Thus in this case the charge is not the determining factor. The previous discussion has centred about chemical shift differences arising from small structural rearrangements, for the data tell us there are no large-scale rearrangements_ However, the additional charge on the metal ion for Co(III)c may induce small electric-field effects in resonances of groups close to the haem which would lead to upfield chemical shifts. This would be predominantly an isotropic effect and thus, resonances of Leu 32 should experience larger shifts due to this mechanism than the para proton resonance of Phe 82. Similarly on this model the Trp 59 resonance at 5.77 ppm (Co(III)c) and the His 18 singlet resonance should experience large shifts compared to their corresponding resonances of ferrocytochrome c. This is not the case and we conclude that electric-field effects are not important in this conqarison. Additionally, the expected decrease in haem ring-current was not observed in the chemical shifts of assigned resonances. Resonance shifts due to modification of the protein other than those associated with the metal replacement have not been observed_ This is not surprising as the ion-exchange and electrophoretic mob&ties of Co(III)c and fenicytochrome c are the same [4] _ Zn(II)-cytochrome
c
The problems concerning the assignrnent of resonances of Zn(II)c have been discussed in the results section. There a list of tentative assignments was constructed (Table 3) based on comparison of the spectrum of Zn(II)c with that of ferrocytochrome c_ Differences in structure are apparent between Zn(II)< and ferrocytochrome c, however; note, for example, the additional two two-proton intensity peaks at 6.23 and
G. R Moore et al.
12
5.48 ppm in the spectrum of Zn(II)-c and the different secondary shifts for the upfield ringcurrent-shifted methyl resonances. These uncertainties prohibit detailed discussion of the structure of Zn(II)-c in terms of the structure of ferrocytochrome c other than to say that immediately on dissolution of Zn(II)-c, the protein conformation closely resembles that of ferrocytochrome c. The spectra of Zn(II)-c given in figure 3 show that, in contrast to ferrocytochrome c and Co(III)-c, there are many, large temperature-dependent effects not all of which se reversible_ In the absence of firm assignments we will confme ourselves to a discussion of the Met 80 resonances-but note that the resonances, in figure 3, disappearing with increasing temperature from the region 8-10 ppm, probably arise from exchangeable NH protons. Shifts of the resonances of Met 80 with temperature are represented in figure 5. These shifts are in the direction of decreasing secondary shift with increasing temperature and they extrapolate at T = m to: for the CH3 and @CH resonances 2.0 f 0.2 ppm and for the flH resonances 2.8 + 02 ppm. Additionally, the intensity of the methyl resonance decreases with increasing temperature. The intensity variation for this resonance implies that ca. 66% of the Zn(II)c molecules possess Met 80 ligation at 27” while ca. 34% do not. At 57O more than ca. 34% of the Zn(II)-c molecules possess Met 80 ligation_ These data are consistent with schemes such as
where bond length b >a_ Kis required to be>103 set-l in order that the resonance condition for fast exchange is met. The extrapolated values for the Met 80 chemical shifts at T = m accord well witk the primary positions for methionine resonances; 2 -12 ppm for the CH, and PCH resonances, and 2.64 ppm for the 7CH resonances_ In fact the spectrum of Zn(II)-c after exposure of a solution to light at 27°C for ca. 3 hr is that of a random coil protein. Crystal structure determinations for Zn(Ii)-porphyrin complexes have shown that the Zn(I1) ions are displaced from the center of the porphyrin plane; for (HaO)Zn(tetraphenylporphyrin) this displacement is 0.35 A [28] _ A similar disphcement for Zn(II)-c, presumably on the side of the His 18 ligand, would lead to a wrtakening of the metal-S bond. This is probably one cause of the relative instability of Zn(II)-c, but note also that Zn(II)-porphyrins are light sensitive [29] _
Co@),
Cu(II), Ni(II), and Mn(II1) Derivatives of Cytochrome
c
We can add nothing new to our previous knowledge concerning the structures of Co(II)c, Cu(II)-c, Mn(III)-c, and Ni(1Q-c as the quality of the spectra was insufficient to obtain assignments. The lack of contact shifted resonances for Mn(III)c and Ni(II)-c presents a problem as for paramagnetic ions with short electron spin-lattice relaxation tirres the resonances of the ligand nuclei should be observable [30]. Thus, nmr studies [3 l] of Mn(III> and NiII) acetylacetonates revealed sharp, strongly shifted resonances of the acetylacetonate ligands. It is feasible that exchange processes such as that occurring for Zn(II)c may operate in these proteins; with K = lo3 to lo4 set-l and a con-
13
Studies of Horse Cytochrome c
6 ppm
-3-5
\
“
-3-O
-2-5
-1-S
’
;7
37
-‘I
47
57
Temperature
“C
FIGURE 5. Temperaturedependenceof the resonancesof Met80 of zinc-cytochromec.
siderably greater chemical shift difference the resonances of the &and nuclei would then be in intermediate exchange and therefore not observable_
General Diiussion Our studies have shown that the structures of Co(III)c and Zn(II)c are similar to that of ferrocytochrome c, although in the case of Zn(1Q-c there are some structural modifications in addition to the exchange processes at the coordination site. Co(II)-c has been shown to be 6 coordinate with Met 80 and His 18 providing the axial ligands [16] and this protein possesses similar pH transitions to ferrocytochrome c; pKa’s at ca. 3 and ca. 12 [32] _ In view of the data presented in this paper it must be the case that only a small conformation change accompanies oxidation-state change for cobalt
G. R. Moore et al.
14
.
cytochrome c. Therefore, these proteins may serve as viable probes for the biological action of native cytochrome c. As discussed in an earlier report [4], there is also a thermodynamic element involved where cobait-cytochrome c undergoes oxidation state change as its Em = -140 mV whereas that of native cytochrome c is + 260 mV. Nevertheless, the lack of activity for cobalt cytochrome c with cytochrome reductase preparations (it is active with cytochrome oxidase) has been deduced as arising from structural modification involving the surface of the protein [4] . This is ofgreat interest as the chemical properties of native cytochrome c also suggest that there is a difference between the structure of the protein surface in its two oxidation states [33] _ An important conclusion from the comparison of Co(III)< and ferrocytochrome c is that, as the additional charge for Co(IIIb has no major effect upon the protein conformation the same should be true for ferricytochrome c, i.e., the oxidation of native cytochrome c is not accompanied by a Zarge conformation change. It has been necessary to establish this by a technique independent of x-ray crystallography because although the most recent x-ray structure determinations have shown that there is no oxidation state linked conformation change, [26,34,35], previous x-ray studies [36, 371 have been interpreted in the light of massive conformation changes. However, the present results show that there are small differences in conformation between the Co(II1) and Fe(I1) proteins. There must be a small change in metal-S bond length on oxidation state change of iron analogous to the difference between ferrocytochrome c and Co(IIl)-c, but as the bond length for Fe(III)-S is likely to be intermediate between that of Fe(II)-S and Co(III)-S the repercussions of this change will not be any greater than those observed for Co(III)c_ Even so we know that this change affects the surface of the protein around IIe 57. We can now proceed in our investigation 1381 of the solution structure of cytochrome c confident that the difference in chemical shifts for corresponding resonances of ferricytochrome c and ferrocytochrome c arise predominantly from contact and pseudocontact shifts, except in one or two regions of the protein. The central role of the metal ion in dete r-mining the stability of the protein is demonstrated by these studies_ It has been known for a long time that ferrocytochrome c is more stable to extremes of temperature than ferricytochrome c [23,33]. This is mirrored by the behaviour of cobalt cytochrome c where Co(III)-c is less stable than ferrocytochrome c. Yet the protein conformations of Co(III)-c and ferrocytochrome c are very similar. Clearly the additional charge for the +3 oxidation state leads to a destabillsation of the protein. We thank the Science Research Council. rhe Medical Research Council and the Royal Society for jkancti support_ R.PW k a member of the Oxford Enzyme Group_
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6.
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7134
Studies of Horse Cytochrome
7. 8. 9_ 10. 11. 12. 13. 14. 1.5. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29_ 30. 31. 32. 33. 34. 35. 36. 37. 38.
15
c
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Received June 6.1979; accepted June II.
1979