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Nuclear magnetic resonance study of cyanoferrimyoglobin; Identification of pseudocontact shifts
J. Mol. Biol. (1970) 53, 35-48
Nuclear Magnetic Resonance Study of Cyanoferrimyoglobin Identification of Pseudocontact Shifts B. SHEARD, T. YAMANEAND
R.G.
;
SHULMAN
Bell TelephoneLaboratories, Incorporated Murray Hill, N.J. 07974, U.S.A. (Received 18 Mwy 1970) The high-resolution proton magnetic resonance spectrum of sperm whale cyanoferrimyoglobin in solution in water was measured at 220 MHz, and it was compared with the spectrum in deuterium oxide. Exchangeable protons gave three peaks with strongly temperature-dependent shifts in the region - 13 to -24 p.p.m., measured downfield from 2:2-dimethyl-2-silapentane-5-sulphonate. They were shifted by interactions with the unpaired electrons. Four other exchangeable peaks, which had shifts with much smaller temperature dependences, were observed in the region - 10 to - 14 p.p.m. In the upfield region, four non-exchangeable peaks which had been thought to have areas corresponding to two protons each, and which had been assigned to methylene protons of the heme propionate sidechains, have been found to have areas corresponding to three protons, and they must be aasigned to methyl groups. Since they are pammagnetically shifted, as are other methyl peaks at low field, there are too many paramagnetically shifted methyl peaks in the spectrum to be accounted for by contact interactions alone. This means that pseudocontact interactions, which do not depend on spin delocaliz&ion through covalent bonds, must be appreciable. Estimates of pseudocontact shifts allow assignments to be made for the three paramagnetically shifted exchangeable peaks, namely, the protons NBH of His E7, N,H of His F8 and the peptide NH of His F8. Since pseudocontact shifts have been found to be appreciable, some of the peak assignments made earlier for non-exchangeable peaks are now uncertain.
1. Introduction In heme proteins, interactions with unpaired electrons from t’he iron atom produce peaks in the high-resolution proton magnetic resonance spectra which are well outside the usual 0 to -10 p.p.mt range of absorption (Kowalsky, 1965; Wiithrich, Shulman & Peisach, 1968; Kurland, Davis & Ho, 1968). Their positions have been used to compare myoglobins (Wiithrich et al., 1970) and hemoglobins (Yamane, Wiithrich, Shulman & Ogawa, 1970) from various species, and to investigate the heme environment in mixed state hemoglobin in studies of the co-operativity of oxygenation (Shulman, Ogawa, Wiithrich, Yamane, Peisach & Blumberg, 1969). By working with solutions of cyanoferrimyoglobin in waterj: it was hoped that an extra peak would be obtained from the proximal histidine NH proton, which could be unambiguously identified, and could then give information about ligand binding. In fact, we have observed several additional para,magnetically shifted peaks at 10~ 7 All shifts are given in parts per million relative to an internal 2.silapentane-5-sulphonate. Low-field shifts are negative. $ H,O is referred to as “water”; D,O as “deuterium oxide”. 33
st,andard
of sodium
2:2-dimethyl-
B.
36
SkIEARl),
T.
YAMANE
AND
Ib.
G.
SHULMAN
field in water solutions. During the course of this work it became clear that pseudocontact interactions (McConnell & Robertson, 1958; Jesson, 1967) are appreciable and that they are particularly important for explaining the shifts of the extra peaks observed in water. We present here experimental evidence for pseudocontact interactions in cyanoferrimyoglobin and calculate some of the pseudocontact shifts.
2. Experimental Sperm whale and porpoise myoglobin were isolated by a combination of the methods of Hapner, Bradshaw, Hartzell & Gurd (1968) and of Hugli (1968). Homo myoglobin was bought from Calbiochem. The pH was adjusted by dialysis under reduced pressure against which was: 0.1 M-sodium acetate below pH 5.8; 0.1 Mthe appropriate buffer solution, sodium phosphate in the pH range 5.9 to 8.0; 0.1 M-sodium borate above pH 8.0. Concentrations were in the range 10 to 15%. The cyanide derivative was obtained by adding an excess of potassium cyanide in the appropriate buffer. The pH quoted is the pH of tho NMRt sample itself, measured at 25 deg. C+ 2 deg. C. The spectra were obtained with a Ovarian HR220 NMR spectrometer, cquippod with a Fabritok 1062 signal-averaging computer. An RF field of 1 milligauss was used, with a sweep rate of 20 Hz sec. - 1 Probe temperatures were measured with a sample of ethylene glycol and the regulation was better than rf: 1 deg. C.
3. Results The original idea was that if the residence time of t’he hydrogen atom nitrogen-l of the proximal histidine residue were long enough, interactions
bound to with the
H2O
LLI~L~II.I~L~I_I..I._L~
-20
-26
-24
-22
-20
Shift (ppm)
-10
-16
from
vi -14
-10
DSS
FIG. 1. A comparison of 220 MHz spectra of sperm whale MbCN in H,O solutions at various values of pH. The spect,rum at pD 7.00 in D,O solutions is also shown. All samples contained 0.1 M-sodium phosphate buffer. The temperature was 29°C. DSS, 2:2-dimethyl-2.silapentane-tisulphonate.
t Abbreviations
used:
NMR,
nuclear
magnetic
resonance;
MbCN,
cyanoferrimyoglobin.
Slll1-L
STUl)Y
OF
3;
CYANO~‘ERRIhlY’oC;I,O~IN
unpaired electrons would shift its NMR peak outside the usual range of absorption, where it could be observed in solutions in HzO, but not in D20. Following this rationale a paramagnetically shifted exchangeable peak had been found in the spectrum of ferricytochrome c (Sheard, results to be published). In sperm whale MbCN, however. four such peaks (labeled A, B, C and D) were found between -12 and -28 p.p.m. (Fig. l), two of which are superimposed near -14 p.p.m. at high temperatures, as shown below. Peaks A, B and C have shifts which vary strongly and linearly with the inverse of the absolute temperature (Fig. 2), which identifies them as being shifted
-6200
/ ‘H,
-6000
-14
-26 -3000
vr
-24
! ’i j/l -2900 I
-23
-2800
-25
-5400 -5200
-27 -3100.
-5800 -5600
-28
A I
I'
CH,
/,,.,Z, /
,,/
-13
/ ,/
E a .!? u c -12 5
-22 -21 -20 -19
-II
-18 -17 -10 -16 5 1/Tx103
32
33
34 VTxlO'
35
5
FIG. 2. Shifts, at pH 7.0, of low-field peaks in the 220 MHz spectrum of sperm whale MbCN, plotted against the reciprocal of the absolute temperature, 1,/T. Note t,he change of frequency scale between the two parts of the Figure. (0) Peaks observed only in H,O; (0) peaks observed also in I>,O.
by the unpaired electron (Wiithrich et al., 1968). Another four “exchangeable peaks” were observed in H,O between - 9.8 and -11.3 p.p.m., but their positions are considerably less temperature dependent. The pH was varied at a constant temperature, 28*5”C, with the results shown in Figure 3. Consider first the peaks A and B. Where the peaks are observable, their positions are independent of pH, except below pH 5, as the point of denaturation is approached. Peak 9 broadens without changing its position as the pH is lowered, and it disappears
38
B.
SHEARD,
T.
-6000.
YAMANE
ANU
1%. G.
-3000-
fl4
-2900-
' o"
SHULMAN
,--27
-5800 --26 -5600 --25 -5400 --24 -2800..
l__lc-% 0 0 %
-2 --22
: -4800 i Lo -4600
0
ii
-2700-
I -4400-
--20
-420000 -4ooo_
0
oo
0 0
a'
W
--I9 -2400 e
1-18 -2300;.
PH FIG. 3. Shifts, at 28.5’C, in the 220 MHz spectrum of sperm whale MbCN, plotted as a function of pH. Buffers are described under Experimental. Note the change of frequency scale between the two parts of the Figure. (0) Peaks observed only in HaO; ( 0) peaks observed in both Hz0 and I&O. Peaks assigned to methyl groups have been marked accordingly.
below pH 7. This is an exchange phenomenon in which the peak suffers lifetime broadening, and as expected the pectk becomes even broader as the temperature rises and exchange becomes faster, in contrast with the other peaks in the spectrum which display the usual narrowing. Although the pK, for this proton&ion is unknown it must be less than 7 since at pH 7 the peak area is still nearly unity. If we take the simple view that the peak observed is from an exchangeable proton in a residue which itself is rapidly gaining and losing a second labile proton, the residue has to be histidine. In an attempt to observe peaks from the protonated form of the histidine a search w&s made at pH 5 and 5”C, where the exchange should be slower, but no extra peaks were observed between -40 and -12 p.p.m. or between +3 and +32 p.p.m. The peak at -13.8 p.p.m. in Figure 1 has an area for two protons at high pH and for one proton at low pH. The explanation is as follows. At 29”C, the temperature of Figure 1, the two exchangeable peaks C and D are superimposed (see Fig. 2). As the pH is lowered below pH7, peak D broadens and disappears, leaving only
NMR
STUDY
30
OF CYANOFERRIMYOGLOBIN
pH 60
PH 7.0
pH 8.0
1 - IS
I
I -I4
I
I -13
I
I -12
Shift (p.p,m) from DSS
FIG. 4. 220 MHz spectra, at 19”C, of sperm whale MbCN in Ha0 with 0.1 M-sodium phosphate buffer. This shows that the peak near - 14 p.p.m. in Fig. 1 is composed of two peaks above pH 7, one of which disappears at lower pH. DSS, 2:2-dimet’hyl-2-silapentane-5sulphonate.
I
-2k%%?l-22
I
I
-20
I
I
-I8
I
I
-16
Shift (p.p.m.1 from
!
I
-14
!
I
-12
z-u
-10
DSS
Fro. 5, 220 MHz spectra, at pH 8.2 and 29°C of sperm whale, horse and porpoise H20, wit,h O-1 M-sodium phosphate buffer. DSS, 2:2-dimethyl-2-silapentane-5sulphonate.
MbCN in
40
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAS
peak C. This can be seen directly at lower temperatures, where the two peaks arc caused to separate (Fig. 4) by the increased paramagnetic shift in peak C. It turns out that the width of peak D increases so rapidly with temperature that even at pH 8 it is too broad to be observed separately at temperatures above the crossover region. The effect of varying the amino acid composition was observed by comparing myoglobins from various species. Figure 5 shows low-field NMR spectra of the cyanide derivatives of sperm whale, porpoise and horse ferrimyoglobins, at 29°C and pH 8.2. The position of peak B is identical, within experimental error, for all three species, while the broad peak, peak A, varies in position among the species. Both horse MbCN and porpoise MbCN have an extra peak near -13.8 p.p.m. but, in contrast to sperm whale MbCN, these have one-proton areas under the conditions of Figure 5. ‘The peak observed is peak C. The positions of the other low-field peaks are shown as a function of pH in Figure 3. Two of the exchangeable peaks which could be clearly followed as a function of temperature, and which converge near -11 p.p.m. at the low-temperature end of Figure 2, are not plotted in Figure 3 because it proved difficult to identify them with certainty over the whole pH range. The other exchangeable peaks in that region, near -11-O and --lo*0 p.p.m. in Figure 3, have little or no pH dependence. Of the peaks observed previously in D,O solution at pD 7-O (Wtithrich et al., 1968) the methyl peak at lowest field shows a large upfield displacement at low pH, with a point of inflection near pH 5.6, and another methyl peak near -13 p.p.m. drifts downfield almost linearly as the pH falls. The other peaks in Figure 3 have little or no dependence on pH, except the peak moving between -115 and -11.0 p.p.m., thought by Shulman, Wiithrich, Yamane, Antonini & Brunori (1969) to be attributable probably to the proximal histidine residue, and the peaknear -12.4p.p.m., which
*\
Fig. 6. 220 MHz upfield spectrum of sperm whale cyanoferrimyoglobin at pH 8.5 and 30°C in H,O solution with 0.1 M-sodium phosphate buffer. In the inset, the peaks at +3.31 and +3.68 p.p.m., and the peak at +9*10 p.p.m., are shown with a 2.5.fold horizontal magnification. Their vertical enlargements are all equal. DSS, 2 :2-dimethyl-2-silapentane-B-sulphonate.
NMR
STUDY
OF
41
CYANOFERRIMYOGLOBIN
has an area corresponding to two protons and which has been thought to arise, along with its nearest upfield neighbor, from resonance of a porphyrin methyl group (Wiithrich et al., 1968). Figure 6 showsthe upfield region of the spectrum, which was accumulated for six hours. The inserts show expanded versions of the pair of peaks between $3 and +4 p.p.m., labeled R and S, and the peak near +9 p.p.m. Integration: shows that areas are in the ratio 3 :3: 1. Furthermore, each peak of the pair has the samearea as the low-field methyl peak near -27 p.p.m.
4. Discussion The resonanceposition AvObS.can be described by Avcms.
=
A”,,,,
+
Avac.
+
A+.
+
A+.,.
(1)
where Av,,,, . is the position at which resonance would occur in a diamagnetic compound in the absenceof ring current shifts, Av, ,c. is the ring current shift introduced by proximity to the porphyrin ring, Av,. is the contact shift and Av, .p. is the pseudocontact shift. A semi-empirical description of porphyrin ring current shifts, based upon measurementsmade in diamagnetic model systemswith calculable geometries, is now in the press (Shulman, Wiithrich, Yamane & Blumberg, 1970). Contact shifts, Ave., arise when unpaired spin is delocalized from the iron through a sequenceof covalent bonds and has a finite probability of being found in the atomic orbital of the hydrogen atom in question (Eaton & Phillips, 1965; McConnell & Chesnut,, 1958). Unpaired spin in a 1s hydrogen atomic orbital produces at the proton an isotropic magnetic field which is invarient during molecular rotation. This field adds to or subtracts from the external magnetic field, depending upon the direction of polarization of the unpaired spin in the Is orbital, and shifts the resonance to lower or to higher magnetic fields. It has been shown that in some circumstances (Eaton & Phillips, 1965; McConnell & Chesnut, 1958) contact shifts can be used to determine how much of the unpaired spin is delocalized to t’he hydrogen atom and to it,s adjacent carbon atom. (a) Pseudocontactshifts In order to determine the contact shift Av,. from vObS.it is necessary to evaluate all the other terms on the right hand side of equation (1). While Avdlam. and Av,,,. present no difficulties, evaluation of Av,,,. has been hindered by ambiguities which we shall try to resolve here. Electrons in u bonds are localized and in consequencea u bond attenuates unpaired electron spin density by about an order of magnitude, but in n-bonded systems the electrons are more free and there is no strong attenuation with distance. Unpaired electrons can therefore be delocalized to the periphery of the porphin ring of MbCN, and also to the aromatic ring of the proximal histidine ligand. Appreciable contact interactions can then occur with protons directly linked, or contained in groups directly linked, to r-bonding atoms of the porphyrin and the proximal histidine ring, i.e. meso, vinyl and methyl protons may have appreciable contact interactions as may the protons of the propionate methylene groups attached to the porphyrin skeleton and the C,H, C,H, N,H and ,!lCH, protons of the proximal histidine ligand
42
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAN
However, contact interactions with t’he more remote propionate methglene groups should be small. An experimental estimate of the magnitude of pseudocontact shifts was sought (Wiithrich, Shulman, Wyluda & Caughey, 1969) in the methyl peaks of heme methyl esters dissolved in pyridine-water mixtures containing an excess of potassium cyanide. Contact shifts of the ester methyl peaks were expected to be negligible, and since the total paramagnetic shift, Av,. + Av~,~,, was found to be close t’o zero, the pseudocontact contribution had also to be close to zero. From this result it was inferred that pseudocontact shifts could be neglected at all positions in hemes and heme proteins. This neglect is not justified in MbCN, however, for the following reasons. Figure 6 contains a pair of peaks between + 3 and + 4 p.p.m. which have temperaturedependent shifts, indicating that they are shifted by the unpaired electrons (Wiithrich et al., 1968). These peaks, together with the pair of peaks near +1.3 p.p.m., have been assigned in the past to the four methylene groups of the heme propionic acid side-chains, on the presumption that their areas correspond to two protons each, but as explained in the experimental section their integrated intensities correspond to three protons and they are both methyl peaks (see Fig. 6). Paramagnetically shifted methyl peaks are also observed downfield, and since contact interactions alone will not account for the paramagnetic displacement of more than four methyl group resonances, pseudocontact interactions have to be invoked. Furthermore, as is shown below, it is extremely difficult to account for more than two paramagnetically shifted exchangeable peaks unless pseudocontact interactions are appreciable. Indeed, these observations on MbCN prompted a re-examination of paramagnetic shifts in free hemes, and it was found that inclusion of pseudocontact terms helped the spectral interpretation (Shulman, Glarum & Karplus, 1970). In fact pseudocontact interactions were used by Hill, Mann & Williams (1967) to explain paramagnetic shifts in molecular complexes of 1,3,5trinitrobenzene with cobalt(H) mesoporphyrin IX dimethyl ester. The expression for a pseudocontact shift, where the g-factors gZ, gy and g, are unequal and where the rotation time is long (Jesson, 1967 ; Kurland & McGarvey, 1970), is Av,,,. = 106C{[g2 - fr(g” + gz)] (1- 3 cos2Q) + s(gi - gz) sin2.Q cos ZY} (2) in which Q is the polar angle between the iron-proton vector and the z-direction. and Y is the angle between its projection in the xy plane and the x axis. With the (2), factor IO6 included, Av,.,. is expressed in p.p.m. In equation
(3) where /I is the Bohr magneton, S is the total electron spin, k is the Boltzmann constant, T is the absolute temperature, R is the distance of the proton from the centre of the iron, and K is the fraction of the unpaired spin on the iron atom. The g-factors in powdered samples of cyanoferrihemoglobin (HbCN) have been measured in electron spin resonance experiments (Blumberg & Peisach, personal communication) as 3.4, 1.89 and 0.74 at 1*4”C, and MbCN g-factors are presumably very similar. Preliminary electron spin resonance measurements on single crystals of MbCN have shown that the largest g-factor is 3.4 and that the principal g-axis is perpendicular to the heme plane (J. C. Hensel t R. G. Shulman, unpublished observations). The directions of the g, and g, axes in the heme plane are unknown, but, ~Ivr,~, can st,ill IF calcnlat~ccl
NMR
STUDY
OF
reasonably well for protons located shown below. Equation (2) can be written A+.,.
CYANOE’ERltlMYOGLOBIN
near
the heme
= lO%‘{P
43
normal
through
the iron,
+ Q COY2Y]
as is
(4)
where, for given g-values, P and Q depend only on the azimuthal angle Sz. With s;! <54”44’ (i.e., negative P) the error from neglecting the Y-dependent term would be maximum if cos2Y were 1.0, and the fractional error would then be Q/(P +Q). With the g-values given above, the error is less than 1 y0 if the proton lies within a 12” cone centred on the z-axis. This rises to a maximum possible error of 3.5% for a 20” cone and to 10~5”/~ for a 30” cone (Fig. 7). Neglect of the Q COH2Y term and substitu-
15
LO
25
30
Q (deg.) FIG. 7. The maximum possible error (4)) in calculations of the pseudocontact
tion
of numerical
values
introduced by neglecting the Y-dependent term shift, dv p c., plotted as a function of the azimuthal
for the physical
A VP.C. =
049
constants
x 106h’ TB3
in C reduces
equation
(equation angle Q.
(2) to
(1 - 3 co&?)
where R is expressed in Angstrom units. K is taken to be 0.75 (Shulman, Glarum & Karplus, 1970). It should be noted that when prot,ons are near the z-axis, making 52 small, A+,,, is negative and the pseudocontact shift is to low field. (b) Exchangeable
protons
A visual inspection of a model of ferrimyoglobin built according to Kendrew & Watson’s co-ordinates (personal communication) reveals six exchangeable protons in positions where their resonance could experience substantial contact or pseudocontact shifts to low field. They are the N,H protons of His FG2 and of His E7, which Bretscher (1968) has suggested might be hydrogen bonded to the nitrogen atom of the cyanide ligand, the N,H proton of His F8, the OH proton of Ser F7 and the peptide NH protons of His F8 and Ala F9. The positions of these protons have been calculated from Kendrew & Watson’s co-ordinates for ferrimyoglobin, and the corresponding pseudocontact and ring-current shifts have been evaluated. Ring-current shifts were estimated from a curve showing the variation of A ysc, with the position of a proton
44
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULXIAN
relative to the heme (Shulman, Wiithrich, Yamane & Blumberg, 1970). For serinc, the position of the OH oxygen atom was used. Table 1 shows the calculated shifts for all but the N,H proton of His FGZ, which is difficult to evaluate because Q is about 55”. Suffice it to say that Av,.,, is unlikely to have a negative value greater than -3 p.p.m. for this proton. Of the six exchangeable protons listed in Table 1, only two, N,H of His E7 and N,H of His F8, can have non-vanishing values of Av,. One, and perhaps both, of these are connected to the iron by a series of bonds through which unpaired spin can be delocalized. TABLE
Amino
His His His Sor His Ala
E7 F8 FG2 F7 F8 F9
acid
Proton
N,H NS,H N,H OH Peptide Peptide
w-4
NH NH
Q (d%.)
4.2
22
5.1
17
5.6
60 28
5.8 6.9 8.8
10 11
1
A W.C. @p.m.) at 300°K - 23 - 16 smalit - 8.5 - 7.1 - 3.4
A R.C. (p.p.m.)
4.0
-
-: 3.0 -j 0.5 -1 2.5
-
-I
-I-
2.0
-:. 1.5
Est~inmtcd A%, (pp.“‘.)
A%,,, (p.p.m.)
- 32 t Av, -26 ,-Av, ?
13.43 13.41
- 13.4$ -3.75
-
-9.0/l - 9q
Estimated contributions to the shift for exchangeable protons near the iron atom cyanoferrimyoglobin. The z-direction is defined from the co-ordinates of the Fe N, atom of His F8. t Av,,,, is in the approximate range 12.5 p.p.m., giving an estimated Avobs of p.p.m. $ The shift for CDCl, solutions of histidine (Varian spectra catalog, no. 20). Effects bonding have not been included. § The shift for CDCI, solutions of the protected dipeptide tBOC-Ser.Ala-OBz personal communication). Ij Approximate shift for peptide NH protons (Glickson et al., 1969).
‘J.7
- 14.1 - 10.9 of sperm whale atom and the -10.4
t,o-15.4
from
hydrogen
(D.
Torchia,
At this point, some of the exchangeable peaks can be identified. Table 1 shows that only two peaks are able to be displaced to extremely low fields, although this can occur only if the contact terms Av, are small or negative. Experimentally two peaks are observed at very low field, and they can be distinguished in two ways. First, peak A broadens and finally disappears as the pH falls, whereas peak B shows no variation in width or position as the pH is changed. Presumably the distal histidine, His E7, must titrate at some point, but the proximal histidine, His F8, need not necessarily do so. Second, if only one of the two protons maintains a fixed position and a fixed paramagnetic shift among myoglobins from various species (Fig. 5), it is more likely to be the proximal His F8 N,H proton than the His E7 N,H proton. The assignments, therefore, are that peak A is from the distal histidine N,H proton, and peak B is from the proximal histidine N,H proton. Since the proximal histidine N,H peak is calculated (Table 1) to appear at -26 + Avc p.p.m., the contact shift Av, is of the order of + 5 p.p.m. Any error in calculating A,.,. (-16 p.p.m.) is exaggerated-in this estimate of Avc, but if K and the g-factors are as quoted it seems reasonable to say that Av, is small and positive. A contact shift of +5 p.p.m. represents an unpaired spin density in the N, r orbital of about +0.2%. Similarly, Av, is calculated to be + 9 p.p.m. for the His E7 proton. The uncertainties in this value are probably larger than for the proximal histidine because the co-ordinat’es of this proton, assumed
NMR
STUDY
OF
CYRNOPERRINYOGLOBIN
45
t’o be 1.8 A away from the cyanide nitrogen at an angle of 60” to the Fe-C-N axis, are less certain. In fact it is not firmly established that the CN axis is t,ruly perpendicular to the heme plane (Bretscher, 1968). However, even if the proton is assumed to be at D =O, R = 5.0 L%the value of Av, is still +6 p.p.m. A value of d yc # 0 would confirm Bretscher’s contention (Bretscher, 1968) that His E7 is hydrogen-bonded to the cyanide ligancl. The paramagnetically shifted peak near -14 p.p.m. in Figures 1 and 4, peak C, is assigned to the peptide NH proton of His F8. This assignment is supported as follows. The shift is so strongly temperature dependent that extrapolation to l/T = 0 gives a shift of -1.7 p.p.m. This alone is probably enough to eliminate consideration of the His FG2 N,H proton, but it does necessitate consideration of the F7 Ser OH proton (Table 1). However such an assignment would require dv, ,c. for the OH proton to be about 40% larger than calculated while at the same time A+.,. for the His F8 peptide NH proton would have to be at least 40% snudler than calculated. since no other exchangeable peak occurs below -11 p.p.m. An assignment to the proximal histidine peptide NH proton is strongly supported by the presence at 300°K of a similar peak at -13.2 p.p.m. in the spectra of human hemoglobin K-subunits (S. Ogawa, personal communication), in which both serine F7 and histicline FG2 arc replaced by leucine. As expected for a His F8 pepticle NH assignment, both horse MbCN and porpoise MbCN spectra contain an extra peak near -14 p.p.m. (Fig. 5). which is present below pH 6. For horse MbCN at least (we have not yet checked porpoise MbCN), its shift has the anticipated strong dependence on temperature. As mentioned above, the peak assigned to the His E7 N,H proton, peak A, broadens below pH 7. This lifetime broadening is probably associated with rapid protonation and deprotonation at the other nitrogen atom of the imidazole ring. If the deprotonation is base catalyzed, as for imidazole in free solution (Eigen, 1964), the best conditions for observing narrow peaks from protonated distal histidine rings are 10~ pH and low temperature. Nevertheless, a careful search at pH 5 and 5°C revealed no additional peaks. The rate of protonation, calculated form the peak broadening as follows, is consistent with values in other proteins, and with the known rate of protonation of imidazole in free solution. Allowing 35 Hz for the &polar width of the NH peaks, the observed width of 235 Hz at pH 6.81 gives a lifetime 7 of 1.6 msec at 28°C for the unprotonatecl species. A rat,e constant, k,, defined in equation (6), can be evaluated as follows: MbCN + H+ 2 MbCNH+. (fj) ka At any instant the rate of disappearance of unprotonated moleculesMbCN which we already present is given by - d[MbCN] = k,[MbCN] [H+] , (7)
at
By definition 1 7
d[MbCN]
at
1 ‘-.-.--* [MbCN]
(8)
Hence
Since 7 is I.6 msec at pH 6.81, k, = 4
x
log lit’res mole-Isec-I.
Such large rat,e
4ti
U.
SHEASI),
T.
YAMAh-E:
AlYL)
It.
G.
SHULMAN
constants are not unexpected in proteins; in fact the fear was that k, might be even larger and so prevent observation of the peak. In ribonuclease, for example, each of four histidine C&H peaks moves about 100 Hz at IO0 MHz as the histidine residues titrate (Meadows, Markley, Cohen & Jardetzky, 1967) and for each peak the system is in a fast exchange region. At the midpoint of the titration, the lifetime of the unprotonated species is Q (100 n)-l set, and for a pK, of 6.7 this means that k, 9 1.6 x lo9 litres mole-’ at 28°C. For imidazole in free solution at zero ionic strength and 25”C, k, = 15 x lOlo litres mole-l set-l (Eigcn, 1964). These rates agree rather well with the value of k, = 5 x lo9 litres mole-l see-l observed for peak A and support the assignment proposed. Another exchangeable peak, peak D, is observed to broaden at low pH. Its position varies little with temperature (Fig. 2). A peak occurs in the same position in diamagnetic oxymyoglobin (Patel, personal communication), and since the position is the same, one can infer that in MbCN the pseudocontact shift is negligible. The peak width has an extraordinarily large temperature dependence in the range 25 to 35°C. The peak was not detected above about 35”C, even though its width at 25°C was only about 30 Hz. A similar peak occurs in the spectrum of horse MbCN at low temperature but this peak has already disappeared at 28°C the temperature of Figure 5. The fact that rapid exchange can eliminate the peak means that an assignment to a tryptophan NH proton is untenable (Glickson, McDonald & Phillips, 1969), and in any case the peak occurs further to low field than would be expected. Imidazole in CDCl, solution has an NH peak at -13.4 p.p.m. (Varian spectra catalog no. 20) but in aqueous solution at pH 7 the proton residence time is too short to allow observation of an NH absorption peak. For the same reason, histidine residues on the surface of a protein, or in internal positions easily accessible to water molecules, will not give separately observable NH peaks. If a histidine residue is buried in the protein, on the other hand, in such a way as to increase the proton residence time to 1O-2 seconds or more, an NH peak is expected in the vicinity of -13.4 p.p.m. Of the 12 histidine residues in myoglobin, three have NH protons close to the iron (E7, F8 and FG2), and these can be eliminated as explained above because in cyanoferrimyoglobin the peak in question has almost no pseudocontact shift. All the other histidine residues are on the surface of the molecule, having their NH protons easily accessible to the solvent, with the single exception of His B5, which is buried inside the protein in a hydrophobic pocket provided by Val A5, Be G12, Val G15 and Leu G16. The NH proton is about 18 A from the iron, and meets t’he requirement that it should bc beyond the range of significant pseudocontact interactions. A good assignment of peak D would therefore be to the NH proton of His B5. In support of this assignment, it has been observed that corresponding spectra from human hemoglobin u-chains do not contain such a peak (S. Ogawa, personal communication), which is to be expected since His B5 is replaced by tyrosine. The unusual temperature dependence of the peak width would then be understood as a change in the accessibility of water to that interior region of the protein. Pate1 suggests, however, that since two of the surface histidines, His CD6 and His EF5, appear to be hydrogen bonded to nearby residues, which could slow their rates of proton exchange, one of these might be a better assignment. At present these alternatives can not be distinguished unambiguously. Little can be said about the remaining exchangeable peaks plotted near -11 p.p.m, and -10 p.p.m. in Figure 2. There are two tryptophan residues in the mole-
NMR
STUDY
OF
CYANOFERRIMYOGLOBIN
4i
cule which ought to produce NH peaks in this region (Glickson et al., 1969), and in Table 1 a prediction is made that the peptide NH peak at Ala F9 ought also to be present. Presumably none of these peaks is the His FG2 NBH peak, since it would have to move downfield with rising temperature towards a position of about -12.9 p.p.m. at at l/T = 0. This proton must either exchange rapidly, or its shift must have an upfield pseudocontact contribution, taking the peak above -10 p.p.m. and beyond observat*ion.
(c) Non-exchungeahleprotom We turn now to peaks from non-exchangeable protons, shown in Figure 2 by open circles and observed earlier in deuterium oxide solutions (Wiithrich et al., 1968). No methyl group is in a position where pseudocontact interactions could displace its resonance as low as -18 p.p.m., and therefore the assignment of the two methyl peaks below -18 p.p.m. to porphyrin methyl protons is almost certainly correct. Porphyrin methyl peaks are expected to have pseudocontact shifts in the range +2.6 to +7*4 p.p.m., depending on the orientation of the in-plane g-tensor axes, and since the total par&magnetic shift of Av,. + A+,,. is much greater than this, the major contribution is from the contact interact,ion. Of the protein methyl groups which are in positions suitable for large pseudocontact shifts, only one methyl group, one of the methyl groups of Val El 1, is a likely assignment for a lotield methyl peak. The centre of rotation of its hydrogen atoms is 4.8 A from the iron, with Q = 25”. A pseudocontact shift Av,,,, of about -15 p.p.m. can be calculated which, together with a ring current shift A+,,-., of +3*5 p.p.m. and an expected Avai,,. of -1 p.p.m. in the absence of these additional interactions (Av,. should be negligible), leads to a predicted Av,,,, of about -12.5 p.p.m. At least one, and possibly, two methyl groups produce peaks in this region, which have been assigned to porphyrin methyl groups (Wiithrich et al., 1968). At present there is not sufficient evidence for confident assignments either to porphyrin methyl groups or to the methyl group of Val Ell. The remaining peaks observed at low field in deuterium oxide solutions were assigned to proximal histidine C,H and C,H protons, and to vinyl protons (Shulman, Wiithrich, Yamane, Antonini & Brunori, 1969). It is certainly likely that one of t)he peaks at either -17 p.p.m. or -18 p.p.m. in Figure 1, and one of the peaks near -11 p.p.m., are vinyl peaks, since two peaks are absent in the spectrum of cyanoferrimyoglobin reconstituted with deuteroporph-yrin, in which the vinyl groups are replaced by h,ydrogen atoms (Shulman, Wiithrich, Yamane, Antonini & Brunori: 1969). However, in addition to the possibility that the C&H and C,H protons of the proximal histidine ring could produce peaks in this range, pseudocontact interactions could displace peaks into this region from the #KHz protons of the proximal histidine, especially with assistance from a contact interaction, from the ortho or meta protons of Phe CD1 or from the C,H proton of His FG2. It is possible also that peaks from propionic acid CH, groups adjacent to the porphyrin ring could be contact shifted into t,his region. The fact that the non-exchangeable peak moving towards -11 p.p.m. at, low pH (Fig. 3) has an appreciably pH-dependent shift, may be an indication that this peak is pseudocontact shifted, since pseudocontact, shifts are expected t,o be extremely sensitive to small movements within the protein. In the upfield region (Fig. 6), we restate that peaks R and S are not propionic acid methylene peaks, since their areas require assignments to methyl groups. This appears to be true also of peaks I’ and Q. whir11 wf’rc similarly axsigned to propionic
48
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAN
acid CH, protons (Wiithrich et al., 1968). There are several amino acid CH, groups near the iron which could account for these peaks, but it will be more profitable to discuss their assignments when the directions of the in-plane g-tensors are known. We wish
to thank
Dr
Seiji
Ogawa
for
many
interesting
and
useful
discussions.
REFERENCES Bretscher, P. A. (1968). Thesis, Cambridge University, England. Eaton, D. R. & Phillips, W. D. (1965). Advance8 in Magnetic Resonance, ed. by J. S. Waugh, vol. 1, p. 103. New York: Academic Press. Eigen, M. (1964). Angewandte Chemie, Intern. Edit., 3, 1. Glickson, J. D., McDonald, C. C. & Phillips, W. D. (1969). Rio&em. Biophys. Res. Comm. 35, 492. Hapner, K. D., Bradshaw, R. A., Hartzell, C. R. & Gurd, F. R. N. (1968). J. B&Z. Chem. 243, 683. Hill, H. A. O., Mann, B. E. & Williams, R. J. P. (1967). Cltem. Comm. p. 906 Hugh, T. E. (1968). Ph.D. Thesis, Indiana University. Jesson, J. P. (1967). J. Chem. Phys. 47, 579, Kowalsky, A. (1965). Biochemistry, 4, 2382. Kurland, R. J., Davis, D. G. & Ho, C. (1968). J. Amer. Chem. Sot. 90, 2700. Kurland, R. J. & McGarvey, B. R. (1970). J. Maq. Res. 2, 286. McConnell, H. M. & Chesnut, D. B. (1958). J. Chem. Phys. 28, 107. McConnell, H. M. & Robertson, R. E. (1958). J. Chem. Phys. 29, 1361. Meadows, D. H., Markley, J. L., Cohen, J. S. & Jardetzky, 0. (1967). Proc. Nut. Acnd. Sci., Wash. 58, 1307. Shulman, R. G., Glarum, S. H. & Karplus, M. (1970). J. Mol. Biol. in the press. Shulman, R. G., Ogawa, S., Wiithrich, K., Yamane, T., Peisach, J. & Blumberg, W. E. (1969). Science, 165, 251. Shulman, R. G., Wiithrich, K., Yamane, T., Antonini, E. & Brunori, M. (1969). Proc. Nat. Acad. Sci., Wash. 63, 623. Shulman, R. G., Wtithrich, K., Yamane, T., Patel, J. & Blumberg, W. E. (1970). J. Mol. Biol. 53,143. Wiithrich, K., Shulman, R. G. & Peisach, J. (1968). Proc. Nat. Acad. Sci., Wash. 60, 373. Wiithrich, K., Shulman, R. G., Wyluda, B. J. & Caughoy, W. S. (1969). Proc. Nat. Acad. Sci., Wash. 62, 636. Wiithrich, K., Shulman, R. G., Yamane, T., Wyluda, B. J., Hugli, T. & Gurd, F. R. N. (1970). J. Biol. Chem. in the press. Yamane, T., Wiithrich, K., Shulman, R. G. & Ogawa, S. (1970). J. Mol. Biol. in the press
Nuclear Magnetic Resonance Study of Cyanoferrimyoglobin Identification of Pseudocontact Shifts B. SHEARD, T. YAMANEAND
R.G.
;
SHULMAN
Bell TelephoneLaboratories, Incorporated Murray Hill, N.J. 07974, U.S.A. (Received 18 Mwy 1970) The high-resolution proton magnetic resonance spectrum of sperm whale cyanoferrimyoglobin in solution in water was measured at 220 MHz, and it was compared with the spectrum in deuterium oxide. Exchangeable protons gave three peaks with strongly temperature-dependent shifts in the region - 13 to -24 p.p.m., measured downfield from 2:2-dimethyl-2-silapentane-5-sulphonate. They were shifted by interactions with the unpaired electrons. Four other exchangeable peaks, which had shifts with much smaller temperature dependences, were observed in the region - 10 to - 14 p.p.m. In the upfield region, four non-exchangeable peaks which had been thought to have areas corresponding to two protons each, and which had been assigned to methylene protons of the heme propionate sidechains, have been found to have areas corresponding to three protons, and they must be aasigned to methyl groups. Since they are pammagnetically shifted, as are other methyl peaks at low field, there are too many paramagnetically shifted methyl peaks in the spectrum to be accounted for by contact interactions alone. This means that pseudocontact interactions, which do not depend on spin delocaliz&ion through covalent bonds, must be appreciable. Estimates of pseudocontact shifts allow assignments to be made for the three paramagnetically shifted exchangeable peaks, namely, the protons NBH of His E7, N,H of His F8 and the peptide NH of His F8. Since pseudocontact shifts have been found to be appreciable, some of the peak assignments made earlier for non-exchangeable peaks are now uncertain.
1. Introduction In heme proteins, interactions with unpaired electrons from t’he iron atom produce peaks in the high-resolution proton magnetic resonance spectra which are well outside the usual 0 to -10 p.p.mt range of absorption (Kowalsky, 1965; Wiithrich, Shulman & Peisach, 1968; Kurland, Davis & Ho, 1968). Their positions have been used to compare myoglobins (Wiithrich et al., 1970) and hemoglobins (Yamane, Wiithrich, Shulman & Ogawa, 1970) from various species, and to investigate the heme environment in mixed state hemoglobin in studies of the co-operativity of oxygenation (Shulman, Ogawa, Wiithrich, Yamane, Peisach & Blumberg, 1969). By working with solutions of cyanoferrimyoglobin in waterj: it was hoped that an extra peak would be obtained from the proximal histidine NH proton, which could be unambiguously identified, and could then give information about ligand binding. In fact, we have observed several additional para,magnetically shifted peaks at 10~ 7 All shifts are given in parts per million relative to an internal 2.silapentane-5-sulphonate. Low-field shifts are negative. $ H,O is referred to as “water”; D,O as “deuterium oxide”. 33
st,andard
of sodium
2:2-dimethyl-
B.
36
SkIEARl),
T.
YAMANE
AND
Ib.
G.
SHULMAN
field in water solutions. During the course of this work it became clear that pseudocontact interactions (McConnell & Robertson, 1958; Jesson, 1967) are appreciable and that they are particularly important for explaining the shifts of the extra peaks observed in water. We present here experimental evidence for pseudocontact interactions in cyanoferrimyoglobin and calculate some of the pseudocontact shifts.
2. Experimental Sperm whale and porpoise myoglobin were isolated by a combination of the methods of Hapner, Bradshaw, Hartzell & Gurd (1968) and of Hugli (1968). Homo myoglobin was bought from Calbiochem. The pH was adjusted by dialysis under reduced pressure against which was: 0.1 M-sodium acetate below pH 5.8; 0.1 Mthe appropriate buffer solution, sodium phosphate in the pH range 5.9 to 8.0; 0.1 M-sodium borate above pH 8.0. Concentrations were in the range 10 to 15%. The cyanide derivative was obtained by adding an excess of potassium cyanide in the appropriate buffer. The pH quoted is the pH of tho NMRt sample itself, measured at 25 deg. C+ 2 deg. C. The spectra were obtained with a Ovarian HR220 NMR spectrometer, cquippod with a Fabritok 1062 signal-averaging computer. An RF field of 1 milligauss was used, with a sweep rate of 20 Hz sec. - 1 Probe temperatures were measured with a sample of ethylene glycol and the regulation was better than rf: 1 deg. C.
3. Results The original idea was that if the residence time of t’he hydrogen atom nitrogen-l of the proximal histidine residue were long enough, interactions
bound to with the
H2O
LLI~L~II.I~L~I_I..I._L~
-20
-26
-24
-22
-20
Shift (ppm)
-10
-16
from
vi -14
-10
DSS
FIG. 1. A comparison of 220 MHz spectra of sperm whale MbCN in H,O solutions at various values of pH. The spect,rum at pD 7.00 in D,O solutions is also shown. All samples contained 0.1 M-sodium phosphate buffer. The temperature was 29°C. DSS, 2:2-dimethyl-2.silapentane-tisulphonate.
t Abbreviations
used:
NMR,
nuclear
magnetic
resonance;
MbCN,
cyanoferrimyoglobin.
Slll1-L
STUl)Y
OF
3;
CYANO~‘ERRIhlY’oC;I,O~IN
unpaired electrons would shift its NMR peak outside the usual range of absorption, where it could be observed in solutions in HzO, but not in D20. Following this rationale a paramagnetically shifted exchangeable peak had been found in the spectrum of ferricytochrome c (Sheard, results to be published). In sperm whale MbCN, however. four such peaks (labeled A, B, C and D) were found between -12 and -28 p.p.m. (Fig. l), two of which are superimposed near -14 p.p.m. at high temperatures, as shown below. Peaks A, B and C have shifts which vary strongly and linearly with the inverse of the absolute temperature (Fig. 2), which identifies them as being shifted
-6200
/ ‘H,
-6000
-14
-26 -3000
vr
-24
! ’i j/l -2900 I
-23
-2800
-25
-5400 -5200
-27 -3100.
-5800 -5600
-28
A I
I'
CH,
/,,.,Z, /
,,/
-13
/ ,/
E a .!? u c -12 5
-22 -21 -20 -19
-II
-18 -17 -10 -16 5 1/Tx103
32
33
34 VTxlO'
35
5
FIG. 2. Shifts, at pH 7.0, of low-field peaks in the 220 MHz spectrum of sperm whale MbCN, plotted against the reciprocal of the absolute temperature, 1,/T. Note t,he change of frequency scale between the two parts of the Figure. (0) Peaks observed only in H,O; (0) peaks observed also in I>,O.
by the unpaired electron (Wiithrich et al., 1968). Another four “exchangeable peaks” were observed in H,O between - 9.8 and -11.3 p.p.m., but their positions are considerably less temperature dependent. The pH was varied at a constant temperature, 28*5”C, with the results shown in Figure 3. Consider first the peaks A and B. Where the peaks are observable, their positions are independent of pH, except below pH 5, as the point of denaturation is approached. Peak 9 broadens without changing its position as the pH is lowered, and it disappears
38
B.
SHEARD,
T.
-6000.
YAMANE
ANU
1%. G.
-3000-
fl4
-2900-
' o"
SHULMAN
,--27
-5800 --26 -5600 --25 -5400 --24 -2800..
l__lc-% 0 0 %
-2 --22
: -4800 i Lo -4600
0
ii
-2700-
I -4400-
--20
-420000 -4ooo_
0
oo
0 0
a'
W
--I9 -2400 e
1-18 -2300;.
PH FIG. 3. Shifts, at 28.5’C, in the 220 MHz spectrum of sperm whale MbCN, plotted as a function of pH. Buffers are described under Experimental. Note the change of frequency scale between the two parts of the Figure. (0) Peaks observed only in HaO; ( 0) peaks observed in both Hz0 and I&O. Peaks assigned to methyl groups have been marked accordingly.
below pH 7. This is an exchange phenomenon in which the peak suffers lifetime broadening, and as expected the pectk becomes even broader as the temperature rises and exchange becomes faster, in contrast with the other peaks in the spectrum which display the usual narrowing. Although the pK, for this proton&ion is unknown it must be less than 7 since at pH 7 the peak area is still nearly unity. If we take the simple view that the peak observed is from an exchangeable proton in a residue which itself is rapidly gaining and losing a second labile proton, the residue has to be histidine. In an attempt to observe peaks from the protonated form of the histidine a search w&s made at pH 5 and 5”C, where the exchange should be slower, but no extra peaks were observed between -40 and -12 p.p.m. or between +3 and +32 p.p.m. The peak at -13.8 p.p.m. in Figure 1 has an area for two protons at high pH and for one proton at low pH. The explanation is as follows. At 29”C, the temperature of Figure 1, the two exchangeable peaks C and D are superimposed (see Fig. 2). As the pH is lowered below pH7, peak D broadens and disappears, leaving only
NMR
STUDY
30
OF CYANOFERRIMYOGLOBIN
pH 60
PH 7.0
pH 8.0
1 - IS
I
I -I4
I
I -13
I
I -12
Shift (p.p,m) from DSS
FIG. 4. 220 MHz spectra, at 19”C, of sperm whale MbCN in Ha0 with 0.1 M-sodium phosphate buffer. This shows that the peak near - 14 p.p.m. in Fig. 1 is composed of two peaks above pH 7, one of which disappears at lower pH. DSS, 2:2-dimet’hyl-2-silapentane-5sulphonate.
I
-2k%%?l-22
I
I
-20
I
I
-I8
I
I
-16
Shift (p.p.m.1 from
!
I
-14
!
I
-12
z-u
-10
DSS
Fro. 5, 220 MHz spectra, at pH 8.2 and 29°C of sperm whale, horse and porpoise H20, wit,h O-1 M-sodium phosphate buffer. DSS, 2:2-dimethyl-2-silapentane-5sulphonate.
MbCN in
40
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAS
peak C. This can be seen directly at lower temperatures, where the two peaks arc caused to separate (Fig. 4) by the increased paramagnetic shift in peak C. It turns out that the width of peak D increases so rapidly with temperature that even at pH 8 it is too broad to be observed separately at temperatures above the crossover region. The effect of varying the amino acid composition was observed by comparing myoglobins from various species. Figure 5 shows low-field NMR spectra of the cyanide derivatives of sperm whale, porpoise and horse ferrimyoglobins, at 29°C and pH 8.2. The position of peak B is identical, within experimental error, for all three species, while the broad peak, peak A, varies in position among the species. Both horse MbCN and porpoise MbCN have an extra peak near -13.8 p.p.m. but, in contrast to sperm whale MbCN, these have one-proton areas under the conditions of Figure 5. ‘The peak observed is peak C. The positions of the other low-field peaks are shown as a function of pH in Figure 3. Two of the exchangeable peaks which could be clearly followed as a function of temperature, and which converge near -11 p.p.m. at the low-temperature end of Figure 2, are not plotted in Figure 3 because it proved difficult to identify them with certainty over the whole pH range. The other exchangeable peaks in that region, near -11-O and --lo*0 p.p.m. in Figure 3, have little or no pH dependence. Of the peaks observed previously in D,O solution at pD 7-O (Wtithrich et al., 1968) the methyl peak at lowest field shows a large upfield displacement at low pH, with a point of inflection near pH 5.6, and another methyl peak near -13 p.p.m. drifts downfield almost linearly as the pH falls. The other peaks in Figure 3 have little or no dependence on pH, except the peak moving between -115 and -11.0 p.p.m., thought by Shulman, Wiithrich, Yamane, Antonini & Brunori (1969) to be attributable probably to the proximal histidine residue, and the peaknear -12.4p.p.m., which
*\
Fig. 6. 220 MHz upfield spectrum of sperm whale cyanoferrimyoglobin at pH 8.5 and 30°C in H,O solution with 0.1 M-sodium phosphate buffer. In the inset, the peaks at +3.31 and +3.68 p.p.m., and the peak at +9*10 p.p.m., are shown with a 2.5.fold horizontal magnification. Their vertical enlargements are all equal. DSS, 2 :2-dimethyl-2-silapentane-B-sulphonate.
NMR
STUDY
OF
41
CYANOFERRIMYOGLOBIN
has an area corresponding to two protons and which has been thought to arise, along with its nearest upfield neighbor, from resonance of a porphyrin methyl group (Wiithrich et al., 1968). Figure 6 showsthe upfield region of the spectrum, which was accumulated for six hours. The inserts show expanded versions of the pair of peaks between $3 and +4 p.p.m., labeled R and S, and the peak near +9 p.p.m. Integration: shows that areas are in the ratio 3 :3: 1. Furthermore, each peak of the pair has the samearea as the low-field methyl peak near -27 p.p.m.
4. Discussion The resonanceposition AvObS.can be described by Avcms.
=
A”,,,,
+
Avac.
+
A+.
+
A+.,.
(1)
where Av,,,, . is the position at which resonance would occur in a diamagnetic compound in the absenceof ring current shifts, Av, ,c. is the ring current shift introduced by proximity to the porphyrin ring, Av,. is the contact shift and Av, .p. is the pseudocontact shift. A semi-empirical description of porphyrin ring current shifts, based upon measurementsmade in diamagnetic model systemswith calculable geometries, is now in the press (Shulman, Wiithrich, Yamane & Blumberg, 1970). Contact shifts, Ave., arise when unpaired spin is delocalized from the iron through a sequenceof covalent bonds and has a finite probability of being found in the atomic orbital of the hydrogen atom in question (Eaton & Phillips, 1965; McConnell & Chesnut,, 1958). Unpaired spin in a 1s hydrogen atomic orbital produces at the proton an isotropic magnetic field which is invarient during molecular rotation. This field adds to or subtracts from the external magnetic field, depending upon the direction of polarization of the unpaired spin in the Is orbital, and shifts the resonance to lower or to higher magnetic fields. It has been shown that in some circumstances (Eaton & Phillips, 1965; McConnell & Chesnut, 1958) contact shifts can be used to determine how much of the unpaired spin is delocalized to t’he hydrogen atom and to it,s adjacent carbon atom. (a) Pseudocontactshifts In order to determine the contact shift Av,. from vObS.it is necessary to evaluate all the other terms on the right hand side of equation (1). While Avdlam. and Av,,,. present no difficulties, evaluation of Av,,,. has been hindered by ambiguities which we shall try to resolve here. Electrons in u bonds are localized and in consequencea u bond attenuates unpaired electron spin density by about an order of magnitude, but in n-bonded systems the electrons are more free and there is no strong attenuation with distance. Unpaired electrons can therefore be delocalized to the periphery of the porphin ring of MbCN, and also to the aromatic ring of the proximal histidine ligand. Appreciable contact interactions can then occur with protons directly linked, or contained in groups directly linked, to r-bonding atoms of the porphyrin and the proximal histidine ring, i.e. meso, vinyl and methyl protons may have appreciable contact interactions as may the protons of the propionate methylene groups attached to the porphyrin skeleton and the C,H, C,H, N,H and ,!lCH, protons of the proximal histidine ligand
42
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAN
However, contact interactions with t’he more remote propionate methglene groups should be small. An experimental estimate of the magnitude of pseudocontact shifts was sought (Wiithrich, Shulman, Wyluda & Caughey, 1969) in the methyl peaks of heme methyl esters dissolved in pyridine-water mixtures containing an excess of potassium cyanide. Contact shifts of the ester methyl peaks were expected to be negligible, and since the total paramagnetic shift, Av,. + Av~,~,, was found to be close t’o zero, the pseudocontact contribution had also to be close to zero. From this result it was inferred that pseudocontact shifts could be neglected at all positions in hemes and heme proteins. This neglect is not justified in MbCN, however, for the following reasons. Figure 6 contains a pair of peaks between + 3 and + 4 p.p.m. which have temperaturedependent shifts, indicating that they are shifted by the unpaired electrons (Wiithrich et al., 1968). These peaks, together with the pair of peaks near +1.3 p.p.m., have been assigned in the past to the four methylene groups of the heme propionic acid side-chains, on the presumption that their areas correspond to two protons each, but as explained in the experimental section their integrated intensities correspond to three protons and they are both methyl peaks (see Fig. 6). Paramagnetically shifted methyl peaks are also observed downfield, and since contact interactions alone will not account for the paramagnetic displacement of more than four methyl group resonances, pseudocontact interactions have to be invoked. Furthermore, as is shown below, it is extremely difficult to account for more than two paramagnetically shifted exchangeable peaks unless pseudocontact interactions are appreciable. Indeed, these observations on MbCN prompted a re-examination of paramagnetic shifts in free hemes, and it was found that inclusion of pseudocontact terms helped the spectral interpretation (Shulman, Glarum & Karplus, 1970). In fact pseudocontact interactions were used by Hill, Mann & Williams (1967) to explain paramagnetic shifts in molecular complexes of 1,3,5trinitrobenzene with cobalt(H) mesoporphyrin IX dimethyl ester. The expression for a pseudocontact shift, where the g-factors gZ, gy and g, are unequal and where the rotation time is long (Jesson, 1967 ; Kurland & McGarvey, 1970), is Av,,,. = 106C{[g2 - fr(g” + gz)] (1- 3 cos2Q) + s(gi - gz) sin2.Q cos ZY} (2) in which Q is the polar angle between the iron-proton vector and the z-direction. and Y is the angle between its projection in the xy plane and the x axis. With the (2), factor IO6 included, Av,.,. is expressed in p.p.m. In equation
(3) where /I is the Bohr magneton, S is the total electron spin, k is the Boltzmann constant, T is the absolute temperature, R is the distance of the proton from the centre of the iron, and K is the fraction of the unpaired spin on the iron atom. The g-factors in powdered samples of cyanoferrihemoglobin (HbCN) have been measured in electron spin resonance experiments (Blumberg & Peisach, personal communication) as 3.4, 1.89 and 0.74 at 1*4”C, and MbCN g-factors are presumably very similar. Preliminary electron spin resonance measurements on single crystals of MbCN have shown that the largest g-factor is 3.4 and that the principal g-axis is perpendicular to the heme plane (J. C. Hensel t R. G. Shulman, unpublished observations). The directions of the g, and g, axes in the heme plane are unknown, but, ~Ivr,~, can st,ill IF calcnlat~ccl
NMR
STUDY
OF
reasonably well for protons located shown below. Equation (2) can be written A+.,.
CYANOE’ERltlMYOGLOBIN
near
the heme
= lO%‘{P
43
normal
through
the iron,
+ Q COY2Y]
as is
(4)
where, for given g-values, P and Q depend only on the azimuthal angle Sz. With s;! <54”44’ (i.e., negative P) the error from neglecting the Y-dependent term would be maximum if cos2Y were 1.0, and the fractional error would then be Q/(P +Q). With the g-values given above, the error is less than 1 y0 if the proton lies within a 12” cone centred on the z-axis. This rises to a maximum possible error of 3.5% for a 20” cone and to 10~5”/~ for a 30” cone (Fig. 7). Neglect of the Q COH2Y term and substitu-
15
LO
25
30
Q (deg.) FIG. 7. The maximum possible error (4)) in calculations of the pseudocontact
tion
of numerical
values
introduced by neglecting the Y-dependent term shift, dv p c., plotted as a function of the azimuthal
for the physical
A VP.C. =
049
constants
x 106h’ TB3
in C reduces
equation
(equation angle Q.
(2) to
(1 - 3 co&?)
where R is expressed in Angstrom units. K is taken to be 0.75 (Shulman, Glarum & Karplus, 1970). It should be noted that when prot,ons are near the z-axis, making 52 small, A+,,, is negative and the pseudocontact shift is to low field. (b) Exchangeable
protons
A visual inspection of a model of ferrimyoglobin built according to Kendrew & Watson’s co-ordinates (personal communication) reveals six exchangeable protons in positions where their resonance could experience substantial contact or pseudocontact shifts to low field. They are the N,H protons of His FG2 and of His E7, which Bretscher (1968) has suggested might be hydrogen bonded to the nitrogen atom of the cyanide ligand, the N,H proton of His F8, the OH proton of Ser F7 and the peptide NH protons of His F8 and Ala F9. The positions of these protons have been calculated from Kendrew & Watson’s co-ordinates for ferrimyoglobin, and the corresponding pseudocontact and ring-current shifts have been evaluated. Ring-current shifts were estimated from a curve showing the variation of A ysc, with the position of a proton
44
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULXIAN
relative to the heme (Shulman, Wiithrich, Yamane & Blumberg, 1970). For serinc, the position of the OH oxygen atom was used. Table 1 shows the calculated shifts for all but the N,H proton of His FGZ, which is difficult to evaluate because Q is about 55”. Suffice it to say that Av,.,, is unlikely to have a negative value greater than -3 p.p.m. for this proton. Of the six exchangeable protons listed in Table 1, only two, N,H of His E7 and N,H of His F8, can have non-vanishing values of Av,. One, and perhaps both, of these are connected to the iron by a series of bonds through which unpaired spin can be delocalized. TABLE
Amino
His His His Sor His Ala
E7 F8 FG2 F7 F8 F9
acid
Proton
N,H NS,H N,H OH Peptide Peptide
w-4
NH NH
Q (d%.)
4.2
22
5.1
17
5.6
60 28
5.8 6.9 8.8
10 11
1
A W.C. @p.m.) at 300°K - 23 - 16 smalit - 8.5 - 7.1 - 3.4
A R.C. (p.p.m.)
4.0
-
-: 3.0 -j 0.5 -1 2.5
-
-I
-I-
2.0
-:. 1.5
Est~inmtcd A%, (pp.“‘.)
A%,,, (p.p.m.)
- 32 t Av, -26 ,-Av, ?
13.43 13.41
- 13.4$ -3.75
-
-9.0/l - 9q
Estimated contributions to the shift for exchangeable protons near the iron atom cyanoferrimyoglobin. The z-direction is defined from the co-ordinates of the Fe N, atom of His F8. t Av,,,, is in the approximate range 12.5 p.p.m., giving an estimated Avobs of p.p.m. $ The shift for CDCl, solutions of histidine (Varian spectra catalog, no. 20). Effects bonding have not been included. § The shift for CDCI, solutions of the protected dipeptide tBOC-Ser.Ala-OBz personal communication). Ij Approximate shift for peptide NH protons (Glickson et al., 1969).
‘J.7
- 14.1 - 10.9 of sperm whale atom and the -10.4
t,o-15.4
from
hydrogen
(D.
Torchia,
At this point, some of the exchangeable peaks can be identified. Table 1 shows that only two peaks are able to be displaced to extremely low fields, although this can occur only if the contact terms Av, are small or negative. Experimentally two peaks are observed at very low field, and they can be distinguished in two ways. First, peak A broadens and finally disappears as the pH falls, whereas peak B shows no variation in width or position as the pH is changed. Presumably the distal histidine, His E7, must titrate at some point, but the proximal histidine, His F8, need not necessarily do so. Second, if only one of the two protons maintains a fixed position and a fixed paramagnetic shift among myoglobins from various species (Fig. 5), it is more likely to be the proximal His F8 N,H proton than the His E7 N,H proton. The assignments, therefore, are that peak A is from the distal histidine N,H proton, and peak B is from the proximal histidine N,H proton. Since the proximal histidine N,H peak is calculated (Table 1) to appear at -26 + Avc p.p.m., the contact shift Av, is of the order of + 5 p.p.m. Any error in calculating A,.,. (-16 p.p.m.) is exaggerated-in this estimate of Avc, but if K and the g-factors are as quoted it seems reasonable to say that Av, is small and positive. A contact shift of +5 p.p.m. represents an unpaired spin density in the N, r orbital of about +0.2%. Similarly, Av, is calculated to be + 9 p.p.m. for the His E7 proton. The uncertainties in this value are probably larger than for the proximal histidine because the co-ordinat’es of this proton, assumed
NMR
STUDY
OF
CYRNOPERRINYOGLOBIN
45
t’o be 1.8 A away from the cyanide nitrogen at an angle of 60” to the Fe-C-N axis, are less certain. In fact it is not firmly established that the CN axis is t,ruly perpendicular to the heme plane (Bretscher, 1968). However, even if the proton is assumed to be at D =O, R = 5.0 L%the value of Av, is still +6 p.p.m. A value of d yc # 0 would confirm Bretscher’s contention (Bretscher, 1968) that His E7 is hydrogen-bonded to the cyanide ligancl. The paramagnetically shifted peak near -14 p.p.m. in Figures 1 and 4, peak C, is assigned to the peptide NH proton of His F8. This assignment is supported as follows. The shift is so strongly temperature dependent that extrapolation to l/T = 0 gives a shift of -1.7 p.p.m. This alone is probably enough to eliminate consideration of the His FG2 N,H proton, but it does necessitate consideration of the F7 Ser OH proton (Table 1). However such an assignment would require dv, ,c. for the OH proton to be about 40% larger than calculated while at the same time A+.,. for the His F8 peptide NH proton would have to be at least 40% snudler than calculated. since no other exchangeable peak occurs below -11 p.p.m. An assignment to the proximal histidine peptide NH proton is strongly supported by the presence at 300°K of a similar peak at -13.2 p.p.m. in the spectra of human hemoglobin K-subunits (S. Ogawa, personal communication), in which both serine F7 and histicline FG2 arc replaced by leucine. As expected for a His F8 pepticle NH assignment, both horse MbCN and porpoise MbCN spectra contain an extra peak near -14 p.p.m. (Fig. 5). which is present below pH 6. For horse MbCN at least (we have not yet checked porpoise MbCN), its shift has the anticipated strong dependence on temperature. As mentioned above, the peak assigned to the His E7 N,H proton, peak A, broadens below pH 7. This lifetime broadening is probably associated with rapid protonation and deprotonation at the other nitrogen atom of the imidazole ring. If the deprotonation is base catalyzed, as for imidazole in free solution (Eigen, 1964), the best conditions for observing narrow peaks from protonated distal histidine rings are 10~ pH and low temperature. Nevertheless, a careful search at pH 5 and 5°C revealed no additional peaks. The rate of protonation, calculated form the peak broadening as follows, is consistent with values in other proteins, and with the known rate of protonation of imidazole in free solution. Allowing 35 Hz for the &polar width of the NH peaks, the observed width of 235 Hz at pH 6.81 gives a lifetime 7 of 1.6 msec at 28°C for the unprotonatecl species. A rat,e constant, k,, defined in equation (6), can be evaluated as follows: MbCN + H+ 2 MbCNH+. (fj) ka At any instant the rate of disappearance of unprotonated moleculesMbCN which we already present is given by - d[MbCN] = k,[MbCN] [H+] , (7)
at
By definition 1 7
d[MbCN]
at
1 ‘-.-.--* [MbCN]
(8)
Hence
Since 7 is I.6 msec at pH 6.81, k, = 4
x
log lit’res mole-Isec-I.
Such large rat,e
4ti
U.
SHEASI),
T.
YAMAh-E:
AlYL)
It.
G.
SHULMAN
constants are not unexpected in proteins; in fact the fear was that k, might be even larger and so prevent observation of the peak. In ribonuclease, for example, each of four histidine C&H peaks moves about 100 Hz at IO0 MHz as the histidine residues titrate (Meadows, Markley, Cohen & Jardetzky, 1967) and for each peak the system is in a fast exchange region. At the midpoint of the titration, the lifetime of the unprotonated species is Q (100 n)-l set, and for a pK, of 6.7 this means that k, 9 1.6 x lo9 litres mole-’ at 28°C. For imidazole in free solution at zero ionic strength and 25”C, k, = 15 x lOlo litres mole-l set-l (Eigcn, 1964). These rates agree rather well with the value of k, = 5 x lo9 litres mole-l see-l observed for peak A and support the assignment proposed. Another exchangeable peak, peak D, is observed to broaden at low pH. Its position varies little with temperature (Fig. 2). A peak occurs in the same position in diamagnetic oxymyoglobin (Patel, personal communication), and since the position is the same, one can infer that in MbCN the pseudocontact shift is negligible. The peak width has an extraordinarily large temperature dependence in the range 25 to 35°C. The peak was not detected above about 35”C, even though its width at 25°C was only about 30 Hz. A similar peak occurs in the spectrum of horse MbCN at low temperature but this peak has already disappeared at 28°C the temperature of Figure 5. The fact that rapid exchange can eliminate the peak means that an assignment to a tryptophan NH proton is untenable (Glickson, McDonald & Phillips, 1969), and in any case the peak occurs further to low field than would be expected. Imidazole in CDCl, solution has an NH peak at -13.4 p.p.m. (Varian spectra catalog no. 20) but in aqueous solution at pH 7 the proton residence time is too short to allow observation of an NH absorption peak. For the same reason, histidine residues on the surface of a protein, or in internal positions easily accessible to water molecules, will not give separately observable NH peaks. If a histidine residue is buried in the protein, on the other hand, in such a way as to increase the proton residence time to 1O-2 seconds or more, an NH peak is expected in the vicinity of -13.4 p.p.m. Of the 12 histidine residues in myoglobin, three have NH protons close to the iron (E7, F8 and FG2), and these can be eliminated as explained above because in cyanoferrimyoglobin the peak in question has almost no pseudocontact shift. All the other histidine residues are on the surface of the molecule, having their NH protons easily accessible to the solvent, with the single exception of His B5, which is buried inside the protein in a hydrophobic pocket provided by Val A5, Be G12, Val G15 and Leu G16. The NH proton is about 18 A from the iron, and meets t’he requirement that it should bc beyond the range of significant pseudocontact interactions. A good assignment of peak D would therefore be to the NH proton of His B5. In support of this assignment, it has been observed that corresponding spectra from human hemoglobin u-chains do not contain such a peak (S. Ogawa, personal communication), which is to be expected since His B5 is replaced by tyrosine. The unusual temperature dependence of the peak width would then be understood as a change in the accessibility of water to that interior region of the protein. Pate1 suggests, however, that since two of the surface histidines, His CD6 and His EF5, appear to be hydrogen bonded to nearby residues, which could slow their rates of proton exchange, one of these might be a better assignment. At present these alternatives can not be distinguished unambiguously. Little can be said about the remaining exchangeable peaks plotted near -11 p.p.m, and -10 p.p.m. in Figure 2. There are two tryptophan residues in the mole-
NMR
STUDY
OF
CYANOFERRIMYOGLOBIN
4i
cule which ought to produce NH peaks in this region (Glickson et al., 1969), and in Table 1 a prediction is made that the peptide NH peak at Ala F9 ought also to be present. Presumably none of these peaks is the His FG2 NBH peak, since it would have to move downfield with rising temperature towards a position of about -12.9 p.p.m. at at l/T = 0. This proton must either exchange rapidly, or its shift must have an upfield pseudocontact contribution, taking the peak above -10 p.p.m. and beyond observat*ion.
(c) Non-exchungeahleprotom We turn now to peaks from non-exchangeable protons, shown in Figure 2 by open circles and observed earlier in deuterium oxide solutions (Wiithrich et al., 1968). No methyl group is in a position where pseudocontact interactions could displace its resonance as low as -18 p.p.m., and therefore the assignment of the two methyl peaks below -18 p.p.m. to porphyrin methyl protons is almost certainly correct. Porphyrin methyl peaks are expected to have pseudocontact shifts in the range +2.6 to +7*4 p.p.m., depending on the orientation of the in-plane g-tensor axes, and since the total par&magnetic shift of Av,. + A+,,. is much greater than this, the major contribution is from the contact interact,ion. Of the protein methyl groups which are in positions suitable for large pseudocontact shifts, only one methyl group, one of the methyl groups of Val El 1, is a likely assignment for a lotield methyl peak. The centre of rotation of its hydrogen atoms is 4.8 A from the iron, with Q = 25”. A pseudocontact shift Av,,,, of about -15 p.p.m. can be calculated which, together with a ring current shift A+,,-., of +3*5 p.p.m. and an expected Avai,,. of -1 p.p.m. in the absence of these additional interactions (Av,. should be negligible), leads to a predicted Av,,,, of about -12.5 p.p.m. At least one, and possibly, two methyl groups produce peaks in this region, which have been assigned to porphyrin methyl groups (Wiithrich et al., 1968). At present there is not sufficient evidence for confident assignments either to porphyrin methyl groups or to the methyl group of Val Ell. The remaining peaks observed at low field in deuterium oxide solutions were assigned to proximal histidine C,H and C,H protons, and to vinyl protons (Shulman, Wiithrich, Yamane, Antonini & Brunori, 1969). It is certainly likely that one of t)he peaks at either -17 p.p.m. or -18 p.p.m. in Figure 1, and one of the peaks near -11 p.p.m., are vinyl peaks, since two peaks are absent in the spectrum of cyanoferrimyoglobin reconstituted with deuteroporph-yrin, in which the vinyl groups are replaced by h,ydrogen atoms (Shulman, Wiithrich, Yamane, Antonini & Brunori: 1969). However, in addition to the possibility that the C&H and C,H protons of the proximal histidine ring could produce peaks in this range, pseudocontact interactions could displace peaks into this region from the #KHz protons of the proximal histidine, especially with assistance from a contact interaction, from the ortho or meta protons of Phe CD1 or from the C,H proton of His FG2. It is possible also that peaks from propionic acid CH, groups adjacent to the porphyrin ring could be contact shifted into t,his region. The fact that the non-exchangeable peak moving towards -11 p.p.m. at, low pH (Fig. 3) has an appreciably pH-dependent shift, may be an indication that this peak is pseudocontact shifted, since pseudocontact, shifts are expected t,o be extremely sensitive to small movements within the protein. In the upfield region (Fig. 6), we restate that peaks R and S are not propionic acid methylene peaks, since their areas require assignments to methyl groups. This appears to be true also of peaks I’ and Q. whir11 wf’rc similarly axsigned to propionic
48
B.
SHEARD,
T.
YAMANE
AND
R.
G.
SHULMAN
acid CH, protons (Wiithrich et al., 1968). There are several amino acid CH, groups near the iron which could account for these peaks, but it will be more profitable to discuss their assignments when the directions of the in-plane g-tensors are known. We wish
to thank
Dr
Seiji
Ogawa
for
many
interesting
and
useful
discussions.
REFERENCES Bretscher, P. A. (1968). Thesis, Cambridge University, England. Eaton, D. R. & Phillips, W. D. (1965). Advance8 in Magnetic Resonance, ed. by J. S. Waugh, vol. 1, p. 103. New York: Academic Press. Eigen, M. (1964). Angewandte Chemie, Intern. Edit., 3, 1. Glickson, J. D., McDonald, C. C. & Phillips, W. D. (1969). Rio&em. Biophys. Res. Comm. 35, 492. Hapner, K. D., Bradshaw, R. A., Hartzell, C. R. & Gurd, F. R. N. (1968). J. B&Z. Chem. 243, 683. Hill, H. A. O., Mann, B. E. & Williams, R. J. P. (1967). Cltem. Comm. p. 906 Hugh, T. E. (1968). Ph.D. Thesis, Indiana University. Jesson, J. P. (1967). J. Chem. Phys. 47, 579, Kowalsky, A. (1965). Biochemistry, 4, 2382. Kurland, R. J., Davis, D. G. & Ho, C. (1968). J. Amer. Chem. Sot. 90, 2700. Kurland, R. J. & McGarvey, B. R. (1970). J. Maq. Res. 2, 286. McConnell, H. M. & Chesnut, D. B. (1958). J. Chem. Phys. 28, 107. McConnell, H. M. & Robertson, R. E. (1958). J. Chem. Phys. 29, 1361. Meadows, D. H., Markley, J. L., Cohen, J. S. & Jardetzky, 0. (1967). Proc. Nut. Acnd. Sci., Wash. 58, 1307. Shulman, R. G., Glarum, S. H. & Karplus, M. (1970). J. Mol. Biol. in the press. Shulman, R. G., Ogawa, S., Wiithrich, K., Yamane, T., Peisach, J. & Blumberg, W. E. (1969). Science, 165, 251. Shulman, R. G., Wiithrich, K., Yamane, T., Antonini, E. & Brunori, M. (1969). Proc. Nat. Acad. Sci., Wash. 63, 623. Shulman, R. G., Wtithrich, K., Yamane, T., Patel, J. & Blumberg, W. E. (1970). J. Mol. Biol. 53,143. Wiithrich, K., Shulman, R. G. & Peisach, J. (1968). Proc. Nat. Acad. Sci., Wash. 60, 373. Wiithrich, K., Shulman, R. G., Wyluda, B. J. & Caughoy, W. S. (1969). Proc. Nat. Acad. Sci., Wash. 62, 636. Wiithrich, K., Shulman, R. G., Yamane, T., Wyluda, B. J., Hugli, T. & Gurd, F. R. N. (1970). J. Biol. Chem. in the press. Yamane, T., Wiithrich, K., Shulman, R. G. & Ogawa, S. (1970). J. Mol. Biol. in the press