Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration

J. iWol. Biol. (1974) 86, 749-756

Assignments of the Paramagnetically Shifted Heme Methyl Nuclear Magnetic Resonance Peaks of Cyanometmyoglobin by Selective Deuteration A. MAYER, S. O~AWA, R. G. SHULMAN, T. YAMANE

Bell Laboratories Murray Hill, N.J. 07974, U.X.A. JO&

A. S. CA~ALEIRO, ANT~NIO M. D’A. ROCHA GONSALVES GEORQE W. KENNER AND KEVIN M. SMITH

The Robert Robinson Laboratories University of Liverpool, Liverpool, England L69 3BX (Received 29 January Three of the four pammagnetically nance peak of cyanometmyoglobin nuclear magnetic resonance spectra

1974)

shifted heme methyl nuclear magnetic resocould be assigned by comparing the proton

of myoglobins reconstituted from selectively deuterated hemes. These spectra indicate that the fourth methyl nuclear magnetic resonance peak has to be looked for outside the region -9 to -43 parts per million.

1. Introduction It is well known that the nuclear magnetic resonance lines from protons near the iron in par&magnetic heme proteins are sometimes shifted far from their positions in diamagnetic heme proteins and, consequently, are well resolved. Resonances of the methyl groups of the porphyrin ring in cyanometmyoglobin and cyanomethemoglobin have been studied intensively (Shulman et al., 1969a,b; Sheard et al., 1970; Wiithrich et al., 1970; Ogawa et al., 1972a,b). Recently, we have chemically synthesized protoporphyrin IX with those four methyl groups selectively deuterated in pairs (Rocha Gonsalves et al., 1971; Cavaleiro et al., 1974a) and have shown how this allows positive assignments of the four methyl resonances observed in the nuclear magnetic resonance spectra of the hemes in pyridine-cyanide solutions (Cavaleiro et al., 19743). In this report we show how these partially deuterated hemes, when combined with apomyoglobin to form reconstituted MbCNT, allow an unequivocal assignment of the heme methyl resonances in MbCN. It is shown that these assignments disagree with those made previously on the basis of adding cyclopropane to MbCN, where the reasonable assumption was made that the most strongly shifted resonance could be assigned to the methyl group nearest the cyclopropane binding site, as determined by X-ray crystallography. t Abbreviations used: MbCN, ferric cyanide derivative of myoglobin; relative to an internal standard of sodium-2,2-dimethyl-2-pentasilane-5-sulfonate from this standard is expressed by a negative sign. 749

p.p.m., parts per million, The

lower

field

A. AlAYER

750

IC:T AL.

2. Experimental Methods Sperm whale myoglobin was prepared by a combination of the methods of Hugli & Gurd (1970) and Hapner et al. (1968). Globin was prepared according to the method of Hill et al. (1962). Deuterated hemes, prepared as described previously (Rocha Gonsalves et al., 1971; Cavaleiro et al., 1974u), were coupled to globin following the procedure described by Antonini et al. (1964). The reconstituted myoglobin solution was purified through DEAE-Sephadex A50, as described by Hapner et al. (1968), except the buffer used was 0.1 M-phosphate, pH 7.0. The myoglobin was converted to the cyanomet form by the addition of sodium cyanide and concentrated by vacuum dialysis to approximately 10e3 M-home. Horse myoglobin was purchased as the ferriaquomet derivative in a lyophilized form and used without further purification. It was dissolved and converted into the cyanomet derivative by treatment wit’h sodium cyanide (buffered at pH 9 with 0.05 Mborate). The cyclopropane (Matheson, Coleman and Bell Inc.) was passed over the surface of the protein solution for 10 min, with mild agitation of the container The proton nuclear magnetic resonance measurements were made with Varian spectrometers at 220 MHz (Varian HR220) and at 300 MHz (Varian HR300). 3. Results Figure 1 shows the protoporphyrin IX iron formula with the notation used to describe the eight /3 positions. The CH, groups at positions 1, 3, 5 and 8 of the

H3C

CH=CH2

H3 C

CH3

H

RO,

c

C”,

CH2

CHz

CHz

Pm. 1. Heme group viewed from the proximal site is marked by X.

side. Projection

-COzR

of the center of the cyclopropane

ASSIGNXENTS

751

SN CYANOMETMYOGLOBIN

(b)

5,8-C2H3 (c) I I@-C’H,

(d)

(e)

I --&+k

I

-24

IlJ

I

-22

-20 Resonance

I

I,

I

-18

-16

I

I

-14

I,

I

-12

I

-10

(p.p.m.)

Fro. 2. The 230 MHz spectra of sperm whale MbCN reconstituted from selectively deuterated hemes in the downfield region of normal MbCN in Hz0 (a); 0.1 M-phosphate buffer (pH 7).reconstituted MbCN with deuterated mesoprotonsin the 01and y positions (b); and with deuterated methyl groups in positions 5 and 8 (c), 1 and 8 (cl), 1 and 3 (B), in 2HZ0, 0.1 M-phosphate buffer (pII 7), all measured at 3 1°C.

752

ASSIGNMENTS

I 0

IN

753

CYANOMETMYOGLOBIN

I

2 Resonance( pp.m.)

I

3

.-l----

4

Fra. 4. The 300 MHz spectra upfield region of (a) normal horse MbCN and (b) with added cyclopropane. In H,O, 0.05 M-borate buffer (pH 9), and measured at 31°C.

porphyrin ring have been replaced in pairs with PH, groups by chemical synthesis (Rocha Gonsalves et al., 1971; Cavaleiro et al., 1974a). Figure 2 shows the downfield region of the 220 MHz spectra of sperm whale MbCN, which has been reconstituted with the selectively deuterated hemes. The spectra of (5,8)-C2H,, (1,8)-C2H, and (1,3)-C2H, MbCN spectra (Fig. 2(c), (d) and (e)) provide 49

A. MAYER

754

ET

AL.

a unique assignment of the shifted resonances of the porphyrin methyls. For comparison, a spectrum of normal whale MbCN is shown (Fig. 2(a)). It should be noted that the normal MbCN is dissolved in H,O, while the reconstituted MbCN species are dissolved in aH,O. The arrows mark exchangeable proton resonances in the H,O sample that have been observed previously. The spectra of the reconstituted myoglobins are not as good as that of the normal myoglobin, probably due to the presence of some denat,ured or other species introduced during the preparation. The spectrum of MbCN reconstituted from hemes deuterated in the a and y meso positions (but with normal CH, groups) is shown in Figure 2(b). Figures 3 and 4 show the effects of binding cyclopropane on the 300 MHz nuclear magnetic resonance spectrum of horse MbCN. In the downfield region (Fig. 3), it can be seen that cyclopropane shifts the methyl resonance near -13 p.p.m. upfield by 280 Hz, and slightly but definitely influences t,he resonance positions of the other two methyl resonances at -27 and -18 p.p.m. (see Table 1). The single proton resonances in the upfield region are influenced by the binding of cyclopropane (Fig. 4): in particular, the resonance at +9 p.p.m. is shifted downfield by 65 Hz (Table 1). In all respects these results are identical with those published previously, in which the spectra were measured at 220 MHz. However, the increased resolution (and signal TABLE 1 Shifts of resonance in response to the addition of cyclopropane to th.e ferric cyanide derivative of myoglobin Shift by addition of cyolopropane (Hz) Horse MbCNf Porpoise MbCN §

Frequenoyt kwm.)

Intensity (no. protons)

- 26.9

3

+ 15

3

+100 + 70 - 20 -150 + 20

- 10 - 160 i- 10

+ 60 + Es-l $280 t 10 -I-150

-t 190 + 10 + 80

-23.2 -21.0 - 18.2 -17.7 - 17.0 - 13.6 - 13.5 - 13.2 - 12.2 -11.4 -+ + + -+ + + + -,!+

1.4 1.6 l-8 2.0 2.2 2.5 3.2 3.6 9.0

1 3 1-t 2

+ 25 - 10 7 - 20 1 - 10 + 75 ll -0 0 - 50 - 65

3 3

t Magnetic field positions measured dimethyl-2.pentasilane&sulfonate. $ From present 300 MHz spectra. 3 From Shulman et al. (1969a,b). 7 In 220 MHz spectra not resolved.

in p.p.m,

with

respect

0 0 -

20

-

“5

+ 20 - 66 - 60 to an internal

standard

of 2,2-

ASSIGNMENTS

IN

CYANOMETMYOGLOBIN

755

to noise) of the present spectra allow us to state that the smaller shifts down to 0.05 p.p.m., which had been reported previously, are detected reproducibly with greater accuracy in the present 300 MHz spectra, as shown in Table 1.

4. Discussion The four porphyrin methyls of the heme in MbCN are shifted downfield by interactions with the unpaired electron. The resonances with the intensity of three protons at -27, -19 and -13 p,p.m. had been attributed to three of the four porphyrin methyls, while the fourth methyl was suggested to be near -12 p.p.m., where a resonance from about three protons was observed in porpoise and horse MYbCN, and two resonances with intensities of one and two protons were observed in sperm whale MbCN. The appearance or disappearance of the downfield shifted resonances with intensities of three protons in the spectra of (5,8)-C?H,, (1,8)-C2H, and (1,3)-C2H, MbCN (Fig. 2(c), (d) and (e)) allows the definite assignment of these resonances. The resonance at -28 p.p.m. originates from CH, in position 5, the one at -18 p.p.m. from CH, in position 1 and the one at -13 p.p.m. from CH, in position 8. A comparison of the spectrum of (1 ,3)-C2H, MbCN with all the other spectra of Figure 2 shows that there is no pronounced variation of intensity in the range between - 13 and -11 p.p.m., as would be anticipated if the CH, in position 3 had its resonance there. Therefore, this is clear evidence that the resonances corresponding to one and two protons near -12 p.p.m. in whale MbCN do not originate from methyl 3. It is concluded, therefore, that the resonance with the intensity of three protons observed in horse and porpoise MbCN at -12 p.p.m. does not originate from methyl 3 either. Since there is no resonance with the intensity of three protons in the region between -13 and -9 p.p.m., as can be seen in Figure 2, the resonance from methyl 3 has to be outside of the region studied between -9 and -43 p.p.m. A tentative assignment of the methyl resonances was attempted previously by Shulman et al. (19693) when studying the effect of xenon and cyclopropane on the nuclear magnetic resonance spectra of cyanomyoglobins, assuming that xenon and cyclopropane bind to the same proximal heme site in MbCN in solution as in the aquomet myoglobin in the crystal state (Schoenborn et al., 1965; Schoenborn, 196’7) (the project’ion of the center of cyclopropane on the heme plane is as shown in Fig. 1). Previously the obvious conclusion was drawn that the resonance near -13 p.p.m. originates from methyl 1, because of its unique large shift. This conclusion disagrees with the assignments for the porphyrin ring methyls given by the present experiments. Thus there is little effect of cyclopropane on the position of the resonance of methyl 1, which should be closest, but a very large effect on the position of methyl 8, which is many Angstrom units away. A reinvestigation of the cyclopropane effect on the nuclear magnetic resonance spectra of IKbCN was carried out at 300 MHz for whale and horse myoglobin (Figs 3 and 4). There is a small but reproducible shift of about 20 Hz of the methyl resonance near -18 p.p.m. Hence methyl 8 is the group most conspicuously affected by the cyclopropane binding but there is evidence that mebhyls 1 and 5 are also affected slightly. The methyl resonance at -13 p.p.m. is extremely sensitive to changes in the pH value (Sheard et al., 1970), being shifted upfield with increasing pH by about 0.6 p.p.m. in the range pH 4.5 to pH 9, while the methyl resonance at -18 p.p,m. is insensitive to variations of pH in the same

A. MAYER

756

ET

AL.

pH range. The methyl resonance at -27 p.p.m. is also insensitive to variation of pH above pH 6, while it is very sensitive to changes below pH 6, being shifted upfield with decreasing pH values. The methyl resonance at -13 p.p.m. assigned to methyl 8 is apparently sensitive to changes in the pH value, which may stabilize the molecule in slightly different conformations. This suggests that the binding of cyclopropane or xenon to MbCN may cause a slight rearrangement in the vicinity of the heme, which mainly affects the methyl resonance in position 8. That widespread changes are occurring on binding of cyclopropane is seen from the extensive shifts listed in Table 1. The reason for the large response of methyl 8 may be connected with its sensitivity to changes in the pH value. Although we cannot eliminate the possibility that the cyclopropane binds at different sites in solution and in the crystal, we consider this an unlikely explanation. More probably, the present experiments illustrate that the residues affected by introduction of a weak perturbation like cyclopropane are not always those closest to the perturbation. One of us (A. M.) was on leave from the Physik-Department der Technischen Universit&t, Munchen, during the course of this work. Two of us (J. A. S. C. and A. M. d’A. R. G.) were on financially assisted leave of absence from the University of Lourenpo Marques, Mopambique. We gratefully acknowledge additional financial support from the Calouste Gulbenkian Foundation, Lisbon, Portugal. REFERENCES Antonini,

E., Brunori,

(1964).

Biochim.

M., Caputo, A., Chiancone, E., Rossi-Fanelli, Biophys.

A. & Wyman,

J.

Actu, 79, 284-300.

Cavaleiro, J. A. S., Rocha Gonsalves, A. M. d’A., Kenner, G. W. & Smith, K. M. (1974a). J. C. S. Perkin I, in the press. Cavaleiro, J. A. S., Rocha Gonsalves, A. M. d’A., Kenner, G. W., Smith, K. M., Shulman, R. G., Mayer, A. & Yamane, T. (19743). J. C. S. Chem. Commun. 392-393. Hapner, K. D., Bradshaw, R. A., Hartzell, C. H. & Gurd, F. R. N. (1968). J. Biol. Chem. 243, 683-689. Hill, R. J., Konigsberg, W., Guidotti, 0. & Craig, L. C. (1962). J. Biol. Chem. 237, 15491554. Hugli, T. E. & Gurd, F. R. N. (1970). J. Biol. Chem. 245, 1930-1938; 1939-1946. Ogawa, S., Shulman, R. G. & Yamane, T. (1972a). J. Mol. BioZ. 70, 291-300. Ogawa, S., Shulman, R. G., Fujiwara, M. & Yamane, T. (19726). J. Mol. BioZ. 70, 301-313. Rocha Gonsalves, A. M. d’A., Kenner, 0. W. & Smith, K. M. (1971). Chem. Commun. 1304-1305. Schoenborn, B. P. (1967). Nature (London), 214, 1120-1122. Schoenborn, B. P., Watson, H. C. & Kendrew, J. C. (1965). Nature (London), 207, 28-30. Sheard, B., Yamane, T. & Shulman, R. G. (1970). J. Mol. BioZ. 53, 35-48. Shulman, R. G., Wiithrich, K., Yamane, T., Antonini, E. & Brunori, M. (1969a). Proc. Nat.

Acad. Sci.,

U.S.A.

63, 623-628.

Shulman, R. G., Peisach, J. & Wyluda, B. J. (19696). J. Mol. BioZ. 48, 517-523. Wiithrich, K., Shulman, R. CT.,Yamane, T., Wyluda, B. J., Hugli, T. E. & Gurd, F. R. N. (1970). J. BioZ. Chem. 245, 1947-1953.