BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35636

MOSSBAUER SPECTROSCOPY OF MESOHAEM AND PROTOHAEM

MYOGLOBINS AND T H E I R FLUORIDE COMPLEXES

38r

GEORGE LANG*, TOSHIO ASAKURA** ANn TAKASHI YONETANI'*

* Nuclear Physics Division, United Kingdom Atomic Energy Research Establishment, Harwell, Didcot, Berks, (Great Britain) and ~*Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. z9Io 4 (U.S.A.)

(Received April 9th, 197 o)

SUMMARY

The M6ssbauer spectra of 57Fe-enriched ferric myoglobins, pH 6, containing both protohaem and mesohaem prosthetic groups, have been measured under a variety of conditions of temperature and field. In applied fields at 4.2°K well-resolved paramagnetic hyperfine spectra are observed. These are consistent with a high-spin model with axial distortion. In small fields internal fields of order 300 kgauss are observed, corresponding to saturation fields of 493 4- 4 and 498 4- 4 kgauss for meso and proto forms, respectively. A quadrupole splitting of 0.65 + 0.05 mm/sec is observed in each implying that the splitting in absence of magnetic interaction should be 1.3 mm/sec. This is in good agreement with the value of quadrupole splitting observed at I95°K where ielaxation effects suppress magnetic interaction. The field dependence of the low temperature spectra are consistent with an axial splitting, 2D, of 20 ± 2 cm -1, in agreement with susceptibility results. The fluoride complexes of these proteins are also high spin and have similar low temperature spectra. The saturation fields are 525 4- 3 kgauss, 2D is 12.6 ± I cm -1, and the quadrupole splitting is 0.40 ± 0.05 mm/sec, implying a splitting of 0.8 mm/sec in the absence of magnetic interaction. At I95°K the spectrum is unresolved as a result of residual paramagnetic effects. Again, measurements suggest that the internal field of proto form may be a few tenths of a percent higher than that of the meso form.

INTRODUCTION

The usefulness of magnetic measurements in characterizing ferric haem proteins has long been recognized. The earliest work involved attempts to identify spin states by determining the effective number of unpailed spins, usually at or near loom temperature 1. More recently the election spin resonance (ESR) work of the Southamp- ton group 2-~ has ushered in an era of much more precise and detailed magnetic studies,

Abbreviations: ESR, electron spin resonance; EFG, electric field gradient.

Biochim. Biophys. Acta, 214 (197 o) 381-388

382 G. LANG dt *tl.

from which considerable information on electronic energy level schemes has been derived. The usefulness of very accuiate susceptibility measurements ow,r wide temperature ranges has been demonstrated s-12. The presence of unusually large zero field splittings in high-spin ferric haem proteins, indicating a large axial distortion of the ion, was first reported by GIBSON et al. 5, who found that the spin sextet was split into three doublets in haemoglobin fluoride, with the first excited doublet lying at an energy 2D ~_- 5 cm-L M6ssbauer spectroscopy verified this result and in fact indicated that 2D ~ 14 cm -1 (ref, 13). No low-temperature susceptibility results are available for this material, but both ESR and susceptibility measurements have been made on the somewhat similar acid metmyoglobin. The ESR result '~ is 2D ~.76 ::t 1.2 cm ', while susceptibility indicates 2D ~- 2o cm l (ref. 9). The present work verifies the lattel value as well as the corresponding measurement on nly(Nlobin fluoride made by the same investigators.

EXPERIMENTAL PROCEI)URE

Samples of sperm whale myoglobin were enriched to about 8o% in '~TFe by removal and replacement of the haem. A typical sample consisted of o. 7 ml of solution containing 1 2 mM concentration of myoglobin, in addition to phosphate buffer to control pH. The fluorides were made by adding an excess of NaF. The sample celt was of polythene with a '/s-inch-thick sample space and a wall thickness of 1/a 2 inch. Measurements were made in a constant acceleration M6ssbauer spectrometer la, which was regularly calibrated using the known spectrum 16 of metallic iron. Isomer shifts are specified relative to this spectrum. Temperatures of I95°K and 77°K were obtained in a cryostat in which the sample was in a transfer gas which was cooled by dry ice and liquid N2, respectively. For He-temperature runs the sample was immersed in liquid He. The o.5-kgauss fields transverse to the y-beam were obtained by placing a permanent magnet around the cryostat, while longitudinal fields of roughly o.I kgauss were obtained by inducing persistent currents in lead rings placed in a Helm- holtz arrangement about the sample. High fields in transverse direction were obtained by a superconducting solenoid.

EXPERIMENTAL RESULTS

Similar results have been obtained for tile acid metmyoglobin in o. I M phosphate buffer (pH 6) and in 0.5 M phosphate buffer (pH 6). At 4.2°K and in zero magnetic field the M6ssbauet spectra ofmeso- and protomyoglobin are not observably different. Both are very broad and diffuse with only poorly resolved peaks. Application of a small transverse external magnetic field, however, gives rise to a well-defined six-line spectrum, characteristic of an effective magnetic field at the nucleus of order 300 kgauss (Fig. Ib). In longitudinal field Lines 2 and 5 become very weak, indicating that the effective field tends to be nearly parallel to the applied field (Fig. Ia). At 14. 5 kgauss (c) the spectrum is a superposition of two sextets, with the wider one having greater intensity. Finally with 29 kgauss applied (d) a single sextet is seen with effective field about 15 % greater than at (b). Protomyoglobin spectra are shown in Fig. I. The meso- myoglobin spectra under the same conditions differ only in having very slightly larger apparent effective magnetic fields. At 77°K the spectra are featureless and diffuse

Biochim. Biophys. Acta, 2I 4 (I97 o) 381-388

MOSSBAUER SPECTRA OF MYOGLOBINS 383

SOURCE VELOCITY (mr~/N¢l

Fig. z. M(~ssbauer spectra of protometmyoglob in in pH 6, 0. 5 M phosphate buffer at a temperature of 4.2°K. Appl ied magne t i c fields are (a) o. I kgauss parallel to the ~,-beam, (b) o.5 kgauss t r ansve r se to t he T-beam, (c) 14.5 kgauss t ransverse , and (d) 29 kgauss t ransverse . The veloci ty scale is measu red rela t ive to t he cen te r of an iron foil absorber . The solid curves are c o m p u t e d us ing an axial field model wi th 2D ~ 20 cm -1, Hs ~ 498 kgauss , QVz~]4 ~ 0.65 mm/sec , i somer shi f t 0. 4 mm/sec , and a l inewidth of 0.3 m m / s e c full w id th a t ha l f m a x i m u m .

presumably because of thermally induced electron spin relaxation. At I95°K they begin to approach an asymmetric doublet. Application of a small field gives rise to a larger relaxation rate and further sharpening of the spectrum. The quadrupole split doublet, however, remains asymmetric because of incomplete quenching of magnetic hyperfine interactions (Fig. 2c). The isomer shift of 0. 4 mm/sec is characteristic of high-spin haem proteins. The spectra of both proto and meso forms at pH 6 in 0.5 M buffer are not observably different from those of the proto at pH 6 in o.I M buffer shown in Figs. 2c and 2d. One mesomyoglobin sample was accidentally diluted with distilled water to about 0.05 M in buffer..Its low temperature small field spectrum (Fig. 2b) is altered in that it no longer returns to near the background level in the region between the strong absorption lines. At I95°K it is entirely different, consisting of a quadrupole pair whose splitting (2.1 mm/sec) and isomer shift (0.2 mm/sec) are characteristic of low-spin ferric haem proteins. It appears that at low temperature part of the myoglobin is in a low-spin form, giving rise to a broad but not well-resolved component of the spectrum. At I95°K the equilibrium has shifted strongly in favour of the low-spin form, and only a trace of the high-spin component is seen in Fig. 2a.

Biochim. Biophys..~cta, 214 (197o) 381-388

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V E L O C I T Y

lqg. 2. Spectra of metmyoglobin, pH 6, in 0. 5 kgauss transvcrso fields. At (a) is the r95 'K spec t rum of the meso-form in buffer of s t rength about o.o 5 M, while (b) refers to the same sample at 4.2'q(. At (c) is the proto-form at I95°K in o.i M buffer, while (d) refers to this sample at 4.2°K. At pH 6 in 0. 5 M buffer both meso- and proto-forms have spectra of types (c) and (d).

Such effects were probably present but not recognized in earlier M6ssbauer work ~a on methaemoglobin, and may be the explanation for confusing susceptibility results :~ on the same material.

Spectra of meso- and protomyoglobin fluoride are practically indistinguishable, although the internal fields in the proto-form may be a few tenths of a percent higher. They are similar to the acid met-forms in having diffuse zero field 4.2°K spectra, and spectra which are well-defined in applied fields and have a similar dependence on field strength {Fig. 3). They again become diffuse at 77°K with or without external field. Spin relaxation rates at I95°K are not sufficiently fast to reveal the expected quadrup-

ole doublet, however.

DISCUSSION

At He temperature the electron spins of ferric haem proteins appear to have negligible interaction with the lattice phonons. Their interaction with the awe nucleus has a strength corresponding to about IO gauss at the electrons, and interactions with other nuclei are probably at most of the same order. When an external field of IOO gauss or more is applied, it effectively decouples the electrons from the effects of the nuclei and simplifies the solution of the electronic problem. If we include the possibility that the spherical d 5 subshell is distorted by an axial crystal field, the electronic Hamil-

tonian becomes H ~ - - D S z 2 - - 2 [ J S . H (1)

Biochim. Biophys. Acta, 2t 4 (I97o) 3 S I - 3 ~

MOSSBAUER SPECTRA OF MYOGLOBINS 385

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Fig. 3. M6ssbauer spectra of mesomyglobin fluoride at 4.2°K. Applied fields are (a) o.i kgauss parallel to the v-beam, (b) 0. 5 kgauss perpendicular to the v-beam, (c) lO.2 kgauss perpendicular. The solid curves are computed using an axial field model with 2 D -- i2.6 cm -1, Hs = 525 kgauss, O, V z z / 4 = o.4 mm/sec, isomer shift 0. 4 mm/sec, and a linewidth of 0. 3 mm/sec full width at half max imum.

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Fig. 4- At the left are shown the levels of a high-spin ferric ion in axial crystal field. Solid lines show the effects of a field parallel to the x y plane, while dotted lines show the effects of a field along z. At the right are shown the spin expectation values of the ground doublet for various applied field s t rengths and directions.

B i o c h i m . B i o p h y s . Ac ta , 214 (197o) 381-388

386 {;. LANG et al.

The energy-level scheme is shown at the left of Fig. 4. In the present cases only the ground doublet is occupied at 4.2°K. Application of a small field along ~ splits it into states with spin expectation value 1/, 2 in the -~z and z directions. The same field, applied in the xy plane gives rise to states of tile lowest doublet which haw" spin expectation value -~ a/2 in the field direction, i.e., tile transverse g value is (7. Statistical considerations indicate that in a polycrystal in a magnetic field the transverse field situation predominates, and we may concentrate our attention upon it in deciding the qualitative features of the spectra. In the nearly spherical d s distribution ~f spin we expect magnetic interaction with the nuclear spin only via the contact term. Thus we assume that the effective internal field is proportional to < S ?,- and parallel to it. In small transverse applied field the two members of the ground doublet give rise to internal fields which are of equal intensity but oppositely directed. They therefore give rise to identical M6ssbauer spectra and a single sextet appears {Figs. ia, II), 3a and 3b). The relative intensities of the absorption lines indicate that the internal field direction is highly correlated with the direction of the applied field. For fields such that 6 flH becomes comparable to the zero field splitting ~D, mixing between I Sz! .... ~/~ and I Szl a/2 states occurs, giving rise to a ground doublet whose members have unequal] < S ;> I, with the component lower in energy having the larger i ~:- S :---i" This gives rise to the more complicated spectra (Figs. ic and 3c) in which two sextets of unequal intensity are present. Fig. id is simplified because in the 2~t kgauss field the Boltzmann factor of the upper component of the ground doublet is so small that its contribution to the M6ssbauer spectrum is not noticeable.

The spectra of Figs. I and 3 are asymmetric as a result of quadrupole interaction. We have attempted to reproduce to observed spectra theoretically, using a hyperfine Hamiltonian of the form

I I .4 I . : ~/.! Q I 'z~([z ~ 5/-t), (-')

where an electric field gradient (EFG) of axial symmetry has been assumed. A com- puter calculation has been performed in which Eqn. I is solved in order to obtain < S > , then Eqn. 2 is solved to obtain the nuclear excited and ground states. Next the M6ssbauer line spectrmn is found bv calculating the transition probabilities and energies. Finally, the result is folded into a I,orentzian line shape and a numerical average is taken over all possible field directions relative to the crystal. The solid curves of Figs. r and 3 have been calculated in this way. For the acid metmyoglobin the crystal field splitting is 2D 2o ]- 2 cm .t, and the EFG is such that GV~z/4 o.65 ~- o.o5 ram/see. The isomer shift is a := o.4 -k o.o5 mm/sec. It is convenient to express the A tensor in tern> of Hs, the saturation field which would be produced at the nucleus for spin ~ S - : a/.,. The protomyoglobin appeared to have a slightly higher internal field with H s - : 498 ± 4 kgauss, while for mesomyoglobin Hs 493 -k 4 kgauss. All. of the uncertainties quoted here are estimated. The larger internal field of protomyoglobin relative to mesomyoglobin was observed with a greater consistency than the uncertainties in each would imply. Similar treatment of meso- and proto- fluoride complex spectra yielded the results 2D =-= I2.6 ~ I.o cm-~, tt~ 525 q: 3 kgauss, QVzz/4 o.4o :k o.o5 cm/sec, a 0.4 ~ o.o5 mm/sec. There appeared to be a slightly higher field in the protoform relative to the mesoform, amounting at most to a few tenths of a percent.

Because the internal magnetic field, which essentially determines the nuclear

B i o c h i m . B i o p h y s . Ac la , -'l ] (r~17o) 38J-38>:

MOSSBAUER SPECTRA OF MYOGLOBINS 387

quantization axis, tends to lie near the xy plane of the crystal, the shift of the outer two lines of the magnetic sextet relative to the inher four is determined by the trans- verse components of the EFG tensor. This shift may be expressed as QVxx/2 -- -QVzz /4 . Since it is negative we conclude that QVzz and hence Vzz are positive. In the absence of internal magnetic field the EFG axis (the z axis) is the axis of quanti- zation and the quadrupole splitting is QVzz/2. Thus we expect for metmyoglobin a quadrupole splitting of 1.3 mm/sec at temperatures sufficiently high to suppress strong magnetic hyperfine interaction. This is indeed found to agree with the data of Fig. 2c, where the predicted line positions are marked. No such comparison is possible for the fluoride complexes, for the simple quadrupole pat tern has not been observed.

The sign of Vzz is positive for both our materials. I f we imagine distortion of the d 5 subshell as the source of the EFG, we would conclude that there exists an excess of electron density near the xy plane relative to the z axis. This distortion would give rise to a dipolar contribution to the magnetic A tensor, causing the z component to differ from the x and y components. Because the quanti ty (3z2--r~)/r 5 affects both the EFG and the magnetic dipole interaction, we may determine the latter from the quadrupole splitting. The essential assumption is that charge density is everywhere propoltional to spin density. Using Eqn. 20 of LANG 13 (where, however the sign of A E should be reversed), the quadrupole splitting of metmyoglobin implies that Az/Ax -- 1.2. By means of the program described above we have investigated the effect of changing Az, and we find that the resulting small changes in lineshapes fall well within present experimental uncertainties. This is understandable in view of the predominantly transverse orientation of S in the polycrystal. In a comparison of the metmyoglobin with the fluoride the association of the larger Hs (hence larger Ax) with the smaller quadrupole splitting is qualitatively consistent with the above consider- ations, Hs of the fluoride being about 5 % larger while the model would suggest it should be 3% larger if the contact contribution were the same for both materials. Our a t tempts to measure Az by raising the temperature and populating the upper doublets of haem proteins have not been successful because of the relaxation broaden- ing which occurs.

The rather broad and diffuse spectra observed in zero applied field at 4.2°K may be at t l ibuted to the perturbing effects of the nuclear moments of the ligands. The spectrum of myoglobin fluoride is very similar to that of haemoglobin fluoride, and the t reatment of the tlansferred hyperfine interaction 17 should be similar. The zero field spectrum of metmyoglobin is quite similar to what we have observed from haemin in organic solvents, and we conclude that the four porphyrin nitrogens which are common to both compounds dominate the transferred hyperfme interaction. I t is interesting that, while spin transfer onto the ligands has a large effect upon the zero field spectra of the S ~= 5/2, [Sz I = 1/2 states, in the presumably more covalent low-spin compounds reasonable results are obtained (ref. 18, Fig. 5) by ignoring it.

CONCLUSION

The M6ssbauer spectra of metmyoglobin and myoglobin fluoride indicate tile presence of a strong axial field, with 2D = 20 4- 2 cm -1 and 12.6 4- i cm -1 respect- ively. These agree well with the values 20 cm -1 and 14 cm -~ from susceptibility% Two ESR results on metmyoglobin differ from these but are themselves consistent: 2D = 8.76 4- 1.2 cm -1 (ref. 14); 2D = 8.8 4- 0. 9 cm -1 (ref. 19).

Biochim. Biophys. Acta, 214 (197 o) 381-388

388 6. LANG e ta[ .

R E F E R E N C E 5

I E. F. HARTREE, Ann. Rep. Progr. Chem. Chem. Soc. London, 43 (I946) "87. 2 J. E. BENNETT AND J. E. INGRAM, Discussions Faraday Soc., 19 (I955) I4O. 3 J- E. BENNETT AND J. V. INGRAM, Nature, 177 (1956) 275. 4 J . F. GIBSON AND D. J. v . INGRAM, Nature, 178 (1957) 905. 5 J. F. GIBSON, D. J. b;. |NGRAIvI AND 1). SCHONLAND, Discussions P'aradav Sot., 2o ( ,958) 72 6 D. J. E . INGRAM, J. i:. GIBSON ANt) M. F. PERUTZ, Nature, 178 (1956 ) 906. 7 D. 8 M. 9 A.

I o "F.

I I T . 12 T. 13 G. 14 P. I5 T. I 6 R . 17 G. I 8 G . 19 E.

j . E . INGRAM AND J. C. KENDREW, Nature, I78 (1956) 905 . KOTANI, Progr. Theoret. Phys. Kyoto Suppl., 17 (I961) 4. TASAKI, J. OTSUKA AND M. KOTANI, Biochim. Biophys. Acta, I4O ( t967) 2~ 4. [IZUKA AND M. ]XOTANI, Biochim. Biophys. Acta, I54 (1968) 417 . hZUKA, M. KOTANI AND T. YONETANI, Biochim. Biophys. Acta, 167 (196S) 257. [IZUKA AND M. I~OTANI, Biochim. Biophys. Acta, I81 (I969) 275. LANG AND W *. MARSHALL, Proc. Phys. Soc. London, 87 ( I966) 3. EISENBERGER AND P. S. PERSHAN, J. Chem. Phys., 47 ( t967) 3327 . E. CRANSHA~V, Nucl. lnstr. Methods, 3 ° (1964) IOI. ~. PRESTON, ~. S. HANNA AND J. HEBERLE, Phys. Rev., 128 (I962) 22o 7. LANG, Phys. Letters, 26A (1968) 22.3. LANG, D. HERBERT AND T. YONETAN1, .[. Chem. Phys., 49 (1968) 944- F. SLADE AND D. J. E. INGRAM, Proc. Roy. Soc. London Ser. A, 312 (1969) 85.

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