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Hemoglobin: Resonance Raman spectra
830
BIOCHIMICAET BIOPHYSICAACTA
BBA Report BBA 31134 H e m o g l o b i n : R e s o n a n c e R a m a n spectra
THOMASC. STREKASand THOMASG. SPIRO Department of Chemistry, Princeton University, Princeton, N.J. (U.S.A.) (Received April 4th, 1972)
SUMMARY Raman spectra have been recorded for several hemoglobin derivatives in dilute solution. They exhibit a complex set of bands, arising from vibrations of the heine groups. Two of the bands offer promise as structural probes, inasmuch as their intensities correlate inversely with the degree to which the iron atoms are out of plane.
We have observed resonance Raman scattering 1 from dilute solutions (approx. 10-4 M) of hemoglobin derivatives. The technique may provide a solution structural probe for hemoglobin, and should be applicable to other heme proteins as well. Resonant enhancement makes possible the application of Raman spectroscopy, normally a lowsensitivity technique, to biological chromophores in their usual high dilutions. Recent applications include studies of carotenoid pigments 2 , vitamin A-type molecules 3 and rubridoxin 4 . Our spectra are shown in Fig.1. The exciting wavelength, 5682 A, falls in the middle of the visible absorption envelopes of all the hemoglobin derivatives s . Similar spectra, but of lower intensity, were obtained with the 5308-A and 4880-A laser lines, which lie on the high-frequency side of the visible envelopes, but below the Sorer bands. Excitation at 6471 A, below the visible bands, failed to give observable Raman scattering even at concentrations approaching 10-z M, Several Raman bands have been reported by Longs for aqueous myoglobin, using 6328 A He-Ne laser excitation. They are all below 600 cm-1 , a region which is blank in all of our spectra. Intensity-structure correlation Although the spectra of the various derivatives are similar, some of the bands do show substantial changes in their relative intensities. Of particular interest are the bands observed for oxy- and carboxyhemoglobin at 1638 and 1589 cm-1 , the latter being the Biochim. Biophys. Acta, 263 (1972) 830-833
BBA REPORT
831 MetHbH20
~8, ~
DEOXY Hb
HbCO
~-~ ~ ; ~ ~'~bo ~1~.~ ~ ~ ' ~
T~
~
GM -~
Fig.1. Raman spectra of human hemoglobin derivatives obtained with 5682 ~ excitation (20-60 mW) from an Ar÷/Kr+ mixed gas laser. Aqueous solutions of oxy-, ea~boxy- and deoxyhemoglobin, and aquo-(pH 7) and azidomethemoglobin, were prepared by standard methods. Concentrations were approx. 10-4 M in iron, measured spectrophotometricaUy. The samples were contained in l-ram glass capillary tubes, and spectra were recorded with transverse excitation, Clouding of the solutions was observed with excitation at higher frequencies (5308 and 4880/~) but not at 5682 ~l,. At this wavelength repeated scans gave identical, spectra, Instrument conditions: slit width, 10 cm-1; sensitivity, 10~s A; scan rate, 50 ern-1]min; time constant, 1.0 s. most intense feature of the spectra. These bands are missing for deoxyhemoglobin. Evidently the corresponding vibrational modes go out o[ resonance when hemoglobin is deoxygenated. The two bands reappear at full strength in azidomethemoglobin, a lowspin Fe(III) derivative. Their intensity is appreciably diminished in aquomethemoglobin, which is high.spin. It is tempting to associate these variations with the accompanying structural changes ~'s. The iron atom lies in the plane of the pyrrole nitrogen atoms in the low-spin hemoglobin derivatives, whether Fe(II) or Fe(III). In the high-spin derivatives the iron atom, presumably because of its increased radius, is significantly out o f the plane.
Biochim. Biophys. Acta, 263 (1972) 830-833
832
BBA REPORT
The out-of-planarity is greater for Fe(II) than for Fe(III), m accora with the greater radius of the former. Perutz s estimates the out-of-plane distance as approx. 0.8 A in deoxyhemoglobin and approx. 0.3 ~k in aquomethemoglobin. The 1638- and 1589-cm-1 bands correlate well with these features, the in-plane derivatives giving full intensity, aquomethemoglobin giving diminished intensity and deoxyhemoglobin showing no intensity. Moreover we have found that raising the pH of aquomethemoglobin produces an increase in the intensity of these bands, as the predominantly low-spin hydroxymethemoglobin is formed. Indeed a titration curve of intensity vs pH tracks the corresponding variation in spin state 9, with a pKa of approx. 8.5. The frequencies of these indicator bands are much too high for vibrations involving the iron atoms directly. The main contributors to these modes are probably the C---C double bonds around the periphery of the porphyrin rings. It is not obvious why such modes should be particularly sensitive to the position of the iron atom. There is structural evidence to suggest that movement of the iron atom out of the plane is accompanied by a slight movement of the pyrrole nitrogen atoms in the same direction, leading to a degree of doming and puckering of the porphyrin ring 7. Conceivably structural changes of this kind could force particular vibrational modes in or out of resonance. In any event the effect is clearcut experimentally and could be exploited as a structural probe in solution. Inasmuch as the movement of the iron atom in and out of the heine plane appears to trigger the cooperative binding of oxygen to hemoglobin s, such a probe could be useful in mechanistic studies. Frequency assignments
The spectra exhibit an impressive richness of detail. While resonance Raman spectra sometimes display intense overtone progressions ~'2, these are not apparent in the hemoglobin spectra, All the prominent bands presumably arise from fundamental vibrational modes of the heine groups, since the vibrational modes enhanced by resonance scattering are those associated with the chromophore. The frequencies are all between 600 and 1650 cm-~ and can be assigned to a variety of ring stretching and deformation modes ~°. Such assignments are very approximate in view of the undoubtedly high degree of internal coordinate mixing in these complex molecules. Azidometmyoglobin gives a spectrum (not shown) virtually identical to that of azidomethemoglobin demonstrating, as expected, that vibrations of separate heme groups in hemoglobin do not interact. Another notable feature of the spectra is that the frequencies of corresponding bands do not vary by more than 5 cm-~ for any of the hemoglobin derivatives. Although changes in either spin state or oxidation state of the iron atoms have marked effects on the electronic spectra s, they do not seem to alter significantly the electron-density distribution on the porphyrin ring. Otherwise we would expect more substantial shifts in the vibrational frequencies. Resonance Raman spectra are frequently obscured by fluorescence ~'3. We found this to be the case for cyanomethemoglobin. Weak fluorescence can also be seen in the spectra of aquomethemoglobin (broad peak centered at approx. 5940 A) and of deoxyBiochim. Biophys. Acts, 263 (1972) 830-833
BBA REPORT
833
hemoglobin (base line rises toward longer wavelengths). It is unclear whether these effects are intrinsic to the derivatives or arise from impurities. We had hoped to observe vibrations from the ligands bound to the iron a t o m above and below the plane o f the porphyrin ring, but none can be found in our spectra. Apparently these vibrations do not meet the resonance condition. In the case o f carboxyhemoglobin, the C - O stretching mode has been observed in the 1 9 5 0 - 2 0 0 0 - c m -1 region in infrared spectra 11. We examined this region carefully but failed to detect any Raman emission*. Nor is there any evidence for modes arising from the stretching o f bonds between the iron a t o m and the pyrrole nitrogen atoms, which might be expected between 300 and 500 cm -1 . For all the reported spectra the region below 600 cm -1 is blank. This work was supported b y Public Health Service Grant HE 12526 from the National Heart and Lung Institute. We thank Professor Chien Ho for advice on preparing the hemoglobin samples and Professors J.J. Hopfield and I.D. Kuntz for helvful discussions. REFERENCES 1 Behringer, J. (1967) in Raman Spectroscopy (Szymanski, H.A., exl.), Chapter 6, Henum Press, New York 2 Gill, D., Kilponen, R.G. and Rimai, L. (1970)Nature 227, 743 3 Rimai, L., Gill, D. and Parsons, J. (1971) J. Am. Chem. Soc., 93, 1353 4 Long, T.V., Loehr, T.M., Alkins, J.R. and Lovenberg, W. (1971) Z Am. Chem. Soc. 92, 1809 5 Smith, D.W. and Williams, R.J.P. (1970) in Structure and Bonding, Vol. 7, pp.l-45, SpringerVerlag, Berlin 6 Long, T.V. (1969) in MCJssbauerSpectroscopy in Biological Systems, Univ. Ill. Bull. 67, (41), 59 7 Hoard, J.L. (1971)Science 174, 1295 8 Perutz, M.F. (1970) Nature 228,726 9 Brunoni, M., Amieoni, G., Antonini, E., Wyman, J., Zito, R. and Rossi Fandli, A. (1968) Biochint Biophys. Acta 154, 315 10 Boucher, L.J. and Katz, J.J. (1967) J. Am. Chem. Soc. 89, 1340 11 McCoy, S. and Caughey, W.S. (1970) Biochemistry 9, 2387 12 Noble, R.W., Brunoni, M., Wyman, J. and Antonini, E. (1967) Biochemistry 6, (4), 1216
* In view of the well-known propensity of carboxyhemoglobin to ph0todissociatel2, we were concerned that our laser excitation might produce a substantial degree of dissociation. A rough calculation suggests, however, that the fraction of carboxyhemoglobin molecules dissociated could be no greater than 10%. Biochim. Biophys. Acta, 263 (1972) 830-833
BIOCHIMICAET BIOPHYSICAACTA
BBA Report BBA 31134 H e m o g l o b i n : R e s o n a n c e R a m a n spectra
THOMASC. STREKASand THOMASG. SPIRO Department of Chemistry, Princeton University, Princeton, N.J. (U.S.A.) (Received April 4th, 1972)
SUMMARY Raman spectra have been recorded for several hemoglobin derivatives in dilute solution. They exhibit a complex set of bands, arising from vibrations of the heine groups. Two of the bands offer promise as structural probes, inasmuch as their intensities correlate inversely with the degree to which the iron atoms are out of plane.
We have observed resonance Raman scattering 1 from dilute solutions (approx. 10-4 M) of hemoglobin derivatives. The technique may provide a solution structural probe for hemoglobin, and should be applicable to other heme proteins as well. Resonant enhancement makes possible the application of Raman spectroscopy, normally a lowsensitivity technique, to biological chromophores in their usual high dilutions. Recent applications include studies of carotenoid pigments 2 , vitamin A-type molecules 3 and rubridoxin 4 . Our spectra are shown in Fig.1. The exciting wavelength, 5682 A, falls in the middle of the visible absorption envelopes of all the hemoglobin derivatives s . Similar spectra, but of lower intensity, were obtained with the 5308-A and 4880-A laser lines, which lie on the high-frequency side of the visible envelopes, but below the Sorer bands. Excitation at 6471 A, below the visible bands, failed to give observable Raman scattering even at concentrations approaching 10-z M, Several Raman bands have been reported by Longs for aqueous myoglobin, using 6328 A He-Ne laser excitation. They are all below 600 cm-1 , a region which is blank in all of our spectra. Intensity-structure correlation Although the spectra of the various derivatives are similar, some of the bands do show substantial changes in their relative intensities. Of particular interest are the bands observed for oxy- and carboxyhemoglobin at 1638 and 1589 cm-1 , the latter being the Biochim. Biophys. Acta, 263 (1972) 830-833
BBA REPORT
831 MetHbH20
~8, ~
DEOXY Hb
HbCO
~-~ ~ ; ~ ~'~bo ~1~.~ ~ ~ ' ~
T~
~
GM -~
Fig.1. Raman spectra of human hemoglobin derivatives obtained with 5682 ~ excitation (20-60 mW) from an Ar÷/Kr+ mixed gas laser. Aqueous solutions of oxy-, ea~boxy- and deoxyhemoglobin, and aquo-(pH 7) and azidomethemoglobin, were prepared by standard methods. Concentrations were approx. 10-4 M in iron, measured spectrophotometricaUy. The samples were contained in l-ram glass capillary tubes, and spectra were recorded with transverse excitation, Clouding of the solutions was observed with excitation at higher frequencies (5308 and 4880/~) but not at 5682 ~l,. At this wavelength repeated scans gave identical, spectra, Instrument conditions: slit width, 10 cm-1; sensitivity, 10~s A; scan rate, 50 ern-1]min; time constant, 1.0 s. most intense feature of the spectra. These bands are missing for deoxyhemoglobin. Evidently the corresponding vibrational modes go out o[ resonance when hemoglobin is deoxygenated. The two bands reappear at full strength in azidomethemoglobin, a lowspin Fe(III) derivative. Their intensity is appreciably diminished in aquomethemoglobin, which is high.spin. It is tempting to associate these variations with the accompanying structural changes ~'s. The iron atom lies in the plane of the pyrrole nitrogen atoms in the low-spin hemoglobin derivatives, whether Fe(II) or Fe(III). In the high-spin derivatives the iron atom, presumably because of its increased radius, is significantly out o f the plane.
Biochim. Biophys. Acta, 263 (1972) 830-833
832
BBA REPORT
The out-of-planarity is greater for Fe(II) than for Fe(III), m accora with the greater radius of the former. Perutz s estimates the out-of-plane distance as approx. 0.8 A in deoxyhemoglobin and approx. 0.3 ~k in aquomethemoglobin. The 1638- and 1589-cm-1 bands correlate well with these features, the in-plane derivatives giving full intensity, aquomethemoglobin giving diminished intensity and deoxyhemoglobin showing no intensity. Moreover we have found that raising the pH of aquomethemoglobin produces an increase in the intensity of these bands, as the predominantly low-spin hydroxymethemoglobin is formed. Indeed a titration curve of intensity vs pH tracks the corresponding variation in spin state 9, with a pKa of approx. 8.5. The frequencies of these indicator bands are much too high for vibrations involving the iron atoms directly. The main contributors to these modes are probably the C---C double bonds around the periphery of the porphyrin rings. It is not obvious why such modes should be particularly sensitive to the position of the iron atom. There is structural evidence to suggest that movement of the iron atom out of the plane is accompanied by a slight movement of the pyrrole nitrogen atoms in the same direction, leading to a degree of doming and puckering of the porphyrin ring 7. Conceivably structural changes of this kind could force particular vibrational modes in or out of resonance. In any event the effect is clearcut experimentally and could be exploited as a structural probe in solution. Inasmuch as the movement of the iron atom in and out of the heine plane appears to trigger the cooperative binding of oxygen to hemoglobin s, such a probe could be useful in mechanistic studies. Frequency assignments
The spectra exhibit an impressive richness of detail. While resonance Raman spectra sometimes display intense overtone progressions ~'2, these are not apparent in the hemoglobin spectra, All the prominent bands presumably arise from fundamental vibrational modes of the heine groups, since the vibrational modes enhanced by resonance scattering are those associated with the chromophore. The frequencies are all between 600 and 1650 cm-~ and can be assigned to a variety of ring stretching and deformation modes ~°. Such assignments are very approximate in view of the undoubtedly high degree of internal coordinate mixing in these complex molecules. Azidometmyoglobin gives a spectrum (not shown) virtually identical to that of azidomethemoglobin demonstrating, as expected, that vibrations of separate heme groups in hemoglobin do not interact. Another notable feature of the spectra is that the frequencies of corresponding bands do not vary by more than 5 cm-~ for any of the hemoglobin derivatives. Although changes in either spin state or oxidation state of the iron atoms have marked effects on the electronic spectra s, they do not seem to alter significantly the electron-density distribution on the porphyrin ring. Otherwise we would expect more substantial shifts in the vibrational frequencies. Resonance Raman spectra are frequently obscured by fluorescence ~'3. We found this to be the case for cyanomethemoglobin. Weak fluorescence can also be seen in the spectra of aquomethemoglobin (broad peak centered at approx. 5940 A) and of deoxyBiochim. Biophys. Acts, 263 (1972) 830-833
BBA REPORT
833
hemoglobin (base line rises toward longer wavelengths). It is unclear whether these effects are intrinsic to the derivatives or arise from impurities. We had hoped to observe vibrations from the ligands bound to the iron a t o m above and below the plane o f the porphyrin ring, but none can be found in our spectra. Apparently these vibrations do not meet the resonance condition. In the case o f carboxyhemoglobin, the C - O stretching mode has been observed in the 1 9 5 0 - 2 0 0 0 - c m -1 region in infrared spectra 11. We examined this region carefully but failed to detect any Raman emission*. Nor is there any evidence for modes arising from the stretching o f bonds between the iron a t o m and the pyrrole nitrogen atoms, which might be expected between 300 and 500 cm -1 . For all the reported spectra the region below 600 cm -1 is blank. This work was supported b y Public Health Service Grant HE 12526 from the National Heart and Lung Institute. We thank Professor Chien Ho for advice on preparing the hemoglobin samples and Professors J.J. Hopfield and I.D. Kuntz for helvful discussions. REFERENCES 1 Behringer, J. (1967) in Raman Spectroscopy (Szymanski, H.A., exl.), Chapter 6, Henum Press, New York 2 Gill, D., Kilponen, R.G. and Rimai, L. (1970)Nature 227, 743 3 Rimai, L., Gill, D. and Parsons, J. (1971) J. Am. Chem. Soc., 93, 1353 4 Long, T.V., Loehr, T.M., Alkins, J.R. and Lovenberg, W. (1971) Z Am. Chem. Soc. 92, 1809 5 Smith, D.W. and Williams, R.J.P. (1970) in Structure and Bonding, Vol. 7, pp.l-45, SpringerVerlag, Berlin 6 Long, T.V. (1969) in MCJssbauerSpectroscopy in Biological Systems, Univ. Ill. Bull. 67, (41), 59 7 Hoard, J.L. (1971)Science 174, 1295 8 Perutz, M.F. (1970) Nature 228,726 9 Brunoni, M., Amieoni, G., Antonini, E., Wyman, J., Zito, R. and Rossi Fandli, A. (1968) Biochint Biophys. Acta 154, 315 10 Boucher, L.J. and Katz, J.J. (1967) J. Am. Chem. Soc. 89, 1340 11 McCoy, S. and Caughey, W.S. (1970) Biochemistry 9, 2387 12 Noble, R.W., Brunoni, M., Wyman, J. and Antonini, E. (1967) Biochemistry 6, (4), 1216
* In view of the well-known propensity of carboxyhemoglobin to ph0todissociatel2, we were concerned that our laser excitation might produce a substantial degree of dissociation. A rough calculation suggests, however, that the fraction of carboxyhemoglobin molecules dissociated could be no greater than 10%. Biochim. Biophys. Acta, 263 (1972) 830-833