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Evidence of heme-heme interaction in heme-spin-labeled hemoglobin
Vo[. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCE OF HEME-HEME INTERACTIONIN HEME-SPIN-LABELEDHEMOGLOBIN T. Asakura and H. R. Drott Johnson Research Foundation, U n i v e r s i t y o f Pennsylvania P h i l a d e l p h i a , Pennsylvania 19104
Received J u l y 8 , 1971 Stmnar7: Two kinds o f valency hybrid hemoglobins c o n t a i n i n g s p i n - l a b e l e d ferric heme in one type of subunit were orepared. The electron paramagnetic +++ ~+ 4+ +++ . resonance spectra of the a-(~SLFe -BFe 02) and B-(a-Fe 02-~SLFe ) spznlabeled hemoglobins were different, suggesting their non-equzvalent conformational properties in hemoglobin. Deoxygenation of a-spin-labeled hemoglobin altered the resonance amplitude and the line shape reversibly while that of B-spin-labeled hemoglobin altered the line shape. The change in a-spin-labeled hemoglobin is explained by either change in the heme-label distance or the relaxation time of the heme iron in the a-subunit upon oxygen binding to the B-subunit. INTRODUCTION The cooperative oxygen binding to hemoglobin, where the binding ofoxygen to one subunit of hemoglobin alters the successive oxygen affinities of the other three subunits, has been called for a long time as heme-heme interaction (I).
Since the hemes in hemoglobin are separated by distances of the order
of 25-37 ~ (2) it is believed that the molecular basis for the cooperative interaction is caused indirectly bY ligand-induced conformational changes in the protein (3). Several models for the cooperativity of hemoglobin have been proposed and reviewed by Antonini and Brunori (3).
A very explicit description
of the stereochemical mechanism of heme-heme interaction based on the x-ray study of crystalline deoxy- and met-hemoglobin has been proposed by Perutz (4). 0gawa et al. (5) reported that oxygen binding to a-subunit of B-93-spin-labeled mixed state hemoglobin (aFe++-B*Fe+++CN) alters the electron paramagnetic resonance (EPR) spectrum of the spin label upon binding of oxygen to the a-subunit. Nayashi et al. (6) observed the splitting of the g = 6 BPR iron signal belonging to the abnormal B-chains of Hb-Hyde Park when the normal ~-subunit was deoxygenated However, they did not detect any effect from B to a subunit in the corresponding
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Vol. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hb-M having a similar substitution in the a-subunit.
More recently, Ogawa
and Shulman (7) observed the changes in the NMR spectra of a-met-mixed state hemoglobin upon binding of oxygen to the ~-subunit.
However, no effect of oxygen
bifiding to a-subunit was observed in the E-met-mixed state hemoglobin. All these results support the idea that the binding of oxygen to one type of subunit results in a conformational change in the partner subunit.
Nevertheless
there is a discrepancy in the direction of the ligand-induced interaction between and 8 hemoglobin subunits. Recently, we have developed a new technique of heme-spin-labeling in which the prosthetic groups of various hemoproteins are directly spin-labeled with nitroxyl free radicals at the different positions of the porphyrin ring (8-10). The EPR spectrum of the spin-label attached to macromolecules is sensitive to the local environment of the labels (Ii) and provides strucutral and dynamic information about the conformation of hemoproteins in solution or crystalline states.
EPR spectra of heme-spin-labeled hemoproteins have been used to differ~
entiate the heme environments of various hemoproteins such as hemoglobin, ~yoglobiE cytochrome !peroxidase
and horse-radish peroxidase [8, i0).
purthermore, from
the strength of the magnetic interaction of the labels with the closel Z located heme iron, the distances between the spin-label and the heme iron in dissolved hemoproteins have been estimated
(10). The calculated distance in spin-labeled
hemoglobin was in good agreement with that measured from Perutz's hemoglobin model. In this paper, the heme-spin-labeling technique is applied to studies of heme-heme interaction in hemoglobin.
Since spin-labeled heme can be attached
to either type of hemoglobin subunits selectively, mixed-state hemoglobins can be prepared in which one type of subunit contains spin-labeled ferric heme while the other has native ferrous heme.
Using these valence hybrids, we can investigate
whether or not oxygen binding to one type of suhunit causes a change in the heme environment of the other.
The result shows definite changes in the heme
environment upon binding of oxygen to the partner subunit.
1200
Vol. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Spin-labeled protohemin was prepared from protohemin and 2,2,5,5-tetra-
methyl-S-amino-pyrrolidine-l-oxyl
as described previously (8). Carbonmonoxy-
hemoglobin was separated into its ~ and 8 subunits by the method of Bucci and Fronticelli (12). p-Chloromercuribenzoate was removed from the subunits according to De Renzo et al. (13). Apohemoproteins were prepared by the modified procedure (14) of Teale's acid-butanone method (15). Recombination of the aposubunits with spin-labeled hemin was carried out as described elsewhere (8,16). Spin-labeled e- or 8-subunit was mixed with approximately equal amounts of oxy-form of the partner subunit at pH 7.0 for 10 minutes.
The mixture was then
dialyzed against I0 mM potassium phosphate buffer, pH 6.0, and purified by chromatography on carboxymethyl cellulose.
The hybrid hemoglobin eluted from
the column with 0.2 M potassium phosphate buffer, pH 7.0, showed a single band corresponding to native tetramer hemoglobin by electrophoresis.
The optical
spectrum of the hybrid hemoglobin is that of a typical valency hybrid hemoglobin having an intermediate spectrum between that of oxy- and that of met-hemoglobin (17) RESULTS Electron paramagnetic resonance spectra of e and 8 spin-labeledhemoglobins in 0.i M potassium phosphate buffer, pH 7.0, are shown in Fig. I.
It should be
noted a slight difference in the mobility of the label between the two kinds of mixed state hemoglobins indicating the non-equivalent conformational properties of the two subunits in hemoglobin molecule.
Upon addition of ~luoride which
forms a high spin complex with ferric heme to ~spin~laheled hemoglobin~ the resonance amplitude decreases to less than half without: significant changes in the line shape (i0). Removal of oxygen from this system resulted in the change in the outer line shape as shown by an arrow B in Fig. i.
More drastic changes
in the EPR spectrum occur when oxygen is removed from e-spin-labeled hemoglobin. Figure 2B shows a drastic decrease in the central resonance amplitude relative to IA.
There is also evidence of a slightly less immobilized component in the
spectrum.
The spectral changes were entirely reversible upon re-addition of oxygen
1201
Vol. 44, No. 5, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(aFe't02'3SLFe'*') V
~V//
-- """-
8
Fig. 1. EPR spectra of e- and 8-spin-labeled hemoglobin in 0.I M potassium phosphate buffer, pH 7.0, at 23 ° . Microwave frequency of 9.08 GHz and modulation amplituae of 0.8 G were
.
r
2O G
I
used.
to the System (Fig. 2C). was denatured.
The experiment could be repeated until hemoglobin
The extent of changes in the signal amplitude observed in e-
spin-labeled hemoglobin is dependent on the spin state of ferric heme iron. Addition of fluoride potentiates the oxygen effect while the addition of cyanide abolishes the effect.
Therefore, it is reasonable to attrubute this to the
magnetic dipolar interaction between paramagnetic metal ion and spin labels (10,18,19).
High spin fluoride heme has a larger magnetic moment as well as
longer electron spin relaxation time than the low spin complexes and heme would give a stronger dipolar interaction with closely located spin-labels.
This
interaction is manifested in a decrease in the central resonance amplitude
(18).
Low spin complex of cyanide has a smaller magnetic moment and a shorter relaxation time and is, therefore, expected to show much smaller effects.
The change in the
signal amplitude can be explained either by change in the distance between the heme iron and the spin-label or by change in the relaxation time of the ferric heme iron.
1202
Vol. 44, No. 5, 1971
o(Fe'")SL- .~(Fe")O2 Hb/~
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
( 25 °)
SL
._zj I--1
- 02
SL
SL
~
F-I ,
I
L_J
[-q
, ,
÷ 0 2
SL~p_2j ~
r__l
SL (Fd÷~Fe'~ r--n,O,a''
20
G
Fig. 2. EPR spectra of a-spin-labeled hemoglobin in the presence or absence of oxygen at 25 °. The hemoglobin was dissolved in 0.i M potassium phosphate buffer, pH 7.0. Microwave frequency of 9.08 GHz and modulation amplitude of 0.8 G were used.
Thus, these results indicate that oxygen binding to one type of subunit alters the heine environment of the partner subunit, although the nature of the interaction appears to be different between a and 8 subunits.
It is quite reasonable to
think that such a change in the vicinity of the heme upon oxygen binding to the partner subunit might alter the affinity of the heine for the oxygen.
Further
studies on the mechanism of heme-heme interaction of hemoglobin are under progress using spin-labeled and mixed-state hemoglobins.
ACKNOWLEDGEMENTS
We wish to thank Drs. B. Chance, T. Yonetani and G. Reed for their kind advice and discussions. This work was supported by Research Grant GM 12202 and GM 1543S from the United States Public Health Service. T.A. is a recipient of Career Development Award I-K4-GM-47, 463-01 from the United States Public Health Service. HcR.D. is a recipient of National Institute of Health Fellowship 5-F02 HE 39533 from the National Heart Institute.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
i. 2. 3. 4. S. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19.
Bohr, C., Zentr. Physiol. 17, 682 (1903] Perutz, M. F., Proc. Roy. Soc. B 175, I13 (1969) Antonini, E. and Brunori, M., Ann. Rev. Biochem. 59, 977 (1970] Perutz, M. F., Nature 21, 726 (1970) Ogawa, S., McConnell, H.M. and Horwitz, A., Proc. Nat. Acad. Sci. (U.S.) 61, 401 (1968) Hayashi, A., Suzuki, T., Shimizu, A., Morimoto, H., and Watari, H., Biochim. Biophys. Acta., 147, 407 (1967) Ogawa, S. and Shulman, R. G., Biochem. Biophys. Res. Commun. 42, 9 (1971) Asakura, T., Drott, H.R. and Yonetani, C., J. Biol. Chem. 244, 6626 (1969) Asakura, T., Drott, H.R., and Yonetani, C., in Probes of Enzymes and Hemoproteins, edited by B. Chance, T. Yonetani, and A.S. Mildvan, Academic Press, N.Y., 1971 Asakura, T., Leigh, J.S., Drott, H.R., Yonetani, T., and Chance, B., Proc. Nat. ~cad. Sci. (U.S.) 68, 861 (1971) Hamilton, C.L. and McConnell-~--H.M., in Structural Chemistry and Molecular Biology, A., edited by A. Rich and N. Davidson, W.H. Freeman and Company, San Francisco, 1968, p. 115 Bucci, E. and Fronticelli, C., J. Biol. Chem. 240, PC551 (1965) De Renzo, E.C., loppola, C., Amiconi, G., Antonln--:--i,E. and Wyman, J., J. Biol. Chem. 242, 4850 (1967) Yonetani, T., J. Biol. Chem. 242, 5008 f1967) Teale, F. W. J., Biochim. Biop-~s. Acta. 35, 545 [1959] Asakura, T. and Yonetani, T,, J. Biol Chem. 244, 4575 (1969] Banerjee, R. and Ca~oly, R., J. Mol. Biol. 42~-~51 ~969) Leigh, J. S., J. Chem. Phys. 52, 2608 09707-Taylor, J.S., Leigh, J. S. and Cohn, M., Proc. Nat. Acad. Sei. [U.S.)64, 219 (1969]
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCE OF HEME-HEME INTERACTIONIN HEME-SPIN-LABELEDHEMOGLOBIN T. Asakura and H. R. Drott Johnson Research Foundation, U n i v e r s i t y o f Pennsylvania P h i l a d e l p h i a , Pennsylvania 19104
Received J u l y 8 , 1971 Stmnar7: Two kinds o f valency hybrid hemoglobins c o n t a i n i n g s p i n - l a b e l e d ferric heme in one type of subunit were orepared. The electron paramagnetic +++ ~+ 4+ +++ . resonance spectra of the a-(~SLFe -BFe 02) and B-(a-Fe 02-~SLFe ) spznlabeled hemoglobins were different, suggesting their non-equzvalent conformational properties in hemoglobin. Deoxygenation of a-spin-labeled hemoglobin altered the resonance amplitude and the line shape reversibly while that of B-spin-labeled hemoglobin altered the line shape. The change in a-spin-labeled hemoglobin is explained by either change in the heme-label distance or the relaxation time of the heme iron in the a-subunit upon oxygen binding to the B-subunit. INTRODUCTION The cooperative oxygen binding to hemoglobin, where the binding ofoxygen to one subunit of hemoglobin alters the successive oxygen affinities of the other three subunits, has been called for a long time as heme-heme interaction (I).
Since the hemes in hemoglobin are separated by distances of the order
of 25-37 ~ (2) it is believed that the molecular basis for the cooperative interaction is caused indirectly bY ligand-induced conformational changes in the protein (3). Several models for the cooperativity of hemoglobin have been proposed and reviewed by Antonini and Brunori (3).
A very explicit description
of the stereochemical mechanism of heme-heme interaction based on the x-ray study of crystalline deoxy- and met-hemoglobin has been proposed by Perutz (4). 0gawa et al. (5) reported that oxygen binding to a-subunit of B-93-spin-labeled mixed state hemoglobin (aFe++-B*Fe+++CN) alters the electron paramagnetic resonance (EPR) spectrum of the spin label upon binding of oxygen to the a-subunit. Nayashi et al. (6) observed the splitting of the g = 6 BPR iron signal belonging to the abnormal B-chains of Hb-Hyde Park when the normal ~-subunit was deoxygenated However, they did not detect any effect from B to a subunit in the corresponding
1199
Vol. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hb-M having a similar substitution in the a-subunit.
More recently, Ogawa
and Shulman (7) observed the changes in the NMR spectra of a-met-mixed state hemoglobin upon binding of oxygen to the ~-subunit.
However, no effect of oxygen
bifiding to a-subunit was observed in the E-met-mixed state hemoglobin. All these results support the idea that the binding of oxygen to one type of subunit results in a conformational change in the partner subunit.
Nevertheless
there is a discrepancy in the direction of the ligand-induced interaction between and 8 hemoglobin subunits. Recently, we have developed a new technique of heme-spin-labeling in which the prosthetic groups of various hemoproteins are directly spin-labeled with nitroxyl free radicals at the different positions of the porphyrin ring (8-10). The EPR spectrum of the spin-label attached to macromolecules is sensitive to the local environment of the labels (Ii) and provides strucutral and dynamic information about the conformation of hemoproteins in solution or crystalline states.
EPR spectra of heme-spin-labeled hemoproteins have been used to differ~
entiate the heme environments of various hemoproteins such as hemoglobin, ~yoglobiE cytochrome !peroxidase
and horse-radish peroxidase [8, i0).
purthermore, from
the strength of the magnetic interaction of the labels with the closel Z located heme iron, the distances between the spin-label and the heme iron in dissolved hemoproteins have been estimated
(10). The calculated distance in spin-labeled
hemoglobin was in good agreement with that measured from Perutz's hemoglobin model. In this paper, the heme-spin-labeling technique is applied to studies of heme-heme interaction in hemoglobin.
Since spin-labeled heme can be attached
to either type of hemoglobin subunits selectively, mixed-state hemoglobins can be prepared in which one type of subunit contains spin-labeled ferric heme while the other has native ferrous heme.
Using these valence hybrids, we can investigate
whether or not oxygen binding to one type of suhunit causes a change in the heme environment of the other.
The result shows definite changes in the heme
environment upon binding of oxygen to the partner subunit.
1200
Vol. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Spin-labeled protohemin was prepared from protohemin and 2,2,5,5-tetra-
methyl-S-amino-pyrrolidine-l-oxyl
as described previously (8). Carbonmonoxy-
hemoglobin was separated into its ~ and 8 subunits by the method of Bucci and Fronticelli (12). p-Chloromercuribenzoate was removed from the subunits according to De Renzo et al. (13). Apohemoproteins were prepared by the modified procedure (14) of Teale's acid-butanone method (15). Recombination of the aposubunits with spin-labeled hemin was carried out as described elsewhere (8,16). Spin-labeled e- or 8-subunit was mixed with approximately equal amounts of oxy-form of the partner subunit at pH 7.0 for 10 minutes.
The mixture was then
dialyzed against I0 mM potassium phosphate buffer, pH 6.0, and purified by chromatography on carboxymethyl cellulose.
The hybrid hemoglobin eluted from
the column with 0.2 M potassium phosphate buffer, pH 7.0, showed a single band corresponding to native tetramer hemoglobin by electrophoresis.
The optical
spectrum of the hybrid hemoglobin is that of a typical valency hybrid hemoglobin having an intermediate spectrum between that of oxy- and that of met-hemoglobin (17) RESULTS Electron paramagnetic resonance spectra of e and 8 spin-labeledhemoglobins in 0.i M potassium phosphate buffer, pH 7.0, are shown in Fig. I.
It should be
noted a slight difference in the mobility of the label between the two kinds of mixed state hemoglobins indicating the non-equivalent conformational properties of the two subunits in hemoglobin molecule.
Upon addition of ~luoride which
forms a high spin complex with ferric heme to ~spin~laheled hemoglobin~ the resonance amplitude decreases to less than half without: significant changes in the line shape (i0). Removal of oxygen from this system resulted in the change in the outer line shape as shown by an arrow B in Fig. i.
More drastic changes
in the EPR spectrum occur when oxygen is removed from e-spin-labeled hemoglobin. Figure 2B shows a drastic decrease in the central resonance amplitude relative to IA.
There is also evidence of a slightly less immobilized component in the
spectrum.
The spectral changes were entirely reversible upon re-addition of oxygen
1201
Vol. 44, No. 5, 1971
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(aFe't02'3SLFe'*') V
~V//
-- """-
8
Fig. 1. EPR spectra of e- and 8-spin-labeled hemoglobin in 0.I M potassium phosphate buffer, pH 7.0, at 23 ° . Microwave frequency of 9.08 GHz and modulation amplituae of 0.8 G were
.
r
2O G
I
used.
to the System (Fig. 2C). was denatured.
The experiment could be repeated until hemoglobin
The extent of changes in the signal amplitude observed in e-
spin-labeled hemoglobin is dependent on the spin state of ferric heme iron. Addition of fluoride potentiates the oxygen effect while the addition of cyanide abolishes the effect.
Therefore, it is reasonable to attrubute this to the
magnetic dipolar interaction between paramagnetic metal ion and spin labels (10,18,19).
High spin fluoride heme has a larger magnetic moment as well as
longer electron spin relaxation time than the low spin complexes and heme would give a stronger dipolar interaction with closely located spin-labels.
This
interaction is manifested in a decrease in the central resonance amplitude
(18).
Low spin complex of cyanide has a smaller magnetic moment and a shorter relaxation time and is, therefore, expected to show much smaller effects.
The change in the
signal amplitude can be explained either by change in the distance between the heme iron and the spin-label or by change in the relaxation time of the ferric heme iron.
1202
Vol. 44, No. 5, 1971
o(Fe'")SL- .~(Fe")O2 Hb/~
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
( 25 °)
SL
._zj I--1
- 02
SL
SL
~
F-I ,
I
L_J
[-q
, ,
÷ 0 2
SL~p_2j ~
r__l
SL (Fd÷~Fe'~ r--n,O,a''
20
G
Fig. 2. EPR spectra of a-spin-labeled hemoglobin in the presence or absence of oxygen at 25 °. The hemoglobin was dissolved in 0.i M potassium phosphate buffer, pH 7.0. Microwave frequency of 9.08 GHz and modulation amplitude of 0.8 G were used.
Thus, these results indicate that oxygen binding to one type of subunit alters the heine environment of the partner subunit, although the nature of the interaction appears to be different between a and 8 subunits.
It is quite reasonable to
think that such a change in the vicinity of the heme upon oxygen binding to the partner subunit might alter the affinity of the heine for the oxygen.
Further
studies on the mechanism of heme-heme interaction of hemoglobin are under progress using spin-labeled and mixed-state hemoglobins.
ACKNOWLEDGEMENTS
We wish to thank Drs. B. Chance, T. Yonetani and G. Reed for their kind advice and discussions. This work was supported by Research Grant GM 12202 and GM 1543S from the United States Public Health Service. T.A. is a recipient of Career Development Award I-K4-GM-47, 463-01 from the United States Public Health Service. HcR.D. is a recipient of National Institute of Health Fellowship 5-F02 HE 39533 from the National Heart Institute.
1203
Vol. 44, No. 5, 1 9 7 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
i. 2. 3. 4. S. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16. 17. 18. 19.
Bohr, C., Zentr. Physiol. 17, 682 (1903] Perutz, M. F., Proc. Roy. Soc. B 175, I13 (1969) Antonini, E. and Brunori, M., Ann. Rev. Biochem. 59, 977 (1970] Perutz, M. F., Nature 21, 726 (1970) Ogawa, S., McConnell, H.M. and Horwitz, A., Proc. Nat. Acad. Sci. (U.S.) 61, 401 (1968) Hayashi, A., Suzuki, T., Shimizu, A., Morimoto, H., and Watari, H., Biochim. Biophys. Acta., 147, 407 (1967) Ogawa, S. and Shulman, R. G., Biochem. Biophys. Res. Commun. 42, 9 (1971) Asakura, T., Drott, H.R. and Yonetani, C., J. Biol. Chem. 244, 6626 (1969) Asakura, T., Drott, H.R., and Yonetani, C., in Probes of Enzymes and Hemoproteins, edited by B. Chance, T. Yonetani, and A.S. Mildvan, Academic Press, N.Y., 1971 Asakura, T., Leigh, J.S., Drott, H.R., Yonetani, T., and Chance, B., Proc. Nat. ~cad. Sci. (U.S.) 68, 861 (1971) Hamilton, C.L. and McConnell-~--H.M., in Structural Chemistry and Molecular Biology, A., edited by A. Rich and N. Davidson, W.H. Freeman and Company, San Francisco, 1968, p. 115 Bucci, E. and Fronticelli, C., J. Biol. Chem. 240, PC551 (1965) De Renzo, E.C., loppola, C., Amiconi, G., Antonln--:--i,E. and Wyman, J., J. Biol. Chem. 242, 4850 (1967) Yonetani, T., J. Biol. Chem. 242, 5008 f1967) Teale, F. W. J., Biochim. Biop-~s. Acta. 35, 545 [1959] Asakura, T. and Yonetani, T,, J. Biol Chem. 244, 4575 (1969] Banerjee, R. and Ca~oly, R., J. Mol. Biol. 42~-~51 ~969) Leigh, J. S., J. Chem. Phys. 52, 2608 09707-Taylor, J.S., Leigh, J. S. and Cohn, M., Proc. Nat. Acad. Sei. [U.S.)64, 219 (1969]
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