[19] The study of hemoglobin by electron paramagnetic resonance spectroscopy

[19] The study of hemoglobin by electron paramagnetic resonance spectroscopy

312 SPECTROSCOPICPROPERTIES OF HEMOGLOBINS [19] bis-Tris plus 26 mM DPG in DzO at pH 7.0 and at 24°. This clearly shows that there is considerable ...

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SPECTROSCOPICPROPERTIES OF HEMOGLOBINS

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bis-Tris plus 26 mM DPG in DzO at pH 7.0 and at 24°. This clearly shows that there is considerable improvement in the resolution of the aromatic and ring-current-shifted proton resonances as compared to the spectra obtained at 250 or 360 MHz (for example, see Figs. 2, 3, and 7). It should be pointed out that for some 1H resonances of Hb, e.g., the hfs resonances, the enhancement of the resolution attainable at higher magnetic fields is offset by a corresponding increase in their line widths/r so that no net improvement in the 1H NMR spectra can be achieved using this approach. Other directions for improving the 1H NMR spectra consist of the use of specialized NMR techniques to (a) observe proton resonances closer to the water proton resonance; (b) measure more accurately the intensities of the 1H resonances; and (c) improve the resolution of the 1H NMR spectra by distinguishing among resonances of different line widths. Acknowledgment We wish to thank Dr. Susan R. Dowd for helpful discussionsduringthe preparation of this manuscript. The writingof this chapter was supported by research grants from the National Institutes of Health (HL-24525) and the National Science Foundation (PCM 78-25818).

[19] T h e S t u d y o f H e m o g l o b i n b y E l e c t r o n Paramagnetic Resonance Spectroscopy

By WILLIAM E. BLUMBERG Electron paramagnetic resonance (EPR) has been used in the study of the biochemistry of metalloproteins as a means of elucidating certain physical and chemical properties of the metal sites. Applications of EPR to biochemistry can be divided into two classes. The first includes those studies that give qualitative information concerning the presence of paramagnetic metal atoms and the changes that they may undergo, without consideration of quantitative information concerning the chemical physics relating to their physical environments. In other words, EPR spectroscopy can be used as an empirical spectroscopy, just as optical and CD spectra are most often used by the biochemist. The second class includes those studies that yield information concerning the specific physical environments of metal atoms in metalloproteins. Electron paramagnetic resonance absorption spectra have been observed not only for the iron atom of hemoglobin, but also for hemoglobin substituted with other paramagnetic metal atoms as well as for free radicals attached to the protein. METHODS IN ENZYMOLOGY, VOL. 76

Copyright (~ 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181976-0

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In order to understand the physical interpretation of EPR spectra, there is no substitute for a thorough understanding of the quantum mechanical nature of the phenomenon. Such an exposition is beyond the scope of this chapter, and the reader is referred to an excellent elementary book, 1 to more advanced works, 2-4 and to a book addressed to biological applications, ~ although it specifically avoids the discussion of the EPR of heme proteins. A series of volumes giving literature surveys of applications of EPR spectroscopy appears regularly.6 The cases in which EPR spectra are the most tractable for physical interpretation are those in which no crystal field splitting is present. This means that all such spectra are interpretable in terms of an effective spin of ½. Then the magnetic field H, where resonant absorption occurs, is directly proportional to the microwave frequency v according to the equation hv = g/3H, where h and/3 are Planck's constant and the Bohr magneton, respectively. In solid materials (and proteins qualify for this description) g is formally a second-rank tensor, which means that it can have three different values, gx, gy, and gz, along three mutually perpendicular directions in the solid. If two directions are physically indistinguishable, the symmetry is said to be axial; otherwise it is described as rhombic. When the spin state of the paramagnetic center is greater than ½. there will always be crystal field splittings, and these will make the EPR spectra difficult to interpret or even impossible to observe. In general, paramagnetic centers having an even number of unpaired spins (thus having S = 1, 2, 3, etc.) have no EPR absorption except in cases of high symmetry, such as occur in hard crystals, virtually precluding their study in biomolecules. Any observable features in the EPR spectra in this case may be assigned "effective g values" by using the above field-frequency relationship, although it must be remembered that the numbers so assigned are only empirical descriptions of an imperfectly understood entity.

i C. P. Slichter, "Principles of Magnetic Resonance." Springer-Vedag, Berlin and New York, 1980. z j. S. Griffith, "The Theory of Transition Metal Ions." Cambridge Univ. Press, London and New York, 1961. 3 A. Abragam and B. Bleaney, "'Electron Paramagnetic Resonance of Transition Ions." Oxford Univ. Press, London and New York, 1970. 4 S. Geschwind, ed., "Electron Paramagnetic Resonance." Plenum, New York, 1972. 5 H. M. Swartz, J. R. Bolton, and D. C. Borg, "Biological Applications of Elecron Spin Resonance." Wiley (Interscience), New York, 1972. 6 p. B. Ayscough, ed., "Electron Spin Resonance," Vols. 1-6 Royal Society of Chemistry, London, 1971-1980.

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SPECTROSCOPIC PROPERTIES OF HEMOGLOBINS

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Paramagnetic States of Hemoglobin The possibility of using EPR spectroscopy for the study of hemoglobin will be discussed for three classes of preparations: (a) unmodified hemoglobin in various oxidation states and with various iron ligands; (b) hemoglobin modified by the replacement of the iron atom by another paramagnetic metal atom; and (c) hemoglobin modified by the attachment of a paramagnetic label. The iron atom of hemoglobin, considered along with its ligands and the heine, can exist, at least conceptually, in a wide variety of oxidation states from (I) to (VI), denoted here for brevity as Hb(I), etc. Hb(I) has never been prepared, as the strong reductants required prove to be too disruptive to the protein structure. This is unfortunate, as the EPR spectra of iron at this oxidation state, having the electronic configuration dT(t2g6eg1) and S = ½, would be well resolved and rich in information. Hb(II) is the native deoxy ferrous form of the protein as well as the ligated ferrous forms containing CO, the isocyanides, or NO (with the fiction that NO is at oxidation state zero). The CO and isocyanide forms have the configuration d6(t2g6) and S = 0, and perforce are EPR silent. The NO-ligated form has S = ½, and its EPR spectra provide a wealth of structural information. It will be discussed further in a separate section. The deoxy form has S = 2, d°(t2g4eo2), and thus is expected to be troublesome. Absorptions attributable to this very complex spin state have required fields and frequencies not routinely available to the biochemist. The nature of these absorption features remains, and is likely to remain for some time, uninterpreted. Hb(III) occurs as methemoglobin, with or without exogenous ligands. This five d-electron state can exist in two configurations: dS(t~aeo2), S = ~, or dS(tzos), S = ½. They are referred to as the high-spin and low-spin ferric forms, respectively. Each spin state provides easily observable EPR spectra and will be discussed in two sections below. The high-spin case arises from a ligand field at the iron atom sufficiently weak that electron-electron repulsion dominates, giving rise to maximum spin multiplicity. The low-spin case corresponds to ligand fields strong enough to dominate the electron-electron repulsion, giving rise to minimum spin multiplicity. Changing only one atom of the six iron ligands can be sufficient to switch from one to the other. The situation in which the intermediate spin, S = 9, is stabilized cannot occur in heme proteins without a very large distortion of the more or less planar porphyrin ring toward a pyramid. It has not been observed in any state or compound of hemoglobin. Hb(IV) occurs as the ferryl state with a single oxygen atom ligated to

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315

the iron atom. It is configured d4(t~4), S = 1. Neither Hb(IV) nor any other heme protein in the ferryl form has yielded to EPR spectroscopy. Hb(V) might exist by analogy with other heme proteins having this oxidation state. In catalase and the radish peroxidases this state is referred to as compound I, but its electronic configuration is still somewhat controversial even though an EPR absorption has been attributed to it. r In cytochrome peroxidase it occurs as the ferryl state loosely coupled to a free radical) Electron paramagnetic resonance spectra for a similar, but transient, state have been reported for myoglobin9 but not for hemoglobin. Hb(VI) occurs only as the dioxygen adduct, oxyhemoglobin. There have been a number of proposals for the electronic state, but there is unanimous agreement that the state has S = 0 and thus is EPR silent. Line Shapes and g Values All EPR spectra observed in native hemoglobin arise from a single Kramers doublet. Since 56Fe has no magnetic moment, it provides no hyperfine splitting. Therefore, these spectra can always be described by at most three g values. If two of the g values are the same, the site symmetry is said to be axial and the magnetic absorption distribution will have the shape seen in the top left of Fig. 1. Line broadening will round the theoretically sharp corners as shown by the dashed line. The derivative of this distribution, the usual presentation of commercial EPR spectrometers, will have the shape shown below it. If the broadening is not excessive, the g values may be determined from the positions of the zero crossing point on the left feature and the extreme excursion of the right feature. It must be borne in mind that g values determined in such a manner are merely descriptive of the spectrum, not necessarily the true g values that a physicist would want in order to analyze the complete magnetic behavior. When the site symmetry is rhombic, the magnetic absorption distribution will have the shape seen at the top right of Fig. 1. After the effects of line broadening, the derivative spectrum will appear as shown at the bottom right. Again, provided the broadening is not excessive, the g values may be determined by the positions of the zero crossing point and the extreme excursions of the two end features. If these features are not well resolved one from another, the correct positions for determining the g

7 C. E. Schulz, P. W. Devaney, H. Winkler, et al. FEBS Lett. 103, 102 (1979). 8 T. Yonetani, H. Schleyer, and A. Ehrenberg, J. Biol. Chem. 241, 3240 (1966). a W. E. Blumberg, J. B. Wittenberg, and J. Peisach, Fed. Proc., Fed. Am. Soc. Exp. Biol. 27, 526 (1968).

316

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SPECTROSCOPIC PROPERTIES O F H E M O G L O B I N S

A

¢1 i=1 o

4

i

I

gz

I

gv

I

g,

== ¢1 I=

.o

E.< Q.,, [..U

V

Magnetic Field Magnetic Field FIG. 1. Theoretical EPR absorption (upper) and absorption derivative (lower) curves for axial (left) and rbombic (right) site geometries. In the case of the absorption curves, the effects of line broadening have been indicated by the dashed lines. It is the derivative of this broadened curve that is plotted beneath it. values will be displaced from the aforementioned points, and a spectral simulation would be necessary to determine them accurately.

Low-Spin Ferric [Hb(III)] When the water ligand o f methemoglobin b e c o m e s deprotonated to form hydroxide, or when exogenous ligands such as azide or cyanide are added, or when the protein structure is altered so that electron-rich endogenous ligands can bind the iron atom in the distal position, one will find the low-spin ferric case. T h e s e compounds always exhibit E P R absorption, but in certain cases temperatures lower than liquid nitrogen (77°K) may be required for detection or good resolution of the spectra. An example is shown in Fig. 2. T h e r e is no doubt that the E P R o f low-spin ferric iron arises from a t~u orbital combination relatively isolated from other orbitals. Griffith explains how to treat such spectra quantitatively on p. 363 o f his book. 2 This model assumes that the orbitals o f the iron atom are slightly mixed with ligand orbitals and that the orbital angular m o m e n t u m o f t b e iron electrons would be altered slightly by this admixture. This model leads to an analysis involving three parameters derived from the three g values. These are A/X, the tetragonal field in units o f the spin-orbit coupling parameter h; V / h ,

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STUDY OF HEMOGLOBINS BY E P R

< t~ a.

gz

gy

SPECTROSCOPY

317

gx

Magnetic Field FIG. 2. The EPR spectrum of isolated ferric a chains of hemoglobin, pH 8.7. The three g values are 2.56, 2.18, and 1.88. The vertical lines show where the three g values are measured.

the rhombic field; and K, the orbital reduction factor that contains all the covalency information. A method for solving for these parameters has been given by Griffith, and it is easily implemented on a computer or even a programmable hand calculator if it can solve a cubic equation. Unfortunately, because of ambiguities in the signs of the parameters and of the g values, there are many different solutions (48 in the worst case), some of which correspond to different symmetry conditions and some of which are merely rotations of others. That is, some are chemically or electronically different, and some are geometrically different. Blumberg and Peisach TM have provided a classification of a wide variety of low-spin ferric heme compounds, assuming a particular kind of solution for Griffith's equations. Later they added to this classification,11-1a not always using the same geometrical solution (caveat lector). When carrying through Griffith's method, it is always wise to search around a novel solution for alternative solutions that may have greater similarity to previously observed cases. Taylor t4 has presented another model for the EPR of the isolated t2o orbital. It has the advantage that the solution may be obtained without solving a cubic equation, but it is neither more nor less accurate on physical grounds and does not improve on the problem of the number of solutions. For well behaved cases, Taylor's parameters are similar to those obtained by Griffith's model, but this is not always true. It is best not to 10 W. E. Blumberg, and J. Peisach, In "Probes of Structure and Function of Macromolecules and Membranes (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 2, pp. 215229. Academic Press, New York, 1971. 11 D. L. Brautigan, B. A. Feinberg, B. M., Hoffman, et al. J. Biol. Chem. 252, 574 (1977). 12 C. A. Appleby, W. E. Blumberg, J. Peisach, et al. J. Biol. C h e m . 251, 6090 (1976). 13 M. Chevion, J. Peisach, and W. E. Blumberg, J. Biol. Chem. 252, 3637 (1977). 14 C. P. S. Taylor, Biochim. Biophys. A c t a 491, 133 (1977).

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SPECTROSCOPIC PROPERTIES OF HEMOGLOBINS

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try to compare analyses performed by the different mathematical methods. Bohan 15 has noted that some of the ambiguity in assigning the correct electronic solution can be removed by making an EPR measurement using circularly polarized microwaves. This technique is not commonly used, however, and most commerical EPR spectrometers cannot be adapted to perform it. As with any kind of empirical spectroscopy, one makes the argument that species having similar spectra (or in this case similar derived spectral parameters) are structurally similar. This is the rationale of Blumberg and Peisach's classification. 1° and has been used to assign unknown structures, for example, various compounds of leghemoglobin. TM Three low-spin forms of hemoglobin that may be singled out for discussion are found during the course of the denaturation of hemoglobin. As they are formed without exogenous ligands, they have been termed hemichromes. TM Methemoglobin is in pH equilibrium between the high-spin (water bound) form and the hydroxide-bound form, called O hemichrome. This type is quickly and fully reversible to the native ferrous hemoglobin by the erythrocyte methemoglobin reductase enzyme system or, in vitro, by mild reducing agents. In time the O hemichrome will spontaneously be transformed into the H hemichrome, which consists of the normal porphyrin and proximal iron ligands plus the distal histidine. Some alteration in protein structure must occur in order for the Ntele atom of the distal histidine to move within bonding distance of the iron atom. Nonetheless, this type of hemichrome is reversible, both in vitro and in the erythrocyte, to the native ferrous form, although the process is a slow one. Given more time, the H hemichromes will spontaneously be transformed into the B hemichromes. This type of low-spin compound has nitrogen atoms from histidine residues as the axial heme ligands, just as in the H hemichrome, but in this case the protein structure is disrupted sufficiently that the Npros of each histidine becomes protonated as if it were exposed to solvent. This type of hemichrome is not reversible to the native ferrous form. Electron paramagnetic resonance provides the only way in which these three types of hemoglobin products may be distinguished. Low-spin forms of hemoglobin may be quantitated at any temperature at which their spectral features are well resolved, as there are no lowlying excited states to contend with. The derivative spectra may be doubly integrated to give a number that may be compared with a standard. Another procedure, which the author has used for about 20 years, is 15 T. L. Bohan, J. Magn. Reson. 26, 109 (1977). 16 E. A. Rachmilewitz, J. Peisach, T. Bradley, and W. E. Biumberg, Nature (London) 22,

248 (1969).

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319

simpler and more accurate when there are multiple species present. It involves measuring only selected features of the spectrum and applying a formula 'r taking into account the three g values of the species under study. This method is particularly accurate if a model compound can be quantitatively prepared that is similar in spectral features to low-spin hemoglobin. Such a reference material is easily made by dissolving a known quantity of hemin chloride in molten (90°C) imidazole. The resulting stable solid standard resembles a B hemichrome. By this method the kinetics of the denaturation of hemoglobin through the various hemichromes discussed above can be followed quantitatively. High-Spin Ferric [Hb(III)] The high-spin ferric spin state always gives rise to EPR absorption at low temperature, 77°K being sufficiently low for observation in almost all cases. For high-spin ferric heme, adequate resolution may be obtained at 77°K for purposes of detection of the presence of the species and for observing gross changes in symmetry during the course of the change in experimental conditions. A factor of two or three in resolution of the spectral features will be realized at 4°K or 5°K with respect to observation at 77°K. Such temperatures may be necessary to reveal subtle changes in symmetry. An example of the EPR of high-spin ferric heme iron is given in Fig. 3. In this example, gx and gy are the same, giving rise to an axial spectrum. The analysis of the EPR spectra of the high-spin ferric state is, in principle, much more complicated than that of the low-spin ferric state. The complete spin Hamiltonian for this state has been given by Abragam and Bleaney2 It must be emphasized, however, that no complete description of the magnetic properties of any ferric heme iron species has ever been given. It is experimentally too difficult to obtain all the information required. The EPR of ferric heme iron, therefore, is usually analyzed in a much more empirical manner. All ferric heme iron sites are characterized by an axial splitting term D, which is much larger than the microwave frequency expressed in energy units. Typically, D may range from 5 cm -1 to 30 or more, while x-band spectrometers operate with a microwave quantum energy of 0.3 cm-'. Under these conditions, it is impossible to determine D (or indeed any of the spin Hamiltonian parameters) from the EPR spectrum itself. It is customary to assume that the second-rank axial and rhombic coefficients (commonly called D and E, respectively) dominate ,7 R. Aasa and T. Vanngard,J. Magn. Reson. 19, 308 (1975).

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SPECTROSCOPIC PROPERTIES OF HEMOGLOBINS

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.o= r~ O

a~ B

gffi6

g--2 Magnetic Field

FIG. 3. Electron paramagnetic r e s o n a n c e s p e c t r u m of heroin chloride taken at 1.5°K. T h e lower curve is the absorption, and the u p p e r c u r v e is the absorption derivative. It can be seen that, although absorption e x t e n d s c o n t i n u o u s l y from g = 6 to g = 2, the only features distinguishable in the derivative s p e c t r u m are at the e n d s o f the spin distribution.

all the others. In this approximation, there is only one derived parameter that may be obtained from a s p e c t r u m - - t h e ratio E/D, called the rhombicity. TM As E/D has a maximum meaningful value o f ], some spectroscopists have defined R, the percent rhombicity as 3 E/D, which may have a value ranging from 0 to 100%. R may be computed from the observed features of the spectrum near g = 6, by the following procedure. If the gx and gy features are well resolved, gx may be read as the extreme excursion o f the feature to lowest field, and gy may be taken as the zero crossing point. If, as more commonly occurs, these features are not well resolved one from another, the contribution of the skirts o f one feature at the other may be pencilled in and subtracted by hand or the overall shape may be reproduced by simulation. If the features are not at all resolved, then R may not be zero, but at least it is below the resolution permitted by the line width. In cases like this, the lowest possible measurement temperatures are o f significant value. Some examples o f the features near gx and gy for a variety o f rhombicities are given in Fig. 4. Once gx and gy have been determined, R may be computed from

E/D

= (gx - gy)/48

or

R = (gx - gy)/16 × 100% is j. Peisach, W. E. Blumberg, S. Ogawa, et al. J. Biol. Chem. 246, 3342 (1971).

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STUDY OF HEMOGLOBINS BY

EPR

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321

t

"1:I o

¢

< f f

Magnetic Field FIG. 4. Electron paramagnetic resonance spectra in the region near g = 6 for some highspin ferric heme proteins and their chemical derivatives. The arrows indicate the positions at which gx and gy should be measured. A, Ferrimyoglobin cyanate; B, ferrihemoglobin thiocyanate; C, cytochrome oxidase, partly reduced; D, liver catalase fluoride; E, Escherichia coli sulfite reductase. After Peisach et al. t8

This formula is correct through third order in R and applies even though gx and gy may not be symmetrically disposed about g = 6. If the resulting R is greater than about 25%, a more exact solution is required or a diagram of solutions TM may be consulted. Peisach e t al. is have given a table of the rhombicities of a wide variety of high-spin ferric heme proteins and compounds. Unfortunately, it is impossible to assign structures to unknown species by means of the rhombicity alone. The rhombic distortion could arise from mechanical bending of the heme and its axial ligands in response to protein constraints just as well as from electronic variations in ligand field due to alterations in the chemical nature of the axial ligands. Isolated a chains from hemoglobin A can be converted quantitatively to the high-spin ferric state. Depending on conditions, the EPR spectrum will be axial (R = 0) or slightly rhombic (R = 2.3%). 20 It is possible to observe this transformation only with EPR spectroscopy, but it is not possible to use EPR alone to assign a structure to either form. The hemoglobins M represent a class of abnormal hemoglobins in is W. E. Blumberg, In "Magnetic Resonance in Biological Systems" (A. Ehrenberg, B. G. Malmstrom, and T. Vanngard, eds.), pp. 539-560, 1967. z0 j. Peisach, W. E. Blumberg, B. A. Wittenberg et al., Proc. Natl. Acad. Sci. U.S.A. 63, 934 (1969).

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which either the a or the/3 chains naturally occur in the nonfunctional ferric state as a result of an amino acid substitution at or near the heme. The nonsubstituted chain is oxygenated and does not contribute to the EPR. In four hemoglobins M, a tyrosine is substituted for histidine in the abnormal, nonfunctioning chain at a position lying close to or chemically bonded to the heme. In both hemoglobin M Hyde Park and hemoglobin M Boston, in which the/3 chains are abnormal, the percentage of rhombicity of the heroes is approximately the same. For hemoglobins in which the a chains are abnormal, hemoglobin M Iwate and hemoglobin M Saskatoon, the departure from axial symmetry depends on whether the amino acid substitution is proximal to, as in the former case, or distal to the heme. In the case of hemoglobin M Hyde Park, in which the amino acid substitution of the/3 chain is at the point where the protein binds to the heme, the departure from axial symmetry is dependent on the state of oxygenation of the normal a chain. Reversibly deoxygenating the molecule increases the rhombicity of the hemes of the adjacent abnormal chains by a factor of three. From similar experiments carried out with artificial mixed-state hemoglobins, one concludes that those conformational changes that take place upon oxygenation of the /3 chains may be transmitted or transduced across the interchain contacts of the hemoglobin A tetramer so that there may be a change in the symmetry of the heme of the a chains. A similar transmittal or transduction upon oxygenation of the a chains across the interchain contacts of the tetramer to the heme of the/3 chains is not indicated. These experiments do not permit an interpretation of the nature of the change at the metal atom of the ferric chain. The quantitation of the number of spins in the Kramers doublet giving rise to the EPR absorption can proceed straightforwardly by either of the methods recommended for quantitating low-spin ferric EPR spectra. Metmyoglobin at sufficiently low pH that there is negligible hydroxide or Otype hemichrome (ca pH 6.5) will serve very well as a high-spin standard. Such a preparation, now 15 years old, has been used continuously in the author's laboratory, never having been warmed above 77°K. There is a problem with converting from the number of spins observed in a quantitative EPR experiment to the concentration of high-spin heme iron present. This is because the EPR arises from only the lowest of three Kramers doublets, the other two being EPR silent. If D and E separately are known, then the energy level spacing can be computed. Then the Boltzmann population of each level can be computed, knowing the temperature. The fraction in the lowest doublet then can be used to convert from observed spins to quantity of heme iron present. This fraction varies from 100% at very low temperatures to 33% at high temperatures, as can be seen in Fig. 5. That diagram was computed for a particular value of D;

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STUDY OF HEMOGLOBINS BY

EPR

323

SPECTROSCOPY

1.0

0.9 l/}

0.8 -,I ll}

o (/}

0.7

W

,,s

0.6

I.I.

o 0~ 0.5 Z o I-

e,

0.4

f

o(}.

i

B

O.~ 0.~

O.

O

I

I

I

50 I00 150 TEMPERATURE (DEGREES KELVIN} FIG. 5. Fractional populations of the three Kramers doublets for ferrimyoglobin fluoride as a function of absolute temperature. Curves A, B, C represent the populations of the lowest, middle, and upper Kramers doublets of the S = ~ spin system. Doublets B and C are EPR silent under normal conditions.

unfortunately, one seldom has this information independently of the EPR experiment. M6ssbauer spectroscopy, magnetic susceptibility, Orbach relaxation, and far infrared absorption provide several methods by which D may be determined, but each may be used only under a very restrictive set of circumstances. It is always safe to perform quantitation at 1.5°K, pumped helium temperature, where the fraction is 100%, provided one takes care not to power-saturate the spin system. On the other hand, it is seldom practical to try to achieve the high temperature limit, as the spectra begin to lose resolution at high temperatures. If a rough quantitation is

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SPECTROSCOPIC PROPERTIES OF HEMOGLOBINS

[19]

sufficient in a given case, D can be assumed to have any reasonable value; the greatest error one can make in this way is a factor of three. Sometimes it is possible to use the temperature dependence of the spectra themselves for a determination of D. This has been very successfully used for rhombic mononuclear iron proteins. 2a'22 One measures the intensity of the EPR spectrum over a wide range of temperature and then fits the data with a curve similar to curve A in Fig. 5, but with the value of D as a variable. The equation to use for nearly axial symmetry is I = C / T ( 1 + e -2DIkr + e -rotkr)

where C and D are adjustable parameters. This method runs into the difficulty that the line shape changes in a nonuniform manner starting at about 7°K. This is because there are various spin relaxation processes that are applicable to high-spin ferric heine. At very low temperatures the direct single phonon relaxation prevails, at intermediate temperatures the Orbach process dominates, and at high temperatures one finds relaxation via a Raman mechanism. One must be very careful, therefore, that the EPR intensity at each temperature be integrated taking into account possible variations in line shape--simply plotting the peak amplitude will result in a large error. Nitrosylhemoglobin [Hb(II)NO] Nitric oxide binds reversibly to ferrous heine proteins under strictly anaerobic conditions to form a class of compounds known as nitrosyl heme proteins. Nitrosylhemoglobins have EPR spectra that are easily observed at both room temperature and at 77°K and below. Freely rotating nitroxide radicals in solution exhibit very narrow EPR spectra dominated by the 14N hypedine structure. Hemoglobin molecules in solution tumble too slowly to satisfy the conditions of this motional narrowing, and, therefore, nitrosyl hemoglobin exhibits a complex spectrum at room temperature complicated by various dynamical processes. It is much better for this reason to study nitrosylhemoglobin in frozen solution. For this purpose 77°K is adequate--no advantage is realized by going lower. In the frozen state the EPR spectrum of nitrosylhemoglobin is a powder pattern resulting from the overlapping contributions of g values anisotropy and nitrogen hyperfine interactions. It is usually not possible to read correct g values directly from the spectrum. More often the upfield and downfield extrema are labeled effective or apparent g values, as is the midpoint of the nitrogen hyperfine pattern. This is a valid way of describing a given spectrum, but it must be borne in mind that these effective g 21j. Peisach,W. E. Blumberg,E. T. Lode, and M. J. Coon,./.Biol. Chem. 246, 5877(1971). 23W. E. Blumbergand J. Peisach,Ann. N. Y. Acad. Sci. 222, 539 (1973).

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STUDY OF HEMOGLOBINS BY E P R 2.080

2.007

I

I

SPECTROSCOPY

325

1.969 I

A

o

< oil

I 2.074

2.003

I 1.973

Magnetic Field FIG. 6, Electron paramagnetic resonance spectra of nitrosylhemoglobin Kansas recorded at 1.6°K. Curve A, at pH 8.5, in the absence of phosphates; B, third harmonic spectrum in the central region (expanded 5 ×); C, at pH 7.95, in the presence of inositol hexaphosphate. Apparent g values are marked,

values will change as the microwave frequency changes. A complete analysis of such an EPR spectrum requires spectral simulation. True g values may be reported from a successful simulation. When comparing new results with literature results, it is of importance to take note of whether the results are effective g values or true ones from simulation. Both the g values (true or effective) and the nitrogen hyperfine interaction are sensitive to the heine ligand trans to the NO group. Kon 23 has summarized the characteristics of the EPR spectra from a variety of NOheme model compounds. Only three types of spectra are observed on nitrosylhemoglobins--one being the spectrum of denatured hemoglobin reported by Kon and Kataoka/4 Of the other two native spectra, one is assigned to the R state and one to the T state of the tetramer conformation. The R state spectrum is made up of two almost equivalent spectra from the ot and 13 chains, respectively. These spectra are characterized by effective g values ranging between about 2.08 and 1.95 and a nitrogen hyperfine pattern having contributions from the 14N of the nitrosyl group (about 2.3 mT) and from the Ntele of the proximal histidine (about 0.65 mT). The overlapping nitrogen interactions produce a nine-line hyperfine p a t t e r n - - a triplet of triplets--which is usually only poorly resolved in frozen hemoglobin preparations. Figure 6A shows such a spectrum for hemoglobin Kansas. The resolution of the nine lines may be 2a H. Kon, Biochim. Biophys. Acta 379, 103 (1975). 24 H. Kon and N. Kataoka, Biochemistry 8, 4757 (1969).

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enhanced using a third harmonic detection technique,Z5 which is easily accomplished on any spectrometer having provision for variable frequency modulation and lock-in detection but is somewhat more difficult on commercial instruments having fixed frequencies. Figure 6B shows the nineline pattern with increased resolution using 1 kHz modulation and 3 kHz detection. The T state spectrum for nitrosylhemoglobin is more complicated in that the spectrum arising from the/3 chains hardly changes at all, whereas that of the a chains changes drastically. The resulting spectrum is the sum of the two (see Fig. 6C). The outstanding feature of the t~ chain spectrum is the nitrogen hyperfine pattern, which has changed into a triplet having about 1.8 mT splitting for the 14N of NO and no observable splitting arising from the proximal histidine. More exact parameters for both the a and /3 chains have been obtained from single-crystal studies 26 on hemoglobin Kansas. The parameters for NO-a chains in the T state are quite close to, but not exactly the same as, those for pentacoordinate heme-NO. This observation has led some to postulate that the proximal histidine is removed from the heme iron in the T state. However, an infrared stretching frequency has been assigned to this bond in T state hemoglobin by Raman spectroscopy, pointing up the desirability of using several different physical techniques in conjunction whenever that is possible. Hille e t a l . 27 have shown that, while the R to T state change in quaternary structure is presumed to be fast, the changes in EPR spectrum of nitrosylhemoglobin are slow. Therefore, the structure at the NO binding site is reflecting effects of the quaternary structure at equilibrium, but is not following instantaneously. Only EPR spectroscopy could have permitted the study of this delayed kinetic behavior.

Metal-Substituted Hemoglobins It is possible, by first separating the heme from the globin, to reconstitute hemoglobin with porphyrins containing different metal atoms into one or both kinds of chains. Most notable among them are Mn 2÷, Co 2÷, and Cu 2÷, as they are all divalent, thus mimicking the ferrous state of the protein, and they are paramagnetic, having spins of S = 9, ½, and ½, respectively.

25 M. Chevion, M. M. Traum, W. E. Blumberg, and J. Peisach, Biochim. Biophys. Acta 490, 272 (1977). 2e j. C. W. Chien and L. C. Dickinson, J. Biol. Chem. 252, 1331 (1977). 27 R. Hille, J. S. Olson, and G. Palmer, J. Biol. Chem. 254, 2110 (1979).

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Manganese When substituted into either or both chains, Mn ~÷ is nonfunctional in carrying oxygen, but its EPR spectrum provides some information about the local electronic environment at the metal atom. The electronic state of the Mn 2+ atom is dS(t2g3eg2), the same as for high-spin Fe z÷. As is usual in isomorphous structures of Fe 3+ and Mn 2+, the ligand fields of the latter are about an order of magnitude smaller. The value of D for Mn 2÷ in myoglobin 28 is 0.5 cm -1, a value small enough to permit observation of transitions between Kramers doublets with an x-band spectrometer. Thus more information can be obtained from an analysis of Mn2+-porphyrin EPR spectra than can be obtained from an analogous Fe 3r system. There is still not enough information observable, however, to make a complete solution of the spin HamJltonian for the S = ~ spin system.

Cobalt Co s÷ alone provides a functional hemoglobin upon substitution for the iron. When Co s+ is in a porphyrin structure and at least one axial ligand is present, it will be in the low-spin S = ½ state, d;(t~o%gl). The hyperfine structure and g± are quite sensitive to the electron donating capacity of the axial ligand(s). 29,3° Therefore, the EPR of Co s÷ will reflect changes in protein structure that make themselves felt as changes in electronic structure at the metal atom. The oxygen adduct of Co2+-hemoglobin provides an EPR spectrum with a wealth of information.31.32 The drastic reduction in cobalt hyperfine splitting and the approach of the g values toward g = 2 attest to the localization of the unpaired electron on the dioxygen moiety. As in the deoxy case, the EPR parameters are sensitive to electronic structural changes brought about by changes in protein structure. In addition, it has been observed that the resolution of the hyperfine pattern is increased when the cobalt protein is prepared in D20. This demonstrates the power of nearby proton(s) to broaden the EPR features by virtue of the large magnetic moment of 1H. In the case of oxy-Co2+-hemoglobin, this broadening effect has been interpreted as arising from a proton participating in a hydrogen 28 T. Yonetani, H. R. Drott, J. S. Leigh, et al. J. Biol. Chem. 245, 2998 (1970). 29 S. A. Cockle, H. A. O, Hill, J. M. Pratt, and R. J. P. Williams Biochim. Biophys. Acta 177, 686 (1969). 20 j. H. Bayston, F. D. Looney, B. R. Pilbrow, and M. E. Winfield, Biochemistry 9, 2164 (1970). a~ B. M. Hoffman and D. H. Petering, Proc. Natl. Acad. Sci. U.S.A. 67, 637 (1970). 32 j. W. C. Chien and L. C. Dickson, Proc. Natl. Acad. Sci. U.S.A. 69, 2783 (1972).

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bond between the Ntele atom from the distal histidine and the dioxygen moiety.

Copper Cu 2+ may be substituted into various porphyrins and incorporated into globin to make Cu2+-hemoglobin, where the metal atom has electronic structure dg(hg6ega). It is nonfunctional with respect to oxygen transport but provides typical Cu2+-porphyrin EPR spectra. Unfortunately, in contradistinction to the case of Co s+, Cu 2+ EPR spectra are rather insensitive to changes in axial ligands and therefore are poor probes of changes in electronic structure of the axial ligands brought about by protein structural changes. On the other hand, Cu 2+ EPR spectra are quite sensitive to changes in electronic structure of the equatorial ligands? 3 Models of hemoglobin function that postulate that affinity changes are transmitted via the porphyrin structure itself would predict that such changes would be reflected in the Cu 2+ EPR spectra. Optimizing Observation Conditions Specimens for EPR study must be magnetically dilute in order that unwanted line broadening does not occur. Attainment of this requirement is sometimes a problem with model compounds, particularly those of low solubility. However, this is never a problem with hemoglobin, as the protein serves to keep the hemes well enough separated. Thus, samples may be of any concentration in the liquid state or even composed of microcrystals or in special circumstances single crystals. The minimum concentration required for study of a given hemoglobin preparation will depend on the nature of the paramagnetic species one intends to observe. At 77°K one can detect high- and low-spin ferric species at the micromolar level using sample volumes of about 100/zl. Both these species suffer significant thermal line broadening at this temperature, making detailed analysis difficult. A temperature range of 5-20°K is preferable for such samples. For studies at 1.5-4°K (helium temperatures) a superheterodyne spectrometer or one with an external reference cavity will be required. The NO adduct of heme proteins can be studied at 77°K in submicromolar concentrations. Usually specimens for EPR study are quickly frozen in small-diameter quartz tubes. Alternative sample holders include plastic containers (acrylics and olefins are the least contaminated by paramagnetic impurities) and metal cavity parts, a4 The latter has the advantage of permitting very quick 3a j. Peisach and W. E. Blumberg, Arch. Biochem. Biophys. 165, 691 (1974). a4 j. A. Berzofsky, J. Peisach, and W. E. Blumberg, J. Biol. Chem 246, 3367 (1971).

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freezing. In the case of hemoglobin, however, very quick freezing should be avoided unless it is necessary to trap unstable intermediates. This is because the strain induced on the protein by the rapidly advancing "wall" of ice as the sample freezes will cause broadening of the features of the EPR spectrum. One need not worry that the salting-out effect produced by slow freezing will cause all the protein to move to the last portion to be frozen, as the concentration broadening will not be important. This is not true for all metalloproteins, e.g., cytochrome c. When there is some doubt or a new type of protein is being studied, some experimentation with the freezing conditions may be necessary to obtain optimum resolution of the spectral features. The changes in local pH or ionic strength produced by the salting-out effect during slow freezing may, however, produce unwanted effects on the protein not related directly to its magnetic properties.

[20] Application of M6ssbauer Spectroscopy to Hemoglobin Studies By

BOHDAN BALKO

General Introduction There are many advanced physical techniques that in the course of their development have been initially used to study very simple physical systems. Such studies led to a basic understanding of the techniques, which were then applied with confidence to more complex physical systems and eventually found application in the biological and medical sciences. Techniques such as nuclear magnetic resonance (NMR), electron spin resonance (ESR), X-ray diffraction, electronic absorption, and fluorescence spectroscopy are now used routinely to study biomolecules. None of the techniques alone can give the total picture. Some give information about the structure of the molecule, and others tell us about the interaction of the molecule with its environment. Still others can be used to study specific regions or sites on the molecule that are of primary importance to its biological function. The MSssbauer effect (ME) falls into this category. It allows us to study, for example, the environment of the iron ion in the porphyrin ring in hemoglobin. It allows us to study changes in this environment as the molecule goes through the various steps of fulfilling its biological function. The ME also does not answer all the questions, but it can be used to obtain information not available in any other way. The ME is the recoilless emission and absorption of nuclear radiation. METHODS IN ENZYMOLOGY, VOL. 76

Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0