[6] Magnetic circular dichroism

110

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[6J

tures of metal ion active sites, which make significant contributions to biological reactivity. Acknowledgments This work was supported by the National Science Foundation Biophysics Program (MCB 9019752) and the National Institute of Health (GM 40392). E.I.S. expresses sincere appreciation to all the students and collaborators listed as coauthors in the literature cited for their commitment and contributions to this research. Special thanks to Professor John Lipscomb for the collaborative studies on metapyrocatechase.

[6] M a g n e t i c C i r c u l a r D i c h r o i s m

By JOHN C. SUTHERLAND Introduction Magnetic circular dichroism (MCD) is the differential absorption of left and right circularly polarized light induced by an externally applied magnetic field. MCD is one of the several forms of "higher order" spectroscopy that can augment the information provided by the measurement of the absorption of a sample using unpolarized electromagnetic radiation. The additional information provided by MCD can aid in resolving complex absorption spectra. Some biologically important molecules exhibit particularly strong MCD signals and can thus be detected and quantified even when present in complex mixtures. The most sophisticated applications of MCD involve the extraction of properties of the system under study by comparison of experimental spectral parameters with theoretical models. The theory and some of the biological applications of MCD have been reviewed elsewhere. ~-9

I p. j. Stephens, Annu. Rev. Phys. Chem. 25, 201 (1974). 2 p. j. Stephens, Adv. Chem. Phys. 35, 197 (1976). 3 B. Holmquist and B. L. Vallee, this series, Vol. 49 Part G, p. 149. 4 j. C. Sutherland, in "The Porphyrins" (D. Dolphin, ed.), p. 225. Academic Press, New York, 1978. 5 A. J. Thomson, M. R. Cheesman, and S. J. George, this series, Vol. 226, p. 199. Academic Press, New York, 1993. 6 j. C. Sutherland and B. Holmquist, Annu. Rev. Biophys. Bioeng. 9, 293 (1980). 7 j. H. Dawson and D. M. Dooley, in "Iron Porphyrins" (A. B. P. Lever and H. B. Gray, eds.), p. I. VCH, New York, 1986.

METHODS IN ENZYMOLOGY,VOL. 246

Copyright © 1995by AcademicPress, Inc. All rightsof reproductionin any form reserved.

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111

MCD is closely related to some types of spectroscopy but must be clearly differentiated from others. The relationship between MCD and "natural" circular dichroism 1° (CD) is particularly important. CD is the differential absorption of left and right circularly polarized light resulting from the asymmetry of the absorbing moiety or its immediate environment. CD usually tells about the physical conformation of the absorbing species, whereas MCD usually tells about its electronic state. Although the information provided is quite different, the instrumentation required to perform MCD measurements is, almost always, a superset of that required for CD, hence distributing the cost of a spectrometer over a broader range of applications. In contrast, magnetic optical rotatory dispersion (MORD), the difference between the refractive indices of right and left circularly polarized light induced by an external magnetic field, provides information equivalent to that obtained from MCD, but requires different instrumentation. MCD and MORD are linked by KramersKronig integral transforms, 11 as are CD and ORD. The absorption based spectra are usually easier to interpret, so MCD and CD are the methods of choice. MCD measurements are performed on samples containing randomly oriented absorbing species and with the magnetic field oriented parallel (or antiparallel) to the direction of the probing beam of radiation, so that the field does not introduce a preferred direction compared to the polarization of the beam. A magnetic field can generate sufficient torque to partially orient large macromolecules and macromolecular arrays. ~2,~3 Observations made on samples that are not randomly oriented or that can be aligned by the magnetic field can generate magnetic field-dependent signals that do not fit the definition of MCD as discussed here, but which may provide other forms of useful information. Magnetic fields oriented perpendicular to the direction of the probing beam can produce magnetic linear dichroism that, as a result of the methods usually employed in CD and MCD measurements, can produce apparent MCD signals, the origin and interpretation of which are different from what we call MCD.

8 M. J. Stillman, in "Phthalocyanines, Properties and Applications" (C. C. Leznoff and A. P. B. Lever, eds.), p. 227. VCH, New York, 1993. 9 y . A. Sharonov, in "Soviet Scientific Reviews Section D: Physicochemical Biology Reviews" (V. P. Skulachev, ed.), p. 1. Harwood Academic Publ., London, 1991. 10 R. W. Woody, this volume [4]. 1~ C. R. Cantor and P. R. Schimmel, "Biophysical Chemistry." Freeman, New York, 1980. Jz G. Maret and K. Dransfeld, Physica 86-85B, 1077 (1977). ~3 G. Garab, A. Faludi-Daniel, J. C. Sutherland, and G. Hind, Biochem. 27, 2425 (1988).

112

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Signals directly analogous to MCD have been observed in both fluorescence ~4,~5and Raman scattering, 16,17but the use of these measurements for the study of biological materials has been limited. MCD is usually detected by observing light that has been transmitted through a sample. However, much the same information can be obtained from monitoring fluorescence emitted by a sample, an experiment referred to as fluorescence-detected MCD 18 (FDMCD), which can detect the MCD of both fluorescent and nonfluorescent molecules. Although it has not been used frequently in the ultraviolet (UV), visible, or infrared (IR), FDMCD may be the detection method of choice for the measurement of MCD in the X-ray region. 19Special techniques have been developed to record transient MCD spectra with a temporal resolution of nanoseconds. 2° Basic Principles

Measurement of Magnetic Circular Dichroism Integral versus Differential MCD. The definition of MCD presented above specifies that an external magnetic field causes the absorption of left circularly polarized light to differ from that of right circularly polarized light. In principle, one could measure the absorption spectrum of a sample using first one and then the other circular polarization. MCD is that portion of the difference between the two spectra that is induced by application of an external magnetic field. Such measurements are performed only very rarely in the visible, UV, and IR regions of the spectrum because the field-induced effects are usually too small to be detected in this manner. Most MCD is measured as the difference between the absorption of left and right circularly polarized light using the polarization modulation method described below. The relationship between the CD, MCD, the intensities of left and right circularly polarized light, and other observable parameters are given by the Beer-Lambert law. Suppose that a beam of radiation of wavelength X and intensity Io(h) is incident on a sample of thickness l, t4 R. A. Shatwell and A. J. McCaffery, J. Chem. Soc., Chem. Commun., 546 (1973). t5 R. A. Shatwell, R. Gale, R. A. McCaffery, and K. Sichel, J. Am. Chem. Soc. 97, 7015 (1975). 16 L. D. Barron, Nature (London) 257, 372 (1975). 1~ L. D. Barron, Chem. Phys. Lett. 46, 579 (1977). ~8j. C. Sutherland and H. Low, Proc. Natl. Acad. Sci. U.S.A. 73, 276 (1976). 19j. van Elp, S. J. George, J. Chen, G. Peng, C. T. Chen, L. H. Tjeng, G. Meigs, H.-J. Lin, Z. H. Zhou, M. W. W. Adams, B. G. Searle, and S. P. Cramer, Proc. Natl. Acad. Sci. U.S.A. 90, 9664 (1993). z0 R. A. Goldbeck and D. S. Kliger, this series, Vol. 226, p. 147.

[6]

MAGNETIC CIRCULAR DICHROISM

1 13

and that the intensity of the beam which emerges from the far side of the sample is of intensity I(h), as shown schematically in Fig. 1. The absorbance of the sample at that wavelength, A (h), is defined in terms of lo(h) and l(k), and it is related to the molar concentration, c, and the molar extinction coefficient, e(h), of the absorbing species at wavelength h as shown by Eq. (1). 11 We could perform the same measurement using first A(h) ---- log lo(X)/I(h) = ce(h)l

(t)

left and then right circularly polarized light, hence obtaining AL(h) and

AR(k), respectively. The net circular dichroism, AA(k), is defined by the equation AA = AL -- AR and can be written as the sum of the contributions of the CD and MCD according to Eq. (2). Equation (2) presumes (in

AA(h, H) = AAcD(k) + HAAMcD(h)

(2)

agreement with both theory and experiments) that the contributions of CD and MCD to the total observed differential absorption are independent, and that the observed effect is a linear function of the magnetic field strength, H. In the SI system of units, magnetic field strengths are specified in teslas (1 T = 104 Gauss). As A and AAco are dimensionless, it follows that AAMco is expressed in units of T- 1. Dividing each of the differential absorbances by the product of concentration and path length gives the corresponding differential molar extinction coefficient: Ae(h), AeCD(h), and AeMcO(k). The units of Ae and AeCDare M - l cm- 1, whereas the units of AeMCD are M - 1 cm- 1 T- i. In practice, AeMCD(h), AAMcD, A8, and AA are all used to report MCD spectra. If in doubt, check the units! CD is also reported in units of ellipticity (0 = 33 AA) and molar ellipticity ([0] = 3300 Ae). Although 0 and [0] are used less frequently in MCD studies, they are still used extensively in the biochemical CD literature.

Io

I 1

FtG. 1. Transmissionof an optical beam by a sample of thickness I. The incidentbeam is monochromaticand characterizedby wavelengthh and intensity10, whereasthe intensity of the transmittedbeam is 1.

114

ULTRAVIOLET/VISIBLE SPECTROSCOPY

[6]

E x p e r i m e n t a l s p e c t r a r e p o r t e d in ellipticity units can be s e p a r a t e d into the c o n t r i b u t i o n s o f C D and M C D b y an e q u a t i o n e q u i v a l e n t to Eq. (2).

Instruments for Measuring Magnetic Circular Dichroism MCD Spectrometers for Ultraviolet, Visible, and Near-Infrared Spectral Regions. M o s t M C D s p e c t r o s c o p y is p e r f o r m e d in the spectral region with w a v e l e n g t h s f r o m r o u g h l y 120 n m in the v a c u u m ultraviolet ( V U V ) to 1200 n m (1.2/xm) in the near-infrared (IR) w h e r e light can be d e t e c t e d b y a p h o t o m u l t i p l i e r . Figure 2 s h o w s a s c h e m a t i c d i a g r a m o f such an i n s t r u m e n t , which is basically a C D s p e c t r o m e t e r fitted with a m a g n e t to g e n e r a t e a field at the position o f the sample. A k e y c o m p o n e n t o f such a n i n s t r u m e n t is the p h o t o e l a s t i c m o d u l a t o r (PEM) that c o n v e r t s the polarization o f a linearly p o l a r i z e d m o n o c h r o m a t i c light b e a m into first left and then right circularly p o l a r i z e d c o m p o n e n t s while k e e p i n g the total intensity

light

monochromator

PEM

sample

detector

FIG. 2. Schematic diagram of a CD/MCD spectrometer using a photomultiplier as the detector. A separate polarizer (pol) is needed if, as is usually the case with conventional broad spectrum sources, the light emerging from the monochromator is not linearly polarized. The photoelastic modulator (PEM) must be programmed to provide the correct phase retardation to render the light alternately left and right circularly polarized at each wavelength. The static magnetic field, H, is usually supplied by either an electromagnet or a superconducting magnet. Care is required in modifying a CD spectrometer to measure MCD. In addition to physically fitting a magnet in the sample compartment, it is necessary to prevent the magnetic field from interfering with the operation of the instrument. For example, most photomultipliers will not operate in even a moderate magnetic field, Collection of spectra in digital form, usually with a computer that controls the operation of the spectrometer, is highly desirable, as it greatly facilitates the separation of CD and MCD signals [see Eq. (2)] as well as the subtraction of any instrumental baseline.

[6]

MAGNETIC cIRCULAR DICHROISM

115

of the light constant. 21 The polarization is modulated at a frequency of (typically) 50 kHz. Differential absorption of left and right circularly polarized light by the sample results in an amplitude modulation of the beam reaching the detector. If I0(M is the intensity of the incident beam for both polarizations,/L/R0,) is the intensity of the transmitted beam for the two circular polarizations, A(?`) is the average absorption of the sample at wavelength ?`, and hA(?`) = AL(?` ) - AR(?` ) is the net CD [see Eq. (2)], then A I ( h ) -~ IL(?`) -- IR(?`) = I0 IO-~A+aA/2) -- I010-(a-aa/2)" Series expansion of the exponential terms, discarding of higher order differentials (assuming that AA < A), and solving for the CD gives AA(?`) =

-AI(?`) In(10) I(M

(3)

Thus, to measure the CD, one must obtain the ratio of AI, that small portion of the detected signal that is modulated with the same frequency and phase as the polarization changes introduced by the PEM, divided by I, the far larger unmodulated component of the detected signal. This is conveniently accomplished in instruments that use photomultipliers as detectors by controlling the voltage applied to the detector, and hence its gain, to maintain the average signal, I, at a fixed value. The small modulated signal, AI, is amplified with the same gain as the unmodulated signal, and hence it is a direct measure of the net CD. AI is measured with a phase-sensitive detector (PSD, sometimes called a "lock-in amplifier"), which can extract a small periodic signal of known frequency and phase from a large background of incoherent noise. The proportionality constant relating the measured magnitude of the modulated signal with CD is obtained empirically using a material such as camphorsulfonic acid. z2 Once the CD is calibrated, the strength of the magnetic field at the sample, H, can be determined from the magnitude of MCD signal of a standard compound, or H can be determined independently using, for example, the Hall effect. MCD Spectrometers for the Infrared. There are two distinct types of instruments used for IR CD/MCD. "Dispersive" instruments are similar to the design shown in Fig. 2, except that the detector is a solid state device, the gain of which cannot be controlled as can the gain of a photomultiplier. The ratio of hI/I in such instruments is obtained with some form of electronic divider circuit. The second class of IR CD/MCD instruments use modified Fourier transform spectrometers. 23 The long-wave2I j. C, Kemp, J. Opt. Soc. Am. 59, 950 (1969). 22 G. C. Chen and J. T. Yang, Anal. Lett. 10, 1195 (1977). z3 L. A. Nafie and M. Diem, Appl. Spectrosc. 33, 130 (1979).

116

ULTRAVIOLET/VISIBLE SPECTROSCOPY

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length limit of IR CD/MCD spectrometers is greater than I0/zm (< 1000 cm-1). CD/MCD spectrometers that operate in the near-IR (h < -2/~m) are available from commercial sources, whereas mid-IR instruments must still be built in the laboratory. Magnets. Most MCD spectrometers use either electromagnets or superconducting magnets. Electromagnets typically produce magnetic fields in the range from 1 to 2 T, whereas superconducting magnets used for MCD typically generate a maximum field ranging from 4 to 10 T. As the magnitude of the signal observed in an MCD experiment scales with the strength of the magnetic field [see Eq. (2)], higher fields are desirable. However, electromagnets are far easier and less expensive to operate than superconducting magnets. The choice of magnets for studies in which C-type MCD spectra (vide infra) are frequently observed (e.g., metalloproteins) presents a paradox. At low temperatures, AeMCD will tend to be large, hence reducing the need for the higher fields that are usually generated by a superconducting magnet. However, superconducting magnets are already fitted with cryogenic plumbing capable of reaching temperatures near absolute zero, and hence are frequently used in such studies. The ability to cool samples to near absolute zero is also used in the study of small molecules that can be trapped in a matrix consisting of an inert "gas" such as nitrogen or argon. 24

Magnetic Circular Dichroism Spectra MCD spectra are classified as A-, B-, or C-type, nomenclature that is singularly uninformative. A- and B-type MCD result from the perturbation of the states of a molecule involved in the observed photon-induced transition by the external magnetic field. As the change in energy produced by an external magnetic field is small compared to the intrinsic energy of the electrons that define the energy of the absorbing species, the problem can be treated using quantum mechanical perturbation theory. Typically, B-type spectra are the least intense type of MCD while C-type are the most intense, and I shall discuss them in that order. B-Type MCD Spectra. First we consider B-type MCD spectra that are the only contribution to the MCD when both the initial and final states responsible for a transition are unique in energy. If in the absence of an external magnetic field the energies of the initial and final states involved in the absorption of a photon are different from those of any other states in the molecule (or, more generally, the absorbing species), we say that the states are nondegenerate, as is the photon-induced transition between 24 B. E. Williamson, T. C. VanCott, M. E. Boyle, G. C. Misener, M. Stillman, and P. N. Schatz, J. Am. Chem. Soc. 114, 2412 (1992).

[6l

MAGNETIC CIRCULAR DICHROISM

1 17

them. The magnetic field modifies or perturbs these states slightly. Each perturbed state of the absorbing species can be represented as the sum of the unperturbed state plus small contributions from other states of the unperturbed absorber that are "mixed" with the unperturbed state by the external magnetic field. The symmetry of the energy perturbation introduced by an external magnetic field is such that only states with transition dipoles that are nonparallel are mixed together by the magnetic field. Thus a transition that is linearly polarized in the absence of an external magnetic field will become slightly elliptically polarized in the presence of the field, provided there are one or more transitions that have an absorption dipole that is not exactly parallel. We can decompose the elliptical transition dipole into a sum of the original linear component plus a small circular component which represents the stronger absorption of one circular polarization compared to the other circular polarization, that is, the MCD. The perturbations introduced by a magnetic field affect the pairs of states coupled by the magnetic field in a fashion that is identical in magnitude but opposite in sign, so, in the event that most of the observed MCD is due to the mixing of just two states, the MCD of the corresponding absorption bands will have the same (integrated) magnitude but opposite signs. Thus, B-type MCD spectra are similar in shape to the absorption spectrum for the same transition, can be of either sign, and tend to occur in pairs with opposite signs. An energy level diagram for a system that will exhibit B-type MCD is shown in Fig. 3, and the corresponding absorption and MCD spectra are shown in Fig. 4. A-Type MCD Spectra. A-type spectra occur if either the initial or the final state involved in a transition is degenerate, that is, if there are two or more states with exactly the same energy in the absence of an applied magnetic field. Mathematically, energy degeneracy can occur only if the absorbing species is sufficiently symmetric, possessing at least a 3-fold symmetry axis. An applied magnetic field can remove the degeneracy by shifting the formerly unresolved transitions to slightly higher and lower energies. This problem is treated using degenerate perturbation theory. The component shifted to higher energy will absorb light of the opposite circular polarization compared to the component shifted to lower energy, hence producing MCD, as shown in Fig. 5. The splitting of degenerate levels by an applied magnetic field is well known from atomic spectroscopy as the Zeeman effect. (Thus, it might have been called Z-type MCD, rather than A-type, but was not.) In the case of atomic spectra, however, the widths of the absorption lines are less than the energy splitting that can be induced by a reasonable laboratory magnet, so the effect can be detected without recourse to measurement of circular polarization. Most systems of biological interest, however, involve polyatomic molecules in condensed

I 18

ULTRAVIOLET/VISIBLE SPECTROSCOPY

12>

12'> <01Eyl2>I

II>

[6]

A

<01Eyl2'> If'>

i

I

I <01Exll>

i

l

I

I

I I

<01Exlr>

I

Io>

I

H--0

Io>

I

H>0

FlG. 3. Energy level diagram illustrating B-type MCD. Two nondegenerate excited states, 11) and [2), are mixed by the external magnetic field, H. Transitions from the ground state of the system, to), to the perturbed excited states, I 1') and 12'), are elliptically polarized in opposite senses.

media, so spectral bandwidths are usually much greater than the energy splitting induced by laboratory magnets and are very difficult to observe in absorption spectra measured using unpolarized light. Such splitting may be resolved by MCD. As the components of the transition that are shifted in opposite spectral directions absorb opposite circular polarizations, the MCD resembles the derivative of the absorption spectrum, as shown in Fig. 6. Thus, a quantitative comparison of the MCD and the derivative of the absorption band can be used to determine the magnitude of the field-induced splitting. The magnitude of the splitting per unit magnetic field is related to the angular momentum of the degenerate state and hence can be used to determine this parameter. 1,2 C-Type MCD Spectra. If the initial state involved in a transition is degenerate, a different mechanism can contribute to the differential absorption of left and right circularly polarized light. For a normal absorption measurement, the initial state will be the ground state of the system. The multiple energy levels of the magnetic field-resolved degenerate states will be populated according to a Boltzmann probability distribution. As the lower energy states are more likely to be populated, they will account for a greater share of the absorption by the system, and, if the more populated states absorb one circular polarization preferentially, an MCD signal will be observed. An energy level diagram depicting this situation

[6]

MAGNETIC CIRCULARDICHROISM

119

8000

?

6000

"7

4000 2000

0

320

300

340

360

380

Wavelength (nm) b

~'~

0.5

0

;

I

:

320

340

I

o

~ -0.5 -1 300

360

380

Wavelength (nm) FIG. 4. (a) Absorption spectrum corresponding to the energy level diagram shown in Fig. 3. Within the resolution of a typical spectrophotometer, exactly the same absorption spectrum would be recorded in the presence of a magnetic field as in its absence. (b) MCD spectrum for the energy levels shown in Fig. 3, The two B-type MCD spectra have shapes similar to the corresponding absorption bands, but opposite signs.

is shown in Fig. 7. In contrast to A- and B-type spectra, the C-type MCD will be strongly temperature dependent, particularly at lower temperatures, as illustrated in Fig. 8b. The corresponding absorption spectrum is, however, far less sensitive to temperature than the MCD, as illustrated in Fig. 8a. C-type MCD results from a degenerate state of the absorbing system, and hence it will always be combined with an A-type MCD spectrum. For the case of a simple 2-fold degeneracy, the observed MCD spectrum will approach the A-type spectrum, which resembles the deriva-

120

ULTRAVIOLET/VISIBLE SPECTROSCOPY

[6]

©

I © 10> H=0

H>0

FIG. 5. Energy level diagram illustrating A-type MCD. The first excited state is composed of two states that we label IX) and 1I'3. The transition dipoles from the ground state 10) to IX) and IY) are perpendicular to one another, but in the absence of an external field, the energies of the two states are equal. Linear combinations of IX) and IY) will also be valid descriptions of the system in the absence of an external field, so the system can be described equally well as I+) - = (IX) + ilY))/2 I/2. The transitions from [0) to 1+) and 1-) will be circularly polarized in opposite senses to one another, but, as they have the same energy, there will be no net polarization observed. The magnetic field shifts one component to a slightly higher energy and the other component to a lower energy (the amplitudes of the shifts being equal). For most molecules of biological interest, the width of the absorption bands is greater than the field-induced splitting of the energy levels; hence, the splitting is difficult to observe in the unpolarized absorption spectrum, even if easily observable in the MCD.

tive of the absorption spectrum, at high temperatures, because the two states resolved by the magnetic field will be populated equally. As the temperature approaches absolute zero, however, the lower energy component of the initial state will be preferentially populated, and the MCD spectrum will approach the shape of the absorption spectrum. Summary of Spectral Types. Assuming that the energies of field-induced splittings are small compared to kT, the MCD spectrum displayed as a function of photon energy v (in units of wave numbers, cm- 1), can be expanded as a sum of A, B, and C terms according to the following expression: AeMCD(V)V oc ~i' [

dv + Bi +

fi(v)

(4)

where the summation is over all transitions in the designated spectral domain, and f(v) and df(v)/dv are the shape and first derivatives of the

[6]

MAGNETIC CIRCULARDICHROISM

121

tO000 8000 6000 "7

4000 2000 0 520

5O0

540

560

580

600

Wavelength (rim) 10

b

5

-5 -10 500

520

540

560

580

600

Wavelength (nm) FIG. 6. (a) Absorption and (b) MCD spectra corresponding to the energy level diagram shown in Fig. 5. The slight broadening of the absorption spectrum induced by a magnetic field is usually too small to be observed. However, the corresponding A-type MCD of the magnitude shown would be easily observable using the type of spectrometer shown in Fig. 2.

shape of the absorption of the ith transition, normalized such that f f(v) du = 1. The corresponding expression for the unpolarized absorption

spectrum is

v

i

The coefficients A, B, C, and D for each transition can be expressed in terms of quantum mechanical matrix elements, and hence related to molecular parameters.l'2

122

ULTRAVIOLET/VISIBLE SPECTROSCOPY

[6]

O © H=0

H>0

FIG. 7. Energy level diagram for a system in which the ground state is 2-fold degenerate. The quantum mechanical treatment of the degenerate states is similar to the case shown in Fig. 5; however, differential population of the states resolved in energy by application of a magnetic field results in greater absorption of the circular polarization absorbed by the state shifted to lower energy. Note that this will be the absorption band that appears shifted to higher energy. The result is a temperature-dependent, C-type MCD spectrum.

Information from Magnetic CircularDichroism Spectra MCD can provide several types of useful information, but I must also mention some of the potential traps in the interpretation of MCD spectra. The theory of MCD depends on the degeneracy of the initial and/or final states of the transition(s) involved. Because exactly degenerate transitions can exist only if the absorbing species possesses at least a 3-fold axis of symmetry, it might appear that experimental MCD could be used to determine something about the symmetry, or lack thereof, of the absorber. This does not follow, however, for two reasons. First, symmetric molecules (benzene is a good example) have both degenerate and nondegenerate transitions, so the existence of a B-type MCD spectrum does not imply that the absorbing species is not symmetric. Second, an MCD spectrum that resembles the derivative of the absorption spectrum may either be a true A-type spectrum or result from two closely spaced B-type spectra of opposite sign. For example, the MCD of both the 260 and 200 nm bands of adenine and hypoxanthine resemble the derivative of the corresponding absorption bands, 25'26as illustrated in Fig. 9, but the known symmetry of the molecules proscribes their classification as true A-type spectra. Such spectra have been describedas pseudo-A-type. Similarly, the shape of 25 W. Voelter, R. Record, R. Bunnenberg, and C. Djerassi, J. Am. Chem. Soc. 911,6163 (1968). 26 j. C. Sutherland and K. Griffin, Biopolymers 23, 2715 (1984).

[61

MAGNETIC CIRCULAR DICHROISM

123

tO000 8000 6000 "7

4000 2000 |

400

420

440

460

I

I

480

500

Wavelength (nm) 30

b

[" 20 7

~ e~

~

1o 0

i

;

i

I

~

~..,i..=~I

•

-10 4O0

420

440

I

I

I

°a

460

480

500

Wavelength (nm) Fro. 8. (a) Absorption and (b) MCD spectra corresponding to the energy level diagram shown in Fig. 7. For temperatures at which kT is much greater than the difference in energy of the magnetic field-resolved degenerate states, the MCD will be dominated by an A-type spectrum and will resemble the derivative of the absorption spectrum, as in the case illustrated by the dashed curves. For temperatures at which kT is comparable to the difference in energies of the resolved states (as shown by the solid curves), however, the higher probability of occupation of the lower energy state will result in the preferential absorption of one circular polarization over the other. This produces a dramatic effect on the MCD spectrum but only a minor effect in the net absorption spectrum.

the MCD spectrum alone cannot distinguish A- and C-type spectra, because two closely spaced C-type spectra of opposite sign may maintain the shape of the derivative of the absorption band even as the temperature is lowered, as illustrated in Fig. I0. The change, or lack of change, in the magnitude of the MCD signal at low temperatures is the definitive test for C-type MCD.

124

ULTRAVIOLET/VISIBLE SPECTROSCOPY

[6]

4

E

u "7

0

-T

i

150

200

250

300

X(nm)

FIG. 9. Absorption spectrum (bottom) and MCD spectrum (top), of hypoxanthine in dilute phosphate buffer at pH 6 from 180 to 300 nm. The MCD spectrum is similar in shape to the derivative of the absorption spectrum. The spectra demonstrate the ability of MCD to resolve transitions that are not easily detected in absorption spectroscopy. They also demonstrate pseudo-A-type MCD spectra, since the symmetry of hypoxanthine forbids the existence of a true A-type MCD. This illustrates that information about the symmetry of a chromophore cannot be deduced from a comparison of the shape of the absorption and MCD spectra. (Adapted from Sutherland and Griffin, 26 with permission.)

MCD is, however, an exellent method of detecting the existence of multiple transitions that are not resolved in the absorption spectrum. Thus, if the shape of the MCD spectrum differs from that of the corresponding absorption spectrum, we know that the observed absorption spectrum is the sum of two or more unresolved transitions, as illustrated for riboflavin in Fig. I I. The converse is usually, but not always, true. In other words, if the MCD has the same shape as the corresponding absorption spectrum, there is likely to be exactly one transition involved. The slight reservation expressed in the previous sentence is required because of the possibility that an absorption envelope contains two or more transitions, all of which

MAGNETIC CIRCULAR D1CHROISM

[6]

125

_64o I

~o

---4-----4---------~--+-------+---

350

370 390 WAVELENGTH

410 (nm)

I

430

450

FIG. 10. MCD spectra of ferricytochrome c at + 24° and - 6 4 °, illustrating that closely spaced C-type MCD bands can resemble the derivative of the absorption spectrum. Thus, temperature dependence, and not the shape of the spectrum, is the proper hallmark for C-type MCD. The data also show that temperatures near absolute zero are not needed to demonstrate the temperature dependence of MCD spectra. [Adapted from J. C. Sutherland, Anal. Biochem. 113, 108 (1981), with permission.]

have transition dipoles directed parallel to one another. Such transitions will not be coupled to one another by a magnetic field, and could all give B-type spectra (owing to coupling to transitions at different energies). The resulting sum of the B-type MCD spectra would resemble the shape of the absorption spectrum.

]

~

V

wavelength(rim)

FIG. 11. Absorption ( - - - ) and MCD spectra (--) of FMN in 0.1 M phosphate buffer at pH 7. The negative MCD in the region from 240 to 257 nm in this early MCD spectrum illustrated the detection of electronic transitions not revealed in the absorption spectrum. The existence of a distinct electronic transition centered near 245 nm in the UV spectrum of compounds containing the isoalloxazine ring was critical to the proper interpretation of the UV resonance Raman spectra of these compounds [R. A. Copeland and T. G. Spiro, J. Phys. Chem. 911,6648 (1986)]. [Adapted from G. Tollin, Biochemistry 7, 1720 (1968), with permission. Note that the sign of the MCD is inverted compared to the original presentation of the spectrum.]

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MCD, in combination with the unpolarized absorption spectrum, is a powerful tool for characterizing a chromophore. The diversity of possible MCD spectral shapes provides more qualitative information than is available from absorption alone. The ratio of the magnitudes of the MCD and absorption at defined wavelengths provides an even more informative quantitative criterion. Because both MCD and absorption are proportional to the concentration of the absorbing species, their ratio is independent of concentration and, hence, is an intrinsic property of the material; that is, AAMcD(~-I)

A(h2)

ASMCD(~-I) e(h2)

(6)

This ratio, the MCD/absorption anisotropy, thus provides an intrinsic characterization of the material being studied from spectral measurements alone and without knowing its concentration. The wide range of values of this ratio enhances the utility of this type of characterization. The two wavelengths specified in Eq. (6) would normally locate the peak magnitudes of the MCD and absorption, respectively. One cannot simply specify the measurement of both MCD and absorption at the peak of the absorption band because, in the case of A-type spectra, the MCD may be zero at this wavelength. Alternatively, we could plot the anisotropy ratio as a function of wavelength (or wave number) and use this "spectrum" as the signature of a chromophore. Experimental MCD can be compared with theoretical predictions to determine details of the physical and/or electronic structure of molecules. In the case of cyclic 7r-electron systems, the sign of the longest wavelength A- or B-type MCD spectrum can be related to the differences in energy of the two occupied orbitals that are highest in energy and the two unoccupied orbitals that are lowest in energy (see Mich127 and related papers in the same issue). Detailed comparisons can be made between experimental absorption, MCD, and electron paramagnetic resonance (EPR) spectra and theoretical predictions for metal ions in heme and other quasi-symmetric environments using ligand field theory, particularly for transitions that occur in the near-IR (see, e.g., Refs. 28 and 29). The same may be true of MCD studies using soft X-rays.19 Compared to EPR, MCD has the useful property that signals are observed for systems with no unpaired spins. The near-infrared MCD of heine proteins has been particularly 27 j. Michl, J. Am. Chem. Soc. 100, 6801 (1978). 28 p. A. Gadsby and A. J. Thomson, J. Am. Chem. Soc, 112, 5003 (1990). 29 Q. Peng, R. Timkovich, P. C. Loewen, and J. Peterson, FEBS Letters 309, 157 (1992).

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MAGNETIC CIRCULAR DICHROISM

127 I

500

500

400 7 7

300

00.

/

•

800

1000

1200

1400

1600

1800

;2000

Wavelon~h Into

FIG. 12. Near-infrared MCD spectrum recorded at 4.2 K for six low-spin heme proteins, each containing protoheme IX with histidine as one ligand but with different second ligands. The spectra illustrate the sensitivity of the near-IR MCD to the ligation of the heme ring and demonstrate that MCD in this region can be used to assign axial ligands. The near-IR MCD spectra, in combination with EPR, were also used to determine the electronic structure factors for the proteins. (From Gadsby and Thomson, 28 with permission.)

useful in such quantitative studies because the spectra in this region are due to d ~ d and charge transfer transitions and, thus, are more sensitive to the axial ligands of the heme group, as illustrated in Fig. 12. The ability to quantify a species with a distinct MCD spectrum and large MCD/absorption anisotropy in the presence of other absorbing species with lower anisotropies has also been used to quantify the amino acid tryptophan in proteins containing tyrosine residues that absorb in the same spectral region. 3°-32 Tryptophan exhibits a strong positive signal at 297 nm that is easily distinguished from the MCD of tyrosine at that wavelength. The large anisotropy and distinctive shape of the MCD spec3o G. Barth, R. Records, E. Bunnerberg, C. Djerassi, and W. Voelter, J. Am. Chem. Soc. 93, 2545 (1971). 31 G. Barth, E. Bunnerberg, and C. Djerassi, Anal. Biochem. 48, 471 (1972). 32 B. Holmquist and B. L. Vallee, Biochemistry 12, 4409 (1973).

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trum of porphyrin rings have been used to quantify these materials in urine" and to search for them in samples of lunar dust. 34 The wide range of values of the ratio of MCD to absorption forms the basis of another practical, albeit rarely used, application in biophysical spectroscopy. Molecules with a characteristic MCD spectrum and high MCD/absorption anisotropy in a limited spectral region (heme proteins are a good example) can be detected and quantified in complex mixtures that exhibit continuous absorption and scattering of light across the spectrum, such as crude cell or tissue extracts. 35 This use of MCD is similar to one-beam, two-wavelength absorption difference spectroscopy, which is widely used in studies of metabolism and the biochemistry of heine proteins. 36 Magnetic Circular Dichroism in the Extreme Ultraviolet and X-Ray Regions: Wavelengths Less Than 100 Nanometers Although scarcely used at present, the previously inaccessible extreme-UV and X-ray region of the spectrum, along with the infrared, represents the frontier for MCD spectroscopy. Unlike the case for IR wavelengths, however, the instrumentation required for wavelengths less than 100 nm is radically different from that used in any other spectral region. The development of synchrotron radiation sources has made possible the measurement of MCD in the extreme-UV (XUV, h < 100 nm) and X-ray regions of the spectrum. Three properties of synchrotron radiation are critical in this application: broad, continuous spectral distribution (extending from the IR all the way to X-rays), high intensity compared to conventional XUV and X-ray sources, and the polarization properties of the beam. As in the visible and near-IR regions, much of the interest in biological MCD will involve proteins, and other systems, that contain metals. This conclusion is based on the fact that there are so many carbon, nitrogen, and oxygen atoms in most biologically interesting molecules that one can derive little information from the MCD spectra of these elements. In contrast, there are usually no more than a few metal ions in a given protein. X-Ray absorption spectroscopy of these materials has already 33S. M. Kalman, G. Barth, R. E, Linder, E. Bunnerberg, and C. Djerassi, Anal. Biochem. 52, 83 (1973). 34G. W. Hodgson, E. Peterson, K. A. Kvenvolden, E. Bunnenberg, B. Halpern, and C. Ponnamperuma, Science 167, 763 (1970). 35p. M. Dolinger, M. Kielczewski, J. R. Trudell, G. Barth, R. E. Linder, E. Bunnenberg, and C. Djerassi, Proc. Natl. Acad. Sci. U.S.A. 71, 399 (1974). 36B. Chance, Rev. Sci. lnstrum. 22, 634 (1951).

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129

proved useful as a probe of the environment of the metal component of proteins, and MCD should enhance this information, as it does in other spectral regions. The particular advantage of the XUV and X-ray regions, compared to the UV, visible, and near-IR, is that the range of metals that can be studied is increased to include those that do not absorb at the longer wavelengths. Trends and Predictions MCD is useful in probing the electronic structure of small molecules containing conjugated ~--electron systems. The variety of molecules in this class is limited, however, because biological systems tend to use the same molecules over and over in different combinations, as exemplified by proteins and nucleic acids. Natural CD is useful in probing the huge variety of materials that can be constructed from a defined set of molecular building blocks, whereas MCD is useful in characterizing the "blocks" themselves, a drastically more limited enterprise. This limits the extent to which MCD will be used in the ultraviolet, the region where amino acids and nucleic acid bases absorb. The major opportunity for biological studies in the UV is the extension of MCD studies of such molecules into the vacuum UV (h < -200 nm). Such studies complement CD studies of proteins, nucleic acids, and other macromolecules that can now be extended into the VUV. The opportunities for MCD spectroscopy in the visible region are far more extensive. The MCD of the visible absorption bands of porphyrins have been probed by MCD since the beginning of the field. The sensitivity of the MCD spectra to the environment of porphyrin derivatives, in solution as well as embedded in proteins, provides information on both electronic structure and physical conformation. As an example, Dawson and colleagues have used MCD to probe the identity of the heme-type prosthetic group of myeloperoxidase. 37 Such capabilities combined with the seemingly infinite number of metalloproteins available in nature provide ample opportunities for the productive use of MCD. Prospects for MCD spectroscopy in the near-IR are also bright. The weak bands of heme and other metalloproteins are usually either d ~ d transitions of the electrons on the metal ion or charge transfer transitions involving orbitals of both the metal and its ligands. Thus, this spectral region provides direct information on the state of the metal ion, which usually is of critical importance in the function of the protein. The work ~7 M. Sono, A. M. Bracete, A. M. Huff, M. Ikeda-Sato, and J. H. Dawson, Proc. Natl. Acad. Sci. U.S.A. 88, 11148 (1991).

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of Thomson and colleagues was noted above, 2s but numerous examples could be given of interesting near-IR MCD originating from several laboratories. The introduction of commercial CD/MCD spectrometers that operate up to wavelengths of about 2/zm has made it easier to perform such experiments. Worldwide, there are roughly a dozen well-equipped laboratories that study metalloproteins using MCD spectroscopy in the visible and near-IR. MCD in the mid-IR, where absorption is due to changes in vibrational, as opposed to electronic, energy levels, has lagged far behind the development of natural CD in this region. The work of Keiderling and colleagues38 demonstrated that MCD can indeed be measured in the mid-IR. As has happened in other regions of the spectrum, porphyrins were among the first materials studied and produced a large easily measurable signal. The surprisingly large magnitude of the vibrational MCD of highly symmetric molecules appears to reflect the influence of vibronic coupling. 3s-4° The mid-IR is one of the frontiers of MCD spectroscopy. The other frontier for MCD spectroscopy is at the short wavelength end of the spectrum (h -< I00 nm), where facilities for measuring MCD in the XUV, soft X-ray, and X-ray regions have relatively recently become available or are being developed. As with other spectral regions, metalloproteins are likely to be the major focus, because the L edge absorptions of transition metals and K edge absorptions of lanthanides should show large MCD anisotropies. A particular advantage of X-ray absorption spectroscopy is that metals which are "spectroscopically silent" in the visible and near-IR should be easy to study. Because of the cost and difficulty of operation of advanced CD spectrometers and superconducting magnets, MCD has developed as a field dominated by a relatively small number of specialists. The extension of MCD technology into the X-ray domain may continue this trend, as a "beam line" (experimental station) suitable for MCD spectroscopy may cost several million dollars--exclusive of the cost of the synchrotron itself. However, because of the high cost of such instruments, they must be operated as "user facilities" and hence available for use by many scientists. Freed from the burden of developing and maintaining specialized instrumentation, scientists can focus on the preparation of interesting samples and the interpretation of experimental data. Thus, the move toward "big science" may have the paradoxical effect of opening the measurement of MCD to a wider range of scientists. 3s p. V. Croatto and T. A. Keiderling, Chem. Phys. Lett. 144, 455 (1988). 39 M. Pawlikowski and O. S. Mortensen, Chem. Phys. Lett. 168, 140 (1990). 4o M. Pawlikowski, M. Pilch, and O. S. Mortensen, J. Chem. Phys. 96, 4982 (1992).

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Acknowledgments The preparationof this chapterwas supportedby the Officeof Healthand Environmental Research, U.S. Departmentof Energy. I thank Stephen Cramer, Universityof California, Davis, John Dawson, Universityof South Caroline, LouiseHeusinkveld,BrookhavenNational Laboratory,Timothy Keiderling, Universityof Illinois at Chicago, Yu. Sharonov, Engelhardt Institute of Molecular Biology,Moscow, Peter Siddons, BrookhavenNational Laboratory,PhilipStephens, Universityof SouthernCalifornia,MartinStillman,University of Western Ontario, and Andrew Thomson, Universityof East Anglia, for assistance in obtaining preprints, reprints, and references to the current MCD literature.

[7] Low-Temperature Spectroscopy By ROBERT H. AUSTIN and SHYAMSUNDER ERRAMILLI Introduction This chapter is divided into three parts: some practical advice on how to do low-temperature spectroscopy, a discussion of the "internal" structure of visible absorption spectra and their temperature dependence, and a brief discussion of the utilization of low-temperature spectroscopy to probe protein dynamics. We would also like to go beyond the traditional use of low temperatures to trap intermediate chemical states and to speculate about the insight that low-temperature spectroscopy provides into the role of protein dynamics in controlling chemical reactions. Probably the main utilization of low-temperature spectroscopic studies in biophysics has been to trap short-lived intermediate chemical states of a protein. Typically the chromophore alone is the object of study, and the protein is viewed basically as a structure which simply holds the chromophore in place or may contribute some critical amino acids in close proximity to the chromophore. The protein is not believed to play an overtly dynamic role, a dynamic role being one where the reaction process is steered by time-dependent protein conformational changes. Temperature is used to slow down or arrest the reaction kinetics occurring at the chromophore and perhaps to discover additional chemical states which have too short a lifetime to be observed at room temperature. Such kinds of kinetic arrest studies have been successfully used in studies of heme METHODS IN ENZYMOLOGY, VOL. 246

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