[9] Variable-temperature magnetic circular dichroism

[9]

MAGNETIC CIRCULAR DICHROISM

[9] V a r i a b l e - T e m p e r a t u r e

By

ANDREW

199

Magnetic Circular Dichroism

J. T H O M S O N , M Y L E S R. and SIMON J. GEORGE

CHEESMAN,

Introduction A magnetic circular dichroism (MCD) spectrum is a measurement of the difference between the absorption of left and right circularly polarized light as a function of the wavelength of the measuring beam when a sample is placed in a magnetic field applied parallel or antiparallel to the direction of propagation of the light beam.1 Electric dipole transitions between electronic states can be induced by circularly polarized light provided that the component of the electronic orbital angular momentum in the direction of the applied field changes by either - 1 or + 1 for the absorption of right or left circularly polarized light, respectively. The application of a magnetic field leads to the lifting of electronic orbital and spin degeneracies and to the mixing of electronic states. MCD spectroscopy enables the optical transitions between Zeeman sublevels of ground and excited states to be resolved even though the Zeeman splitting may be orders of magnitude smaller than the line width of the transition. Thus, Zeeman spectroscopy can usefully be performed with molecules that give rise to broad electronic transitions. There are three possible consequences to the application of a magnetic field, namely, Zeeman splitting of ground and/or excited degenerate states, field-induced mixing of states, and a change in the population of molecules over the Zeeman sublevels of a paramagnetic ground state. These give rise to contributions to the intensity of an MCD spectrum called, respectively, the A-term, the B-term, and the C-term. 2 Because the C-term arises from a Boltzmann population of molecules over a set of ground state sublevels, it follows that it is nonzero only for molecules with ground state paramagnetism. Therefore, a C-term becomes very intense when a paramagnet is cooled to ultralow temperature. Furthermore the variation of this component of the MCD intensity with field and temperature can be analyzed to yield the magnetic parameters, such as spin, g value, and zero-field splitting, of the ground state.

1 A. D. B u c k i n g h a m and P. J. Stephens, Annu. Rev. Phys. Chem. 17, 399 (1966); P. J. S t e p h e n s , J. Chem. Phys. 52, 3489 (1970). 2 p. j. Stephens, Adv. Chem. Phys. 38, 197 (1976).

METHODS IN ENZYMOLOGY, VOL. 226

Copyright © 1993by AcademicPress, Inc. All rights of reproduction in any form reserved.

200

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9l

Variable-temperature MCD spectra of transition metal ions in proteins can provide a selective probe of the magnetic properties of individual metal ions within a protein provided that optical transitions unique to that center can be identified. The method also provides a link between parameters identified by other magnetic spectroscopies such as electron paramagnetic resonance (EPR) and M6ssbauer spectroscopy or determined by magnetic susceptibility. This can be especially valuable for the study of metalloproteins containing more than one magnetic center. Furthermore, the method is unaffected by the presence of impurities, such as adventitious high-spin Fe(III) or Cu(II), often present in protein preparations, provided that the impurity ion has no optical transitions in the wavelength region of interest. Because the MCD signal is a signed quantity related to factors such as the absolute sign of the ground state g values and the linear polarization of the optical transition, it can provide an additional and useful assignment criterion. The MCD intensity of a paramagnetic molecule varies linearly with the inverse of the absolute temperature when the thermal quanta, kT, are greater than the overall energy spread of the ground electronic state owing to zero-field and Zeeman e f f e c t s . 3 This is the Curie law regime observed in all forms of spectroscopy that are sensitive to the magnetic state of the molecule. The original theoretical analysis of Buckingham and Stephens 1 is only valid in the Curie law limit. At very low temperatures and high applied magnetic fields, the MCD intensity varies nonlinearly with 1/T an d B. 4 Analysis of the intensity variation in this nonlinear region is much less straightforward. However, the B and 1/T space contains much useful information about the ground-state magnetic parameters. Even if unambiguous analysis is impossible, the method gives a set of useful diagnostic parameters. To obtain this information measurements must be made down to 1.5 K and at magnetic fields up to at least 5 tesla. This necessitates the use of a superconducting solenoid. Measurements on frozen aqueous solutions of proteins under these conditions could not be readily and routinely performed until 1980 but became possible because of the design and construction of a split-coil superconducting solenoid with a sample compartment located between the coils. 5 The sample compartment is accessible from the top of the magnet Dewar flask, and samples can be exchanged readily without breaking the vacuum space of the magnet coils. The sample is held at atmospheric pressure and can be immersed in boiling 3 S. B. Piepho and P. N. Schatz, "Group Theory in Spectroscopy with Applications to Magnetic Circular Dichroism." Wiley, New York, 1983. 4 p. N. Schatz, R. L. Mowery, and E. R. Krausz, Mol. Phys. 35, 15376 (1978). 5 A. J. Thomson and M. K. Johnson, Biochem. J. 191, 411 (1980).

[9]

MAGNETIC CIRCULAR DICHROISM

201

liquid helium. This magnet design gave rapid and ready sample exchange and, most importantly, accurate and uniform control of the sample temperature. The design has overcome the problem of polarization artifacts caused by strain in the windows of the sample compartment, which are held at 4.2 K or lower temperature. This method has become the standard way of collecting variable-temperature MCD spectra. Allied with the development of methods for generating achromatic circularly polarized light over a wide wavelength range, spectra can be measured routinely and rapidly over the wavelength range 200-5000 nm, on samples held at temperatures between 1.5 and 300 K and at magnetic fields up to 10 tesla. This has given rise to reports of the MCD spectra of almost all the transition metal centers identified to date in proteins, including hemes, 6 iron-sulfur clusters, 7 nonheme iron (both mononuclear8 and binuclear, 9 nickel,l° manganese,ll copper, 12and molybdenum. 13The method has potential for the study of lanthanide ion probes, but no low-temperature studies appear to have been reported. This chapter describes in detail for the first time the methodology used in the Norwich laboratory (University of East Anglia), which has become that almost universally employed, for the measurement of MCD spectra in the low-temperature and high-field regions. The analysis of magnetization curves is discussed and examples given briefly of the spectra of some of the main classes of transition metal centers. Principles Magnetic circular dichroism spectroscopy involves the application of two principles: first, a laboratory applied magnetic field induces the splitting of electronic states degenerate in zero field and the mixing of electronic states and, second, a circularly polarized light beam induces electric dipole transitions between the Zeeman components of the ground and excited electronic states. The magnetic field must be applied colinear with the direction of the light beam, the so-called longitudinal Zeeman direction, 6 M. R. Cheesman, C. Greenwood, and A. J. Thomson, Adv. Inorg. Chem. 36, 201 0991). 7 M. K. Johnson, A. E. Robinson, and A. J. Thomson, in "Iron-Sulfur Proteins" (T. G. Spiro, ed.), Wiley (Interscience), New York, 1982. 8 j. W. Whittaker and E. I. Solomon, J. Am. Chem. Soc. 110, 5329 0988). 9 R. C. Reem, J. M. McCormick, D. E. Richardson, P. J. Stephens, R. L. Musselman, and E. I. Solomon, J. Am. Chem. Soc. l l l , 4688 (1989). 10 M. R. Cheesman, D. Ankel-Fuchs, R. K. Thauer, and A. J. Thomson, Biochem. J. 260, 513 (1989). 1i j. W. Whittaker and M. M. Whittaker, J. Am. Chem. Soc. 113, 5528 (1991). 12 D. M. Dooley and J. H. Dawson, Coord. Chem. Reo. 60, 1 (1984). I3 N. Benson. J. A. Farrar, A. G. McEwan, and A. J. Thomson, FEBSLett, 307, 169 (1992).

202

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

Y

hvH

B

~ Ey

hv//

ML=+I 1p

........

B

( o)

hv /B

hv ..L B

,/

ML= 0

/\

ML=-I

RC P/I"

LCP

LCP 1S

(n)

ML=O

~

>~

•

(J

I1

._

o

o171o v

RCP FiG. 1. The longitudinal and transverse Zeeman effect for the atomic transition IS ~ 1p (after R. Grinter). B is an applied magnetic field, and heavy arrows denote monochromatic radiation of energy hv. RCP and LCP denote right and left circularly polarized light; o- and ~r denote linearly polarized light perpendicular (senkrecht) and parallel to the applied field axis, z.

since only this arrangement leads to the absorption of circularly polarized photons. The transverse Zeeman experiment leads to selection rules for the absorption of plane polarized radiation. This can be exploited in magnetic linear dichroism (MLD), but that technique has not been much used and almost not at all in metaUoprotein spectroscopy. Figure 1 illustrates

[9]

MAGNETIC CIRCULAR DICHROISM

203

the principles and selection rules of the transverse and longitudinal Zeeman effect for the simple case of an atomic transition 1S --~ 1p. Zeeman spectroscopy has been used to assign the line spectra of gasphase spectra of atoms and monoatomic ions. Because the electronic spectra of such species are devoid of vibrational and rotational substructure, the bandwidths are narrow (< 10 -z cm-1) compared with the magnitudes of typical Zeeman splittings. The electronic spectra of molecular species, by contrast, contain bands which are 102-103 cm -1 in width because vibrational transitions are coexcited with the electronic transition. This is orders of magnitude greater than Zeeman energies and hence precludes direct observation of Zeeman shifts. However, because transitions between Zeeman sublevels occur with opposite circular polarization in the longitudinal Zeeman mode, it is possible to observe individual transitions to Zeeman sublevels. This is the basis for the sensitivity and utility of MCD spectroscopy. Figure 2 illustrates the operation of the selection rules for an electronic transition in a molecule possessing a ground state with a spin moment, S = ½, but no orbital moment and an excited state with a spin moment S = ½, and a 2-fold orbitally degenerate state, characterized by ME = -+1. Two limiting case are considered: zero spin-orbit coupling in the excited state (Fig. 2a) and spin-orbit coupling greater than the Zeeman splitting (Fig. 2b). The selection rules which operate for transitions between the Zeeman subcomponents are AML = -+1 for the absorption of a left circularly polarized photon and AML = - ! for a right circularly polarized photon. Spin changes are forbidden under the selection rule •Ms

= 0.

In Fig. 2a, the four allowed transitions fall at two energies separated by gL~B where gL is the orbital g factor in the excited state. Pairs of transitions with the same circular polarization originate from the upper and lower Zeeman component of the ground state but have the same energy. As the thermal population of the two levels is varied, the integrated intensity does not change, that is, the sum of the intensities of transitions a plus c is equal to that of b plus d, and both forms are independent of temperature. Hence the form of the MCD spectrum is a derivative shape centered at the energy E 0 of the transition in the absence of the magnetic field. The intensity of the difference spectrum AA depends on the magnetic field and gL, the excited state orbital moment. The transitions are shown as lines broadened by a line shape function. This is called an A-term. Note that the lines are broadened into bands in real systems because of the coexcitation of molecular vibrations. If the band shape is to be analyzed in order to determine gL, then each vibronic component of the band must also undergo a Zeeman shift identical to that of the electronic

204

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

(a)

[9]

(b) ML MS

ML

MS

±1

±112

+1

0

.,~

/ 2(gL+2) P.B.B

T +1

+ 112

• 112

-1

-112

±1

~I12

0

.I/2 2V.B.B

+_.112 0

-1/2

-,L-

bcad b

AA

I C

d a

FIG. 2. Circularly polarized transitions between 2A and 2E states in the presence of a magnetic field parallel to the light beam when spin-orbit coupling is (a) zero and (b) nonzero. The lower half of the figure shows, in stick form, the allowed transitions, and hence the form of the MCD and absorption spectra.

c o m p o n e n t . T h i s is c a l l e d a rigid shift, a n d it is m o s t u n l i k e l y to o c c u r , p a r t i c u l a r l y w h e n t h e e x c i t e d s t a t e is e l e c t r o n i c a l l y d e g e n e r a t e . I n this c a s e v i b r o n i c e f f e c t s l e a d to t h e b r e a k d o w n o f t h e B o r n - O p p e n h e i m e r a p p r o x i m a t i o n , a n d t h e rigid shift a p p r o x i m a t i o n is no l o n g e r valid. H o w -

[9]

MAGNETIC CIRCULAR DICHROISM

205

ever, a rigorous analysis can still be carried out by computing the areas of the curves, or the moments. This analysis is described in detail elsewhere, z When spin-orbit coupling in the excited state becomes significantly large compared with Zeeman energies a different picture emerges (Fig. 2b). Spin-orbit coupling splits the 2E excited state into a pair of doubly degenerate states defined by Mj values of -+~-and -+½. The separation is equal to the spin-orbit coupling constant. The state defined by Mj = _½, does not undergo a first-order Zeeman splitting when a magnetic field is directed along the axis of quantization, whereas the other pair, defined by Mj = -+~, does split into two components separated by 2(gL + 2)/tZBB. The absorption spectrum will contain two transitions, one to each of the excited state spin-orbit components. The MCD spectrum contains, as before, four circularly polarized transitions, two of one sign (a, c) and two of the opposite sign (b, d). However, the pairs of transitions separated by spin-orbit coupling which are oppositely circularly polarized originate from the upper and lower components of the ground state. Hence the MCD spectrum is strongly temperature dependent, with each spin-orbit component giving rise to an MCD band of opposite sign that increases in intensity as the temperature is lowered. These are known as C-terms, and this particular case has been termed "spin-dependent" C-terms. The moment of the total bands depends on, inter alia, the excited state spinorbit coupling. The third contribution to an MCD spectrum arises from the fieldinduced mixing of the wave function of states (Fig. 3). Two perpendicularly polarized transitions are coupled by a magnetic field applied normal to the plane of the transitions. This results in a small Zeeman shift. The mixing of the wave functions ensures that each transition acquires a small percentage of the perpendicular polarization. Hence the polarization of the transitions acquire a circular component, the magnitude of which depends on the strength of the applied field and inversely on the energy separation between the two states being mixed. The contribution to the MCD spectrum is called a B-term. B-Term intensity will arise from the mixing between all states, although the more remote in energy the state, the smaller will be its contribution to the B-term. The contribution of each term A, B, and C to the total MCD intensity can be written thus 3 A A = Aecl = 3 2 6 . 6 t X B B c l [ A l ( - r f / r e ) + (B o + C o / k T ) f ] E

(1)

The line-shape function f = f ( E ) , where E is the energy (in cm-1), is introduced. This expression is derived using the rigid shift and BornOppenheimer approximations and assumes the linear limit, namely, k T >>

206

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

.~--~---~

[9]

X + kY

B/AE

Bz=0

__It BZ#0

) ENERGY

A

Y

X

I

I

>ENERCy

FIG. 3. Origin of an MCD B-term for the case of two optical transitions of different energies that are linearly polarized perpendicular to one another and to the applied magnetic field, B. g/XBB where g is the ground state g factor,/J'B is the Bohr magneton, and B the applied magnetic field. Equation (1) shows that the A-term line shape is the first derivative of the absorption band shape with respect to energy, whereas the Band C-term line shapes are identical to the associated absorption band. The three contributions are additive. Because the C-term depends inversely on the absolute temperature, its contribution is readily extracted from an experimental spectrum by variable-temperature studies. Note that if another state becomes thermally accessible over the temperature range of the study then a B-term contribution can appear from this state. The experimental quantity measured is AA = A L - A R where A L and A R are the absorbances in left and right circularly polarized light. AA is related to As ( = e L - eR) , the differential extinction coefficient, by the B e e r - L a m b e r t Law, AA = Aecl. The molar ellipicity, [0] is related to A¢ by the expression [0] = 3300 Ae.

[9]

MAGNETIC CIRCULAR DICHROISM

207

The quantum mechanical expressions for the terms AI, Bo, and C Ohave been described by Stephens,2 and Piepho and Schatz.3 The contributions to the A 1 and Co term can be described in a qualitative way. Consider a trasition a --* j that has intensities polarized along the three Cartesian coordinates proportional to Mi2 where i = x, y, and z. If both states a and j have a spin S = ½ the system is anisotropic, and then the Zeeman splitting of both states will be anisotropic. This can be represented by the components of the g tensor along the three Cartesian directions, gi and gi' referring to the ground and excited states, respectively. The co-term is given by an expression which is the sum of the products of the ground state g tensor components with the components of the electric dipole transition moment perpendicular to the direction of the g tensor component: Co oc gzMxMy + gyMxMz + gxMyMz

Hence, for a sample that contains randomly oriented molecules, such as a liquid or frozen glass, Co will have contributions from all three components. However, if the optical transition a ---~j is polarized only in the x,y plane, then the value o f g z only dictates the magnitude of the Co term. This simplified expression emphasizes that nonzero MCD C0-term intensity requires a pair of perpendicularly polarized components in the transition. The MCD spectrum will select only those molecules lying with nonzero values of these two components perpendicular to the magnetic field direction. An analogous expression can be written for the Ax-term, which demonstrates that the A-term line shape can arise from a degeneracy either in a or inj. Again, the requirement for the components of the dipole strength to be perpendicular to the g tensor component and hence the applied magnetic field is evident: A, ~ (g'z - gz)MxMy + (g'x - gx)MyMz + (g'r - gy)Mz

The absorption spectrum measured in isotropic light has an intensity dependent on the dipole strength, D, and this is given by the sum of the transition moments along the three directions x, y, and z for a randomly oriented sample. By evaluation of C Oand D it is possible to calculate the ratio Co/D and also to obtain the ground state g value by experiment: D = Mx2 + My2 + Mz2

and

Co/D ocg

This has been carried out for many cases. Consider, for example, an octahedral molecule (point group Oh) having a ground state which is orbitally degenerate, Txu, and in which two transitions Tlu ~ Alg and Tlu --->

208

SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S

[9]

Eg can be observed. The ratio of ColD for these transitions can be shown to be 2g and - g respectively. Hence, a study of the temperature dependence of the MCD intensity of these bands over the linear region will give Co and the signs will give a distinction between the assignment of the two bands. However, the ground state g value can only be determined if there is knowledge of the excited state symmetry and an accurate determination of the dipole strength. It turns out that a more accurate determination of ground state g values can be obtained by extending measurements into the nonlinear, low-temperature, high-field regime. 5 This involves studies of MCD magnetization properties.

Magnetic Circular Dichroism Magnetization The MCD intensity associated with the C-term contribution increases linearly with B/T provided that kT is larger than the ground state Zeeman splitting. However, if B/T is increased further, a limit will be reached beyond which the MCD intensity no longer increases. The system is then said to be saturated. The paramagnetic assembly is fully magnetized. This behavior can be understood by recalling that a C-term arises from the unequal population distribution over the ground state Zeeman sublevels. At sufficiently large values of B/T all paramagnetic molecules will be in the lowest Zeeman sublevel, and further increases in B or decreases in T will produce no further population changes. Increases in the A-term and the B-term contributions will occur if the magnetic field is increased, but these changes are usually, but not invariably, a small contribution when a system is close to the saturation limit. Ground State S = ½, Isotropic. It can be shown that, if the ground state consists of a Kramers doublet with an isotropic g value gs, the MCD intensity varies as

&A = K tanh(gj~B/2kT)

(2)

where K is a constant. 4 The shape of this curve yields the ground state g value, gj, readily. The curve can be fitted to Eq. (2). However, there is a simple useful procedure that indicates how closely the curve approximates to a given g value. In the high temperature region the Curie-Law slope of &4 against ~BB/2kT is Kgj. The magnetization limit occurs when &4 = K. Hence, the ratio of the Curie-Law slope to the magnetization limit is gj. The intercept value is 1/gj as shown in Fig. 4a. 5 For electronic ground states with spin S -> ½which are isotropic, the magnetization properties depend on the ground state S value. In zero field the state of spin S comprises (2S + 1) degenerate components. On

[9]

209

MAGNETIC CIRCULAR DICHROISM

a

~n

ii /I I I / I

___

j

,,,7,,

// %1

I / ! ! /'

I I I I I

law region I I 11-

13B/2kT

1O0

6O

%I

2kT

2(J+l)gj

40 I1~ 20

0

~ 1 ~

2~'~3

gj = 2 I 0.5 0.375 0.3 0.25 0.21 0.188

0.0

I

I

0.2

i

I

0.4

i

0.1

6

I

I

0.8

i

.!

10

i

I

1.2

13B/2 kT FIG. 4. (a) MCD magnetization curve of a paramagnetic molecule with a ground state spin S = ½ having an isotropic g value. (b) MCD magnetization curves of molecules with ground state spins o f ½, 1, a, 2, and ~ in the absence of ground state zero-field splitting and for gj = 2.0. The intercept values I are given by a(JH)gj.

210

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

application of a magnetic field the levels with highest and lowest Ms values diverge most rapidly with field, and these dominate the population difference. Figure 4b shows a set of MCD magnetization curves for S = ½, 1, ~, 2, and ~. The Boltzmann populations over the Zeeman sublevels of the (2S + 1) ground state components can be expressed as a simple summation function involving the temperature, magnetic field, and magnetic components. These are the Brillouin functions encountered in the theory of magnetic susceptibility. 14 These principles have been put to the test using species isolated in rare gas matrices. 15It was shown that the intercept values satisfy the relationship tzBB/2kT = ~/(J + 1)gj

(3)

where J is the ground state total angular momentum quantum number and gj = 2.0 for a spin-only ground state. 16 The intercept values marked in Fig. 4 agree with those predicted from Eq. (3). Ground State S = ½, g Value Anisotropic. If the ground state of a Spin S = ½ paramagnet is isotropic, the MCD magnetization characteristics are the same for all molecules in a sample regardless of their orientation relative to an applied magnetic field because the Zeeman splitting is isotropic. However, when the ground state g value is anisotropic, the magnetization properties of an individual molecule depend on its orientation with respect to the applied field. To simulate the MCD magnetization curve of anisotropic paramagnets randomly distributed, it is necessary to average over all orientations. Furthermore, if the optical transition between the ground state and an excited state is strongly polarized, the MCD will selectively detect certain subpopulations. Schatz et al. 4 gave a theoretical treatment of this problem for the particular case of an isolated Kramers doublet ground state with an axially symmetric g tensor and having MCD transitions to excited states either z or xy polarized. They derived an expression for the MCD intensity, Ae, in molecule-fixed coordinates and showed the angular dependence of each term with respect to the applied magnetic field. The sum of this expression over one angle, 0, between the unique molecular axis, z, and the direction of the applied magnetic field is required for an axially stimulated case. The resulting expression contains integrals which have to be evaluated numerically: 14 R. L. Carlin and A. J. van Duyneveldt, "Magnetic Properties of Transition Metal Compounds." Springer-Verlag, New York, 1977. ~ R. G. Graham, R. Grinter, and R. J. Singer, Chem. Phys. Lett. 133, 196 (1987). 16 R. G. Graham, Chem. Phys. Lett. 133, 193 (1987).

[9]

Ae

~ - = M2y

MAGNETIC CIRCULAR DICHROISM

211

[ fr/2cosaOsinO . (FflH~ dO p gNtanh \2kT/ o 2,/2Mz (~/2sin30 (rflH~ dO] Mxy oj ---F~-g ±tanh \ 2kT /

(4)

F = (sin 2 0 g±2 + cos 2 0 gll2)~/2 Equation (4) contains the sum of two terms, one of which depends on gll and the intensity of the transition dipole in the x,y plane and the one which depends on g_ and transition dipole moments polarized either yz and xz. K is a constant, and Mx, My, and M z are the transition dipole moments along the molecular axes. This expression has been generalized to the case of a rhombic ground state where two angles 0 and 4~ need to be averaged. ~b is the angle in the x,y plane between the x axis and the plane containing the field direction. 5 There are a n u m b e r of special cases which may be met in practice that enable simplified rules to be stated and intercept values predicted as follows: (1) F o r xy-polarized transitions only the second term of Eq. (4) is required. (2) If g± = 0 and gll ~ 0 only xy-polarized transitions will contribute to the MCD intensity. Intercept values are readily calculated from Eq. (4) as follows:

I = 3/Dglr where

and where o- = gig± and o- = cosh a. For an isotropic ground state gll = g±, o- = 1, a n d D = 3.0. H e n c e I = 1/g in agreement with the result from Eq. (2). In the case of a ground state where gll ¢ 0 and g± = 0, D = 2.0 and then I = ~g[l" The expression derived by Mowery, Schatz, and Krausz [Eq. (4)] is valid only for an isolated K r a m e r s doublet ground state or for an isolated effective electronic doublet where -+Ms are degenerate. When the ground state spin is greater than S = ½, there will generally be a zero-field splitting that separates the energy levels. In the case of odd spin states the zerofield levels will be pairs of Kramers doublets with M s values of +S, -+(S - 1), + ( S - 2 ) . . . separated by the zero-field splitting energy. For an even spin state degeneracies will remain only ff the zero-field distortion has axial symmetry. In the general case an applied magnetic field will mix the zer0-field spin states, and this will give rise to B-terms. These contributions to the MCD intensity can be large because the field-induced

212

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

mixing is inversely dependent on the energy separation between zerofield levels. This separation is likely to be of the order of a few wave numbers, and hence the B-term condition will not be negligible. Furthermore, the contribution may be temperature dependent if the thermal population of the zero-field levels mixed by the field varies over the temperature range of the experiment. Several different approaches have been taken in an attempt to overcome this complexity of analysis. One way is to make temperature-dependent measurements of the MCD intensity at sufficiently low magnetic fields that the intensity remains linear in field, a7 Then the equations of Buckingham and Stephens are valid. The MCD intensity &4 contributed by each component of the ground state contains an A-, B-, and C-term weighted according to the fractional thermal population of that component. For example, in the case of three pairs of Kramers doublets separated in energy by 2D and 4D, as for S - ~, 3

AA/K = ~ ( A i + B i + Ci/kT)otiB i=1

where ~; is the fractional population of each Kramers doublet and al = 1 / Q , o~2 = e-ED/kUQ, and a3 = e-6D/kT/Q. Q, the partition function, is equal to [1 + e -20/kZ + e +6D/kr]. The curve of~A against 1/Tis then fitted to this expression. The unknowns are D and three values of A, B, and C. The A values may be ignored since they are usually small. If it is known that the optical transition is x y polarized, the B-terms will be zero. The xy-polarized transition selects the subset of the population of molecules which is lying with the z axis, the axis of distortion, parallel to the applied field. The Kramers doublets are not mixed by the magnetic field along this axis. This method of analysis has been applied to the case of high-spin ferric heme groups. The visible region of optical transitions are known, from polarized single-crystal spectra, to be polarized in the heme plane and the axis of distortion of the zero-field splitting that lies perpendicular to this plane. Good agreement was found between D values obtained from analysis of the MCD intensity variation with 1 / T in the linear limit. 15When the zero-field splitting is large, say, 10-100 cm -1, this method can also work well. An example is the analysis of the zero-field splitting of the S = 1 Fe(IV) ferryl form of the heme group. The simulation of the 1 / T variation of the MCD intensity gave values of D for the zero-field splitting of the I7 W. R. Browett, A. F. Fucaloro, T. V. Morgan, and P. J. Stephens, J. Am. Chem. Soc. 105, 1868 (1983).

[9]

MAGNETIC CIRCULAR DICHROISM

213

S = 1 state. No determination of the rhombic distortion parameter was possible. ~8 A more general and rigorous method of fitting magnetization curves over the entire range of B and T has been developed in the Norwich laboratory. 19The approach is to describe a general ground state nS, using a spin Hamiltonian, and to consider transitions from this state to selected components of an excited state np. The excited state is not intended to represent the true excited state. However, it allows the choice of transitions with a defined linear polarization and with selected components of spin. The spin Hamiltonian used to model the ground state comprises terms representing the Zeeman effect and the zero-field splitting D and E: H = tZBBgS + D[Sez - S ( S + 1)/3] + E ( S ~ - S 2)

This Hamiltonian is expressed as a function of the Euler angles 0 and q5 between the molecular axes and the direction of the magnetic field. The excited state, nP, is subject to a Hamiltonian H = ~ L S + V cv

where the first term represents the action of spin-orbit coupling and V CF, the crystal-field term, comprises two parts, the tetragonal (~-)and rhombic (p) components. This Hamiltonian is not angular dependent since the principle of spectroscopic stability removes the need for it to be so. The intensity of the MCD transition between each component of the "S ground state and the np excited state is given by the expression derived by Schatz et al. 4 [Eq. (4)]. This expression is also dependent on the Euler angles 0 and ~b. A computer program enables the MCD intensity to be calculated at each value of B and T for each transition between the ground state components and selected excited state components at chosen values of 0 and ~b. The results are then subjected to a powder averaging process. The linear polarization is selected by the choice of excited state components. Because the ground and excited state spin Hamiltonians are diagonalized, the offdiagonal Zeeman terms are present; in other words, the intrastate B-terms are incorporated. The philosophy behind this approach is to measure the MCD magnetization curves at several different wavelengths for a given paramagnetic center. Because the ground state is common to measurements made at 18 N. Foote, P. M. A. Gadsby, C. Greenwood, and A. J. Thomson, Biochem. J. 261, 515 (1989). 19 M. R. Cheesman, Ph.D. Thesis, University of East Anglia, Norwich, U.K. (1988).

214

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

different wavelengths, only the linear polarization of the transition can vary. If the MCD magnetization curves are independent of wavelength of measurement, the linear polarizations must be the same at all wavelengths. H e n c e , a set of wavelength-dependent magnetization curves enables a smaller number of theoretical parameters to be fitted to a large array of experimental numbers. The ground state parameters are n, the spin, the true g value, and the zero-field parameters D and E. These must remain the same for e v e r y set of curves at all wavelengths. The linear polarization is varied to fit all the curves. This is a nonlinear fitting method and has only been used to fit curves by eye. In principle it could be adapted to use nonlinear statistical methods with a fast digital computer. MCD magnetization curves at a given wavelength represent a three-dimensional surface of MCD intensity against B and 1/T. The object is to simulate this surface. There will be the usual dangers o f false minima being mistaken for a fit. H o w e v e r , this method does demonstrate the overriding significance of intrastate B-term intensity to obtaining a satisfactory fit. As an illustration of the use of the fitting of a set of wavelengthdependent MCD magnetization curves, we give the example of the reduced three-iron cluster [3Fe-4S] ° which was first shown by MCD magnetization to have a ground state spin of S = 2, with a negative value o l D . Magnetic susceptibility studies of the same protein, ferredoxin II of Desulfovibrio gigas, confirmed a spin of S = 2 with a D value of - 2 c m - 1.20The MCD spectrum is shown in Fig. 5, and the magnetization curves recorded at several wavelengths are given in Fig. 6. The MCD magnetization curves vary with the wavelength of measurement. H o w e v e r , all curves can be fitted to a ground state spin S = 2 with D = - 2 c m - 1 and E = 0 c m - 1 with various linear polarizations. This method of analyzing MCD magnetization curves uses one computer program for any spin quantum number and is capable of handling the general case as well as the various limits such as the high temperature case or the linear field approximation. H o w e v e r , the method does not add a contribution from B-terms arising from field-induced mixing with states b e y o n d the ground state. This contribution will appear as a diamagnetic term and will simply be linear in field. H e n c e , a correction can readily be added. The method calculates the zero-field and Zeeman energies and wave functions for the ground electronic state as a function of the Euler angles 0 and q5 using a spin Hamiltonian. This will be valid for ground states 20 E. P. Day, J. Peterson, J. J. Bonvoisin, I. Moura, and J. J. G. Moura, J. Biol. Chem. 263, 3684 (1988).

[9]

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gigas,

with spin-only moments. It may be necessary to add higher terms to the spin Hamiltonian when spin states greater than 2 are considered. Careful consideration must be given to the occurrence of an orbital moment in the ground state. In this case a spin Hamiltonian formalism is unlikely to generate an accurate distribution of energy levels. It has been shown in the case of the high-spin Fe(II) ion in nonheme iron proteins that the levels of the S = 2 state are not well described by a spin Hamiltonian. g In this case it is necessary to designate the ground state as "P and the excited state as nS and to use the Hamiltonian described earlier. By choice of the parameters of spin-orbit coupling and axial (~-) and rhombic (p) crystal fields, any desired order and spacing of ground state energies can be set up. Experimental Methods

Magnets and Cryostats Application of the MCD method to the study of metalloproteins requires the ability to control the sample temperature accurately between ultralow (-1.5 K) and high temperatures (up to 300 K) and to vary the

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[9]

MAGNETIC CIRCULAR DICHROISM

217

magnetic field at the sample between zero and the highest possible field (currently up to 10 tesla). For convenient operation sample exchange should be rapid. These needs are readily fulfilled by a superconducting magnet of the split-coil design, first introduced by Oxford Instruments PLC for the study of the MCD spectra of matrix-isolated species. An outline diagram of the essential features is given in Fig. 7. There are two liquid helium reservoirs, one controlling the sample temperature and the other providing cooling for the superconducting magnet. The sample reservoir is filled by means of a narrow, thin-walled stainless steel tube from the magnet reservoir. A needle valve, operated from outside the cryostat, controls the flow between the two reservoirs. The magnet field is provided by two superconducting solenoids separated along the field axes to permit the sample chamber to fit between them. A split pair of solenoids has a field profile along its axis such that the magnetic field is a maximum in the centers of the two coils. The overlap region where the sample sits has a lower field. Hence, obtaining a high field at the sample requires about 20% higher fields in the solenoid center. This fact, together with the split between the two coils, gives high stray magnetic fields around the magnet and consequently severe problems of shielding components external to the magnet from the field. Current superconducting technology can provide split-pair coils generating a magnetic field between the split of 10 tesla. However, it should be noted that the higher the field, the larger is the mass of the magnet. This demands a considerably more bulky cryostat and larger quantities of liquid helium to fill the magnet and to maintain it cold. These disadvantages are not negligible when a magnet is to be operated routinely for weekly MCD runs. A magnet with a maximum field at the sample of 5-6 tesla is ideal for routine MCD studies, even though a field of 5 tesla and temperature of 1.5 K will only achieve 98% saturation of a paramagnet with a ground state spin S = ½ and g = 2.0. The sample compartment can be fitted with windows to permit illumination of the sample parallel to the magnetic field axes (the Faraday mode) or transverse to the field direction (for magnetic linear dichroism) or to allow photoexcitation of luminescence or photolysis. The windows in the

FtG. 6. MCD magnetization curves of the reduced cluster, [3Fe-4S] °, of Desulfovibrio gigas ferredoxin II recorded b e t w e e n 0 and 5 tesla, at temperatures of 1.55, 4.2, 11, and 28 K, and at wavelengths o f (a) 315, (b) 383, (c) 450, (d) 570, and (e) 707 nm. Data points are indicated. The continuous lines are curves simulated with a ground state spin S = 2 subject to an axial zero-field splitting of D = - 2 cm -1 and to different excited states with varying linear polarizations as indicated.

218

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

Sample introduction port

j---Variable temperature insert vacuum port High vacuum valve Liquid nitrogen can Main helium can boil off port Liquid nitrogen can filling port

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[9]

MAGNETIC CIRCULAR DICHROISM

219

sample cell must remain totally free from strain at all temperatures down to pumped liquid helium. This achievement by Oxford Instruments was the secret to permitting routine low-temperature MCD spectroscopy to be carried out. Although the method of sealing the windows to the mounts is proprietary, it is clear from inspection that the windows are fixed to a metal foil ring with a cement that can withstand temperature cycling and using foil that remains flexible to 1.5 K. These windows have undetectable strain, except sometimes at the edges. In the experience of our laboratory, the windows can be thermally cycled several hundred times before leaks occur. Failure is inevitable and normally occurs as a vacuum leak across the cement. The window material is most conveniently fused quartz of Spectrosil or Infrasil quality, depending on whether optimal transmission is required in the far-ultraviolet or the near-infrared. Use of single-crystal windows such as sapphire should be avoided. Such a window must be cut perpendicular to the unique optic axis. However, convergent light along the axis will undergo birefringence and hence give rise to baseline artifacts.

Temperature Range Using the standard SM-4 system with a double-stage rotary pump, the liquid helium bath in the sample space can be pumped down to approximately 1.5 K within 20 min. A radiation shield at 77 K can be fitted between the sample compartment and the magnet helium reservoir. This permits good temperature stability in the region 4.2-200 K and, at the higher end of this scale, avoids the risk of causing the magnet to go resistive (with total loss of liquid helium in the reservoir). However, the shield does cause larger helium losses from the sample compartment. For routine measurements at 1.5-200 K the installation of a radiation shield is recommended. Temperature stability in the range 1.5-4.2 K is best provided by control of the vapor pressure over the helium bath. This is conveniently done with a flutter valve between a reservoir of preset helium gas pressure and the helium bath. This simple device, called a manustat, is available from Oxford Instruments. At temperatures above 4.2 K, liquid helium is removed from the sample compartment by forcing it into the magnet bath, and a flow of cold helium gas is established from the magnet reservoir into the sample compartment. As an additional aid to temperature stability a small heater, positioned on the sample rod, provides a balance between heat agent and cooling to establish good stability. The sample itself and the sample mounting block constitute a large thermal mass, and the establishment of accurate sample temperatures above 4.2 K is not straightfor-

220

SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S

[9]

ward. Because a gas flow is used, a temperature gradient can be established over the sample itself. To monitor this thermometers should be placed below and above the sample itself. This was carried out for the measurements of the temperature dependence of the cytochrome-c oxidase MCD, and, indeed, temperature gradients of up to 2 K were observed between 4 and 15 K.

Temperature Measurements Sample temperature measurement in the presence of intense magnetic fields requires a thermometer with negligible magnetic field sensitivity. Thermocouples and diodes are therefore not suitable. Carbon-glass resistors have small but negligible field effects, as do strain gauges [i.e., cryogenic linear temperature sensor (CLTS) devices]. Carbon-glass resistors (Lake Shore Cryogenics, Westerville, Ohio) are available for temperature measurement over various ranges depending on the resistance selected. Calibrated resistors are available from Oxford Instruments.

Magnetic Field Control and Measurement The intensity of the magnetic field provided by a superconducting solenoid depends on, inter alia, the current flowing in the magnet. Manufacturers provide a calibration curve of field at a defined point in space versus current in persistent mode. However, it is frequently more convenient, especially for automated measurements of magnetization curves, to measure the field with a Hall probe mounted close to the sample. Calibrated Hall probes are available from Lake Shore Cryogenics. For highly accurate determination of magnetic fields nuclear magnetic resonance (NMR) probes are recommended. Because superconducting magnets have high inductances, the time to increase the current to give full field can be of the order of tens of minutes. However, at the design stage it is possible to specify a magnet which can be run to full field in less than 5 min. The ability to reverse field, and to run baselines at zero and full field, is an important check required for measurements using glasses.

Sample Handling Variable-temperature MCD of proteins down to temperatures of 1.5 K requires, in principle, the formation of a strain-free glass without imperfections such as precipitation, cracks, or bubbles. This is a highly demanding ideal. It turns out that the method is much more forgiving of sample imperfections than might have been expected. This is for two reasons.

[9]

MAGNETIC CIRCULAR DICHROISM

221

First, the use of a single optical beam modulated from left to right circular polarization from which the differential absorbance is recorded means that some baseline and depolarization artifacts are subtracted, provided that both forms of circularly polarized light are scattered or partially depolarized to the same extent. Second, because the effect is induced by an externally applied field and because the sign of the effect depends on the direction of the magnetic field relative to the light beam, quite large baseline rolls or signals can be tolerated. For example, the natural CD is always present and can in some cases be comparable in intensity to the MCD signal. The effect is allowed for by carrying out measurements in forward, zero, and reverse field. Measurements have been made using highly scattering media including membrane fragments. The intense signals from heme proteins especially can sometimes be detected in this environment. The sensitivity of the method can be poor because of the low light levels reaching the detector. To avoid field effects on photomultiplier detectors, it is necessary to have a long distance between the sample and the photocathode. Scattering samples cause large photon intensity losses. It is difficult to recollect the scattered photons using lenses or light pipes. However, this development has not apparently been much explored and would no doubt benefit from a systematic study. The ability to make measurements on whole cells, membranes, or suspensions of scattering materials would considerably widen the scope of the method and, for example, put it on a par with EPR and M6ssbauer spectroscopy. With genetically engineered overexpressing strains of cells becoming commonplace, there are now reasons to investigate the potential of whole-cell MCD. For quantitative, routine measurements on frozen aqueous solutions of proteins, it is necessary to add a hydroxylic solvent to form a good quality glass (see Fig. 8). Solvents such as glycerol, ethanediol, and 2-methyl-2,4-pentanediol mixed with aqueous buffer to at least 50% (v/v) form good-quality optical glasses. Polyethylene glycol (PEG) 200 and sucrose are also effective but are rather viscous. Glycerol and ethanediol do not change the viscosity of the samle substantially and hence permit solutions to be handled with fine-needled syringes. For model studies other mixtures including organic solvents may be needed. Tables of suitable mixtures are available. 21 Hydroxylic solvents do not change volume significantly when cooled from 300 to 1.5 K, but this is not the case for organic solvents, where volume contractions of 25% or more are observed. Care should be taken to allow for changes in concentration owing to solvent volume changes. 21 B. Meyer, " L o w Temperature Spectroscopy," p. 203. Elsevier, New York, 1971.

222

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

.

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[9]

MAGNETIC CIRCULAR DICHROISM

223

Addition of nonaqueous solvents to proteins may change conformations or enzymatic activity. Checks of the effects of added agents on these properties should always be made by activity assays or by measurement of the CD spectrum before and after addition of the glassing agent. Other spectroscopic measurements such as EPR can also be invaluable. Further changes are possible on cooling a protein, and these include appropriate pH changes. Douzou discusses these factors. 22 Because it is rarely possible to prepare a frozen glass perfect in all respects, it is convenient to use sample cells of short path length, 1-3 mm typically. A fiat-faced cell of this thickness usually freezes rapidly and hence gives rise to good-quality glasses. In addition, a high concentration of solute ensures a large MCD signal relative to artifacts arising from imperfections in the glass. The rule is to use the thinnest sample consistent with obtaining MCD signals of acceptable signal-to-noise (S/N) ratio. Also, for measurements in the near-IR (1400-3000 nm), where solvent and protein vibrational overtones contribute to absorbance, the path length must be kept to a minimum in order to permit transmission of acceptable light levels. Because optical cells are subjected to considerable mechanical strain on cooling, the windows may fracture. Hence, cells can be homemade simply using two thin-walled (1 mm) silica plates (10 x 10 mm) separated by a rubber gasket (1-3 mm thick). The windows are glued to the gasket. These cells tend to come apart on warming back to room temperature. This can lead to loss of a valuable sample. Commercial spectrophotometric cells (1-2 mm path length) can be shortened in length to provide more robust cells that, in our experience, undergo many thermal cycles before cracking. Anaerobic cells are made by gluing a rubber septum over the opening to the cells. This can be pierced with a fine syringe needle for filling under anaerobic conditions. These cells remain anaerobic in air at room temperature for approximately 30 min. Cells that are free from rubber can fit into a wide-bore EPR cryostat and cavity to enable EPR studies to be made of the MCD sample without allowing the sample to warm to room temperature. Optical cells are mounted on the end of a thin-walled stainless steel tube, which is lowered into the sample compartment of the magnet (Fig. 7). Temperature field sensors are attached to the sample block. There is no method yet devised capable of freezing MCD samples as rapidly as the rapid quench method developed for freeze-trapping intermediates for EPR studies. We have, however, been able to load samples of peroxidases after reaction with H202 into precooled MCD cells using a 22 p. Douzou, "Cryobiochemistry." Academic Press, New York, 1977.

224

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[9]

pair of syringes driven as in a stopped-flow apparatus. The MCD cell is then subsequently transferred cold into the magnet. This requires precautions against frosting of the MCD cell in the ambient atmosphere.

Quality of Optical Glasses The MCD cell is loaded directly from ambient temperature into the liquid helium bath in the sample reservoir. This leads to a relatively rapid freezing process. The visual quality of the glass thereby formed varies widely, and our experience is that the protein solute itself plays an important role. For example, high molecular weight proteins such as cytochrome-c oxidase invariably form crack-free samples resembling pieces of light green stained glass. Low molecular weight proteins such as ferredoxin (Mr - 5000-10,000) are the most difficult to glass. The optical quality of the glass is assessed by measuring the depolarization of the light beam which has passed through it. In zero magnetic field a sample of an optically active solution is placed between the magnet cryostat and the detector. The magnitude of the CD signal is measured before and after the light has passed through the sample. Any decrease in the intensity of the CD signals is a measure of the extent of depolarization of the light beam. Because depolarization can be wavelength dependent, a full assessment requires measurements of the depolarization at several wavelengths. Suitable solutions that have a high optical stability are stored in the laboratory ready for use. Solutions of resolved [Co(en)3] 3+ (en-ethylenediamine) are ideal for the visible region and are stable over many years. For the near-infrared region, solutions of NiSO 4 in D- or L-tartaric acid 23 or in o- or L-glutamine have been used, but fresh solutions must be made each day. Circular Dichroism Instruments The wavelength range of greatest interest for the study of the lowtemperature MCD spectra of transition metal ions in proteins is currently from 3000 to 200 nm. The protein itself provides absorbing chromophores at wavelengths shorter than around 300 nm owing to the ~'-Tr* transitions of aromatic residues and above 250 nm owing to the increasingly strong absorption from amide residues. This effectively limits measurements to wavelengths longer than 300 nm unless the protein has a rather low molecular weight or a high metal content. In this case the MCD C-term intensity of the paramagnetic metal ion may be detectable above the diamagnetic protein background, which will give rise to weaker B-terms. The protein 23 G. A. Osborne, J. C. Cheng, a n d P. J. Stephens, Rev. Sci. Instrum. 44, 10 (1973).

[9]

MAGNETIC CIRCULAR DICHROISM

225

and aqueous solvent provide an effective cutoff in the near-infrared region at 3000 nm. The absorption from vibrational overtones of protein and solvent such as C - - H , O - - H , and N - - H vibrations becomes sufficiently intense to prevent light from being passed through the sample at around 2000 nm and at wavelengths longer than about 2800 nm. The use of D20 as a solvent for proteins in the near-infrared region decreases substantially the absorbance. Exchange of protein protons where possible to deuterons facilitates measurements. The absorption spectrum of D20 shows that a window of useful transmission exists between 2000 and 3000 nm (Fig. 8). Optical devices for the generation of circularly polarized light use the photoacoustic effect, that is, a bar of an isotropic material such as fused silica is strained by coupling it to a piezoelectric crystal which vibrates at the resonant frequency of the whole assembly. This enables the optical element to be stressed along an axis perpendicular to the optical axis and for birefringence to be induced. The amplitude of the strain can be controlled to ensure that the bar becomes a quarter-wave retardation plate. The applied stress is varied with the wavelength of the monochromatic light passing through the bar, so that the device can be driven as an achromatic quarter-wave plate. The bar oscillates at a high frequency, typically 50 kHz, and undergoes compression and extension on each cycle. Hence, if plane-polarized light is passed along the optic axis of the bar with the plane of polarization at 45 ° to the axis of compression, circularly polarized radiation modulated from right- to left-handedness at the oscillation frequency is generated. This simple device is a most convenient source of alternating circularly polarized light over the range 200-2000 nm with a fused silica bar or from 200 to 8000 nm using a fluorite (CaF2) crystal. All commercial CD instruments use this device for the generation of achromatic circularly polarized light. There are three commercial CD instruments available for the wavelength range 185-1000 nm, made by Jobin-Yvon (Stanmore, Middlesex, UK), JASCO (Tokyo, Japan), and AVIV (Lakewood, New Jersey). These instruments use photomultiplier detection systems. For the wavelength range 1000-2000 nm, two commercial instruments are now available, one from JASCO (Model J-730) and one built to customer specifications by AVIV. The detector system used is a liquid nitrogen-cooled InSb single crystal which has both high detectivity for photons in this wavelength range combined with the rapid response time needed to follow the high modulation frequency of photoelastic modulators. Several homebuilt instruments have been developed, primarily for measurement of vibrational CD spectra in the range 1000-5000 nm. 23'24 These instruments employ a variety of materials for photoelastic modula24 D. G. Eglinton, Ph.D. Thesis, University of East Anglia, Norwich, U.K. (1981).

226

SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S

[9]

tion and a range of first-response, high-detectivity infrared detectors including InSb, InAs, HgCdTe, and others. The sensitivity of these instruments is very high: they are capable of measuring hA values of approximately 1 x 10 - 6 with good S/N ratios. This sensitivity is required for the detection of weak vibrational CD signals. Although it is not needed for MCD studies, the high sensitivity is always valuable, requiring less sample. When used to carry out MCD measurements commercial CD instruments require modification of the light beam. The circularly polarized optical beam must be led through the sample compartment provided (for CD measurements) so that the magnet can be located distant from the magnetic field-sensitive electronics of the instrument. A photomultiplier detector must also be stationed at least 2 m from the center of a splitcoil superconducting solenoid to avoid unacceptable effects of the high magnetic fields on the photomultiplier. Magnetic shielding using mu-metal can be fitted to the photomultiplier. It is not advisable to attempt to shield the field at the magnet. High stray field levels readily saturate highpermeability shielding materials such as mu-metal (77% Ni, 16% Fe, 5% Cu, 6% Mo from Goodfellow Metals, Cambridge, UK). In addition, a shield places a strong force on the magnet unless the field is perfectly centered within the shield. The JASCO CD instrument has proved to be the most easy to modify for use with a high-field magnet. The optical beam emanating from the sample compartment of the JASCO instrument is not highly divergent but requires refocusing before the window of the photomultiplier to avoid diminution of S/N ratios. Examples of Low-Temperature Studies of Metalloproteins Examples have now been reported of the MCD spectra of many transition metal ions and clusters so far identified in proteins. The field has become a large one, and in this section only a selection of examples drawn from the Norwich laboratory are given.

Hemoproteins An extensive review of the area of MCD of hemoproteins has been published. 6 The paramagnetic oxidation states of the heine group are highspin Fe(II) (S = 2), low-spin Fe(III) (S = ½), high-spin Fe(III) (S = ~), and the ferryl state, Fe(IV) (S = 1). Also, states are known in which the porphyrin ring itself becomes oxidized, generating a radical cation with S = ½. The temperature dependences of the MCD spectra of all of these forms of heine have been reported and analyzed in some detail to enable

[9]

MAGNETIC CIRCULAR DICHROISM

227

ground state spins and zero-field splitting parameters to be determined. The optical transitions of the heme group can be divided into three classes, ring-localized transitions involving 7r-Tr* electrons, metal-localized d - d transitions, and charge transfer transitions either from porphyrin orbitals to iron d orbitals [ligand-to-metal charge transfer (LMCT)] or from metal d to porphyrin 7r * orbitals [metal-to-ligand charge transfer (MLCT)]. These classifications are imprecise. All states become extensively mixed because they are relatively close together in energy, and hence all the optical bands, whatever their orbital origins, show strongly temperature-dependent MCD bands. Thus, the unpaired electrons, which are primarily located on the Fe ion, cause even the 7r-Tr* transitions of the porphyrin ring to show paramagnetic MCD effects. The porphyrin 7r orbitals also carry, unusually for organic metal ligands, significant orbital angular momentum. This gives rise to extremely intense MCD signals. Even in the case of low-spin Fe(II) heme, which is diamagnetic, the MCD intensity of the A-terms detected in the visible region 7r-Tr* transitions is extremely high. This makes detection of the heme ring by MCD spectroscopy extremely sensitive. The optical spectra of heme groups contain a large number of transitions, in the case of low-spin Fe(III) protoporphyrin IX ranging from 2000 to 200 nm. Iron-Sulfur Proteins The iron-sulfur class of proteins is very large, and the range of structural variations in the metal site is also wide. The structurally defined metal centers consist of a mononuclear site, a single iron ion coordinated in a pseudotetrahedral geometry by four thiolate groups (the side chains of cysteine residues), a binuclear cluster [2Fe-2S] with bridging sulfide ion (S 2-) that can be ligated by four cysteine ligands, a [3Fe-4S] core that requires three cysteine ligands, and a [4Fe-4S] core that binds four protein ligands, usually cysteine thiolate residues. All of these sites have been characterized crystallographically in proteins, and all except the [3Fe-4S] core have been made as synthetic inorganic model compounds. This subject area has been reviewed extensively in a recent volume of Advances in Inorganic Chemistry. 25 In addition to these clusters, there is clear evidence of more complex iron-sulfur clusters containing more than four iron atoms. EPR spectroscopy has been historically the most convenient way of observing the presence of iron-sulfur clusters in proteins and, indeed, 25 R. Cammack (ed.), Adv. Inorg. Chem. 38 (1992).

228

[9]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

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-0.2

Reduced[2Fe-25S] Sp. maxima Fd

~u I

300

i

I

400

i

I

I

I

500

600

700

l

I

800

Wavelength (nm) FIG. 9. M C D spectra at 1.6 K and 5 tesla of (a) oxidized and (b) reduced rubredoxin from Desulfovibrio gigas and (c) reduced ferredoxin from Spirulina maxima. M C D intensity is given in units of m M -1 cm -1.

[9]

MAGNETIC CIRCULAR DICHROISM I

l

I

!

!

a Oxidized

200

I

FdIII

D. africanus

0

229

i'

-200~

250

125 -400 :

=

b Reduced D. gigas Fd]]

I

300

400

,

i

i

500

600

700

-125

,

I

800

-250

Wavelength (nm) FIG. 10. MCD spectra at 1.6 K and 5 tesla of the three-iron cluster, [3Fe-4S], in (a) oxidized ferredoxin III from Desulfovibrio africanus and (b) reduced ferredoxin II from D. gigas. MCD intensity is given in units of M -1 cm -I.

paramagnetic states of iron-sulfur clusters are, in many cases, highly structured and surprisingly informative. In addition, the MCD magnetization properties have provided an additional useful method of characterizing the spin and zero-field parameters of electronic ground states. The MCD spectra at 1.6 K and 5 tesla of all the paramagnetic states of the structurally well-characterized iron-sulfur clusters are given in Figs. 9-11.

i

300

IA

150

v

i

F

kY

a

[ 4 F e - 4 S ] 3+

Ch. vinosum HIPIP

,3/

V /•

-150

!

b

[4Fe--4S] 2+

-300 I

~

I

I

I

60

I

I

c

'

I

'

I '-'~

[4Fe-4S]1+

D. africanus F d I -4

30 -8

-\ -30

-60

' 300

' 400

~ 500

' 600

,

' 700

Wave ] engt...h

' 800 (nm)

FIG. l l . MCD spectra at 1.6 K and 5 tesla of the three oxidation states of the four-iron cluster, [4Fe-4S] 3+/2+/i +, in (a) oxidized high-potentialiron protein (HiPIP) from Chromatium uinosum, [4Fe-4S] 3+, S = ½, (b) oxidized ferredoxin from Clostridium pasteurianum, [4Fe-4S] 2+, S = 0, and (c) reduced ferredoxin I from Desulfovibrio africanus, [4Fe-4S] +, S = ½. MCD intensity is given in units of M -1 cm -I. The spectrum in (b) is independent of temperature and weak because the cluster is diamagnetic at low temperature, and hence the MCD spectrum consists only of B-terms.

[9]

MAGNETICCIRCULARDICHROISM

231

in whole cells. The optical spectra are rather broad and uninformative. However, it has turned out that the low-temperature MCD spectra of the Only in the case of the single-iron protein rubredoxin do we have any detailed understanding of the electronic states of iron-sulfur proteins. In this case the iron sits at a pseudotetrahedral site and hence is high spin in both the ferrous (S = 2) and ferric (S = ~) states. Hence, the optical transitions consist of d - d transitions in the near-IR for both oxidation states. The visible region is devoid of intense transitions, but transitions between 200 and 360 nm are intense and can be assigned as thiolate-toFe(II) charge transfer transitions. The spectrum of the Fe(III) state is dominated throughout the near-infrared region and the visible region by intense thiol-to-Fe(III) charge transfer transitions. The MCD spectra between 300 and 600 nm are exceedingly intense, testifying to the high orbital angular momentum arising from the metal ion and sustained since it is on a single center. The MCD spectra of iron-sulfur clusters are complex because of the number of electronic states and because the exchange coupling of the spin on two or more centers may relax the spin selection rules. A dense manifold of d states associated with high-spin Fe(III) and with high-spin Fe(II) can be generated by exchange coupling if the cluster contains one Fe(II) and one Fe(III) ion. When three or four iron ions are involved in the cluster, the density of d states becomes high. The allowed optical transitions from an exchange-coupled ground state spin manifold will be sparse at very low temperatures, where the cluster will be frozen into a single ground state component. Hence, the fact that the MCD spectra contain such relatively well-resolved features may be in part due to this. In the case of a cluster the charge transfer states will involve both thiolate-to-Fe(III) and S2--to-Fe(III) transitions. Hence, the complexity of the charge transfer region will also increase. The MCD spectra show that in the case of all iron-sulfur clusters measured so far there are optical transitions detectable over almost the entire accessible wavelength range from 200 to 2000 nm. Spin-coupling theories have been applied to analyze ground state magnetic properties, but little theoretical attention has been given to calculations of excited states and their MCD properties.

Acknowledgments MCDspectroscopyat the Universityof East Anglia(Norwich,U.K.) has beengenerously supported since 1967by the Science and EngineeringResearch Council,the RoyalSociety, the Agriculturaland Food Research Council, and, more recently, by the WellcomeTrust.

232

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[10]

The work described here has benefited from the help and support of Dr. R. Grinter and Professor C. Greenwood and from discussions with Professors P. J. Stephens, P. N. Schatz, E. R. Krausz, and P. Day. Many students and postdoctoral workers have carried out the work, including Dr. D. G. Eglinton, Dr. J. Springall, Dr. M. K. Johnson, Dr. J. Peterson, Dr. M. Stillman, Dr. A. E. Robinson, Dr. A. J. M. Richards, and Dr. D. J. Robbins.

[ 1 O] L i n e a r D i c h r o i s m

By

ALISON RODGER

Introduction Linear dichroism (LD) is the anisotropic absorption of plane or linearly polarized light. As most commonly implemented, LD spectroscopy probes electronic transitions using visible or ultraviolet light. 1.2 Every electronic transition of a molecule has a well-defined polarization (direction of net linear electron displacement). When linearly polarized light strikes the molecule, if its polarization direction is the same as that of the electronic transition of the molecule, then a maximum absorption signal is observed. However, if the polarization of the light is perpendicular to that of the transition, no signal is observed (see Fig. 1). Thus, the LD may be analyzed to give the orientation of the transitions of a molecule with respect to the polarization of incident light. If the polarizations of the transitions of a molecule are known, then the molecular orientation can be determined from the LD. In solution, molecules are usually randomly oriented so the net LD, (LD), equals 0. The basic requirements for experimental implementation of LD spectroscopy are therefore a plane polarized light source, a photon detection system, and a method for orienting the sample. To interpret the data fully, the degree and mechanism of orientation, the polarizations of transitions within the molecule, and the maximum possible LD signal must be known. LD spectroscopy is a powerful and simple geometric tool in many applications. However, it is not widely used. One of the main reasons for this is that a complete LD system is not currently commercially available. The aim of this chapter is to enable the reader unfamiliar with LD spectroscopy to understand the principles of the technique (see Section I); to 1 The increasing number of infrared applications that are being developed (see, e.g., Ref. 2) are outside the scope of this chapter. 2 j. Breton and A. Verm6glio (eds.), "The Photosynthetic Bacterial Reaction Centre: Structure and Dynamics," NATO ASI Series, Plenum, New York and London, 1988.

METHODS IN ENZYMOLOGY, VOL. 226

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