[13] Infrared circular dichroism

306

SPECTROSCOPIC

METHODS

FOR METALLOPROTEINS

[13]

[13] I n f r a r e d C i r c u l a r D i c h r o i s m B y TERESA B . F R E E D M A N a n d L A U R E N C E A . N A F I E

Introduction A dissymmetric (chiral) molecule or molecular conformation, that is, one which is not superimposable on its mirror image, exhibits a differential interaction with left and right circularly polarized radiation. Circular dichroism, the difference in absorbance of left versus right circularly polarized light, can occur for both electronic and vibrational excitation of a chiral molecule. Although the infrared spectral region primarily encompasses vibrational transitions, low-lying electronic transitions in metal complexes can also occur in the infrared. We use here the terms infrared circular dichroism (IRCD) to refer to both types of transitions and vibrational circular dichroism (VCD) to distinguish purely vibrational transitions. The first measurement of IRCD was reported in 19721 and that of VCD in 1973. 2 Since that time, numerous advances in experimental techniques have expanded the region over which IRCD can be measured, and advances in interpretational and calculational approaches have provided a framework for correlating the observed spectra with specific molecular conformations and configurations. 3-6 Metal complexes, metalloproteins, and metalloenzymes provide a particularly interesting area for IRCD investigation, because both conformational and configurational chirality can be present in the vicinity of the metal ion. As current instrumentation precludes observation of IRCD bands below approximately 600 cm -1, metal-ligand vibrations cannot yet be observed directly with this technique. However, the rich vibrational CD spectra of the ligands provide a unique probe of the chiral environment of the metal ion. I I. Chabay, E. C. Hsu, and G. Holzwarth, Chem. Phys. Len. 15, 211 (1972). 2 E. C.Hsu and G. Holzwarth, J. Chem. Phys. 59, 4678 (1973). 3 T. B. Freedman and L. A. Nafie, in "Topics in Stereochemistry" (E. L. Eliel and S. Wilen, eds.), Vol. 17, p. 113. Wiley, New York, 1987. 4 p. j. Stephens and M. A. Lowe, Annu. Rev. Phys. Chem. 36, 213 (1985). 5 T. A. Keiderling, in "Practical Fourier Transform Infrared Spectroscopy" (J. R. Ferraro and K. Krishnan, eds.), p. 203. Academic Press, San Diego. 1990. 6 p. L. Polavarapu, in "Vibrational Spectra and Structure" (H. D. Bist, J. R. Durig, and J. R. Sullivan, eds.), p. 319, Elsevier, New York, 1989.

METHODS IN ENZYMOLOGY, VOL. 226

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

[13]

INFRARED CIRCULAR DICHROISM

307

Absorption and Circular Dichroism Intensity Infrared absorption intensity is expressed as absorbance, A = -log(// I0), where I and I0 are the intensities of the light reaching the detector in the presence and absence of sample, respectively. Alternatively, the intensity can be expressed in terms of the absorption coefficient or molar absorptivity, e = A/cl, where c is the molar sample concentration and l is the path length in centimeters. A molecule with N atoms has 3N - 6 fundamental normal modes of vibration (3N - 5 modes for a linear molecule). In the quantum mechanical treatment, infrared absorption for a fundamental normal vibrational mode corresponds to a transition between the ground vibrational state, a, and the first excited vibrational state, b, of the ground electronic state of a molecule. The intensity of the transition is proportional to the dipole strength, Dab, the absolute square of the electric dipole transition moment, 2

Dab =

f XIrb~t~a dg

~

[(b I ~ I a)[ 2

(1)

where ~ is the electric dipole moment operator, ~ = £i eir~i, for all charged particles in the molecule with charge ei and position r~, and ~Ita (~ [a}) and 0b (=-- I b}) are the wave functions for the ground and first excited vibrational states, respectively. The dipole strength can be obtained from the integrated intensity of an absorption band, 7 e(p) dv Dab = 9.184 x 10 -39 1 Jband

(2)

where Dabis in units of esu 2 cm 2 (esu, electrostatic units). In practice, the wave number frequency v can be considered constant over the absorption band and removed from the integration, Dab

9.2 x 10

39 (b,0)-I f /3(/,')dv

(3)

where v0 is the frequency (cm -1) at the band maximum. For a Lorentzian band shape with a molar absorptivity e0 at the band maximum and halfwidth A at 1/2 of the maximum absorption, the dipole strength can be further approximated as A D ~ 9.2 x 10-39 e 0 - 7r /"0 7 T. R. Faulkner, Ph.D. Dissertation, University of Minnesota, Minneapolis (1976).

(4)

308

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[13]

For a Gaussian band shape with half-width h at 1/e of the maximum absorption, the corresponding expression for the dipole strength is D ~ 9.2 x 10-39e0~Tr 1/2

(5)

P0

For a normal mode to exhibit nonzero dipole strength, that is, for an allowed vibrational transition, a net linear oscillation of charge (equivalent to an oscillating electric dipole moment) must occur within the molecule during the vibrational excitation. These same expressions [Eqs. (1)-(5)] apply to dipole allowed electronic transitions, where a and b are the ground and excited state electronic wave functions. Infrared circular dichroism is the difference in absorption of left and right circularly polarized infrared radiation during vibrational or electronic excitation, expressed as either AA = AL -- AR or Ae = e L - e R. In the quantum mechanical treatment, electronic CD or VCD intensity is proportional to the rotational (or rotatory) strength, Rab , the scalar product of the electric dipole transition moment and the magnetic dipole transition moment. For a transition from a to b,

Rab = Im(f q~a~tObdZ" f tkb~ad'r) =--Im[(a[~[b)'(b[Tn[a)]

(6)

where ~ is the magnetic dipole moment operator, ~ = E i (ei/2mic)r ~. x if;, rni is the mass of the ith particle, and ~ x Pe --= -ihr-~ x O/Or~is the angular momentum operator for the ith particle. Since ~ is a pure imaginary operator, the imaginary part of the scalar product is taken to produce a real quantity Rab. The rotational strength (in units of esu 2 cm 2) can be obtained from the integrated intensity of an IRCD band, or it can be estimated from the maximum band intensity and half-widthT: Rab = 2.296 X 10 -39 fband

Ae(P)I"dv ~

2.3 × 10-39(v0)-' fbandAe(v)

A R ~ 2.3 × 10 -39 Ae o - 7r v0 R - ~ 2 . 3 x 10 39Ae0Z~ 7rl/2

dv

(7)

(Lorentzian band) (8) (Gaussianband)

V0

For IRCD to be observed, the electric and magnetic dipole transition moments must each be nonzero and they must be nonorthogonal to one another. Thus, for a vibration, excitation of that mode must simultaneously produce a net linear oscillation of charge and a net angular charge oscillation about the direction of the linear charge

[13]

INFRARED CIRCULAR DICHROISM

309

oscillation. An electronic transition must be both electric dipole and magnetic dipole allowed for the same Cartesian components of the two dipole moment operators. The anisotropy ratio, g = Ae/e = 4R/D, is a dimensionless parameter that connects the theoretical expressions for IRCD to experiment. For VCD, measured anisotropy ratios are typically 10 -3 to 10 -5, whereas for electronic CD anisotropy ratios of the order of 10-1 to 10-4 are typically seen. We have presented expressions for the dipole and rotational strengths in the same (Gaussian) units; expressions for absorption intensities in km/mol (SI units) can be converted to esu 2 cm 2 by multiplying by 3.987 x 10-37/p, where v is the frequency in wave numbers.

Measurement of Circular Dichroism Intensity with Photoelastic Modulator Infrared CD is measured primarily on two types of instruments, namely, a scanning dispersive spectrometer 8-n or a Fourier transform spectrometer.12'13 In practice, the Fourier transform CD instruments are restricted to the region 2000-600 cm -1 (which we refer to as the midinfrared), whereas dispersive CD instruments provide excellent spectra in the 4000-2000 cm -1 region (which encompasses hydrogen stretching and triple-bond stretching modes) 8 and have been constructed 11to operate down to approximately 600 cm -1. In the region covered by both types of instruments, the Fourier transform CD spectrometers provide much higher resolution, although high signal-to-noise ratios (S/N) are more readily achieved with a dispersive CD instrument. No complete commercial IRCD instruments are as yet available, but the components necessary to modify existing infrared spectrometers can be readily obtained. Fourier transform instruments from several manufacturers have been modified for CD measurement. 5'12'13 Most of the dispersive VCD instruments currently in use have been assembled in-house. 8-11 8 M. Diem, P. J. Gotkin, J. M. Kupfer, A. G. Tindall, and L. A. Nafie, J. Am. Chem. Soc. 99, 8103 (1977); M. Diem, P. J. Gotkin, J. M. Kupfer, and L. A. Nafie, J. Am. Chem. Soc. 100, 5644 (1978); M. Diem, E. Photos, H. Khouri, and L. A. Nafie, J. Am. Chem. Soc. 101, 6829 (1979). 9 T. B. Freedman, S. J. Cianciosi, N. Ragunathan, J. E. Baldwin, and L. A. Nafie, J. Am. Chem. Soc. 113, 8298 (1991). l0 M. Diem, G. M. Roberts, O. Lee, and A. Barlow, Appl. Spectrosc. 42, 20 (1988); O. Lee and M. Diem, Anal. Instrum. 20, 23 (1992). 1i F. Devlin and P. J. Stephens, Appl. Spectrosc. 41, 1142 (1987). 12 E. D. Lipp and L. A. Nafie, Appl. Spectrosc. 38, 20 (1984). 13 p. L. Polavarapu, Appl. Spectrosc. 43, 1295 (1989).

310

.................... FOURIER TRANSFORM

I : t-- i

. . . . . . . . . . . . . . . . . . . . .

I

LIGHT CHOPPER, (oe , AND SOURCE

POLARIZER

PEM ~ M [--1

DETECTOR SAMPLE

I

IJ

GRATING M O N O C H R O M A T O R

I

r-!

I

J Id

VCDD,SPLA~ NORMA',~AT,ONTO/ A A = A L- A R

[13]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

o)M

........... FOURIER

TRANSFORM

I

..........

OVERo,

t| ..... '

j

I

!=, J

AMP'IF,d~.

S A M P L E TRANSMISSION IAC/IDC

FIG. 1. Block diagram of a double modulation vibrational circular dichroism instrument employing either a dispersive monochromator or a Fourier transform spectrometer.

The IRCD instruments operate on a double-modulation principle. A block diagram of both types is provided in Fig. 1. The infrared source for the dispersive instruments is a xenon arc lamp for the 4000-2000 cm -1 region or a carbon rod or Nernst glower source in the mid-infrared region. The Fourier transform instruments employ a silicon carbide rod or Nernst glower source. In the dispersive instrument, the first, low-frequency modulation is provided by a mechanical light chopper, operating at approximately 50-80 Hz, which is placed before or immediately after the entrance slit to the monochromator. This modulation provides a reference for defining zero light level at the detector and determining the single-beam transmission of the sample. Both 750 and 320 mm focal length monochromators have been employed. The blaze angle and density of grooves for the grating is optimized for the region of interest and dispersion desired. The resolution, determined by the grating and slit settings, is typically set at 7-15 cm -1 in the OH-, NH-, and CH-stretching regions and 4-8 cm -I in the mid-infrared to keep noise levels within an acceptable range. In the Fourier transform instrument, the low-frequency modulation is provided by the Fourier transform spectrometer itself, which introduces a sinusoidal oscillation of each wave number frequency, v, at its Fourier frequency, o~F = 2 v V , where V is the mirror velocity of the interferometer. A 4 cm -1 resolution is typically employed, although spectra at 1 cm -1 resolution have been obtained for gas-phase samples.

[13]

INFRARED CIRCULARDICHROISM

311

The light emerging from the monochromator passes through a filter to remove wavelengths diffracted by the grating higher than first order; that emerging from the interferometer passes through a high-frequency cutoff filter to prevent detector saturation. In both instruments, the light is then linearly polarized with a wire-grid polarizer at 45 ° with respect to the stress axes of a photoelastic modulator (PEM), which provides the highfrequency sinusoidal modulation of the beam between left and right circular polarization states at angular frequency toM- We employ a PEM with a ZnSe crystal modulated at 36.7 kHz in the Fourier transform instrument and a PEM with a CaF2 crystal modulated at 57 kHz in our dispersive instrument, both from Hinds International (Hillsboro, OR). The low frequency cutoff of the ZnSe modulator (600 cm -1) places a lower limit on the frequency range over which IRCD can presently be recorded. The sample is placed immediately after the PEM. For liquid and solution samples, 2.5 cm diameter, fixed path length (0.015 mm to 2 cm) cells with BaF2 (-800 cm -1 low-frequency cutoff) or CaF2 (-1200 c m - 1 lowfrequency cutoff) windows are ordinarily employed, as these materials can be used for aqueous samples. The beam emerging from the sample is focused on a liquid nitrogen-cooled semiconductor detector; typically, InSb is employed above 2000 cm -1 and HgCdTe (MCT) is employed for the 2000-600 cm -1 region. The response time of the detector must be less than 1 /~sec for the changes in intensity at the PEM frequency to be followed accurately. The detector signal is amplified by a preamplifier and must then be demodulated to recover the transmission and CD intensities. In the Fourier transform instrument, the signal for the transmission spectrum is sent through a low-pass electronic filter to remove frequencies at toM, through an analog-to-digital (A/D) converter, and then to a computer where the transmission spectrum, which we denote IDC, is recovered by Fourier transformation. For the CD intensity, the detector signal is filtered at tom and sent through a lock-in amplifier tuned to and in phase with the PEM modulation frequency toM. The resulting signal is digitized and Fourier transformed to produce IAC. As described below, the CD spectrum can be obtained from the ratio IAC/IDc. Typically, about 12,000 AC scans and 800 DC scans are recorded in blocks of 256 AC scans and 16 DC scans, ratioing the block of Fourier-transformed AC scans to the DC block that precedes and follows and then coadding the ratioed blocks to obtain the final spectrum. In the dispersive CD instrument, part of the signal is sent to a lockin amplifier tuned and phased to the chopper frequency toc to obtain the transmission spectrum IDC" The CD spectrum is obtained by demodulation of the detector signal by sequential lock-in amplifiers tuned to toM and toc

312

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[13]

to yield IAC, followed by normalization to the transmission spectrum. In practice, normalization on the dispersive instrument is obtained with a negative-feedback normalization circuit and lock-in amplifier which maintain the IDC signal at a constant value. The signal is sent to a computer for further manipulation and display. The computer is also used to control the scanning of the monochromator. It is also possible 1° to employ computer control of the PEM retardation level in order to eliminate the calibration procedure described below. In our laboratory we use an IBM PCtype computer with SpectraCalc software (Galactic Industries, Salem, NH) for data acquisition and manipulation by employing a program written in SpectraCalc Array Basic language. Typical spectra on the dispersive instrument are obtained with a 10 sec time constant on the coc lock-in amplifier and a 3 sec delay between data points collected. Two to four scans are recorded and averaged. A third type of IRCD instrument employing polarization modulation interferometry (PMI) with a polarizing beam splitter has been developed as an alternative to the double modulation methods. 14 This method has seen only limited application. For optimal signal-to-noise ratios, sample absorbances between 0.2 and 0.6 should be employed. However, the range of usable concentrations is often limited by overlapping solvent absorbances, which can obscure features arising from the sample or degrade the signal because of the amount of light removed by an intense, broad solvent background. This problem is particularly acute for aqueous solutions, where high concentrations in D 2 0 ( > 0 . 5 M ) and short path lengths (50-100/xm) are required for the CH-stretching region. Similar concentrations and 15 to 30/zm path lengths are required for the 1500-1100 cm -1 region for samples in H20 or the 1750-1250 cm -1 region for D20 solutions. In nonaqueous solvents, good-quality VCD spectra have been measured at concentrations as low as 5 X 10 -4 M in regions of low solvent background. Mathematics of Circular Dichroism Measurement with Photoelastic Modulator In this section we outline the mathematical procedure by which the CD intensity hA is related to the intensities IDC and IAC obtained experimentally. ~s'16 The IDC signal is the average of the intensity levels for left 14 N. Ragunathan, N. S. Lee, T. B. Freedman, L. A. Nafie, C. Tripp, and H. Buijs, Appl. Spectrosc. 44, 5 (1990). 15j. C. Cheng, L. A. Nafie, and P. J. Stephens, J. Opt. Soc. Am. 65, 1031 (1975); L. A. Nafie, T. A. Keiderling, and P. J. Stephens, J. Am. Chem. Soc. 98, 2715 (1976). 16 L. A. Nafie and M. Diem, Appl. Spectrosc. 33, 130 (1979).

[13]

INFRARED CIRCULAR DICHROISM

313

and right circularly polarized light, IDc = ½(IR+ Ic)

(9)

The retardation angle of the PEM is given by aM = a ° sin OJMt, where coM = 2ZrVMfor modulator frequency ~'M. The IAC signal is IAC = ½(IR-- IL) sin a M

(10)

The intensity can be written in terms of the absorbance of the sample as I =/010 -a. We then have l A G - 10 -AR -- IO-AL

IDC

10_ARq- 10_ALsin o/M tanh[ln 10(AA/2)]sin

OLM

(11)

where the fraction has been rearranged into the form of a hyperbolic tangent involving AA = A L - AR. By invoking the approximation that, for small values of AA, the hyperbolic tangent is equal to its argument, and by expanding sin a Min a series of odd-order spherical B essel functions, we find IAC = 2Jl(aO)l. 1513AA

IDc

(12)

for the final signal processed by the lock-in amplifiers, which eliminates higher order Bessel function terms. The final expression for this intensity ratio for the Fourier transform CD instrument contains an additional factor of exp(-2Vv'r) owing to the electronic effects on the Fourier frequencies of the time constant, z, of the lock-in amplifier. 16 Both this term and the Bessel function dependence for both instruments are removed by carrying out a calibration procedure in which a birefringent plate and a second polarizer are inserted in place of the sample to simulate unit AA. The final CD spectrum is obtained by dividing (IAc/IDc) by 1.1513 times the calibration curve, which is taken to be a straight line or a single value if the spectral region investigated is fairly limited. A calibration spectrum is obtained each time a spectrum is recorded to minimize the effects of day-to-day instrumental drift. With extremely careful alignment, absorption and baseline artifacts can be minimized. However, the best CD spectra are obtained by correcting for such artifacts with a racemic sample or a pair of enantiomers. Spectral Interpretation The interpretation of IRCD spectra is complicated by the fact that, unlike ordinary electronic or vibrational excitation, VCD depends on the correlation of electron velocities with nuclear velocities, a non-Born-Oppenheimer effect. Exact theoretical expressions for the magnetic dipole transition moment contribution to the rotational strength involve either a

314

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[13]

summation over all excited electronic states of the molecule, 17 electronic wave functions perturbed by nuclear displacement and by a magnetic field, TMor electronic wave functions with nuclear velocity dependence. ~9 Although the methodology is available to calculate VCD intensity with molecular orbital programs for small molecules with some of the more exact theoretical approaches, ls'2° practitioners in the field still rely on simplified intensity models and empirical correlations for the interpretation of IRCD spectra in complex systems, particularly for large biomolecules. Electronic CD effects for metal complexes in the infrared region are identified by their perturbing effect on VCD intensities, or by their extremely large bandwidth coupled with a large anisotropy ratio. Two main contributions to VCD intensity have been identified: (1) the coupling of two or more chirally oriented local electric dipole oscillators 9'21'22 and (2) the chiral environment of a local, isolated oscillator. 3 For a set of N identical local oscillators Sj that are coupled to form a set of normalized modes 9 Li =

(13)

ai:S: J

the rotational strength for the ith coupled mode is given by DT

N

°^

R i = - r r u i - y i ~ ai:aikRjk Uj × fik iv k>j

(14)

where v~ is the frequency (cm l) of the ith mode, DT is the total dipole strength for the set of coupled modes (obtained by integrating the area under the absorption bands for the set of modes as described above), Rjk is the j --~ k separation vector between oscillators j and k, and fii is a unit vector along the ith oscillator in the direction of positive electric dipole moment change. The summation is taken over all pairs of oscillators; only chirally oriented pairs will have a nonzero contribution to the sum. This model predicts VCD intensities that are conservative over the range of coupled modes, that is, with equal net positive and negative VCD intensities. For the Ce symmetry (A and B symmetry species pair) and C3 and D 3 (A and E, A2 and E symmetry species pair) symmetries often encountered in metal complexes, the coupled modes give rise to a VCD couplet with two lobes of equal intensity and opposite sign. The model is strictly 17 L. A. Nafie and T. B. Freedman. J. Chem. Phys. 78, 7108 (1983). 18 p. j. Stephens, J. Phys. Chem. 89, 784 (1985). 19 L. A. Nafie, J. Chem. Phys. 96, 5687 (1992). 2o R. Dutler and A. Rauk, J. Am. Chem. Soc. U l , 6957 (1989). 2I G. Holzwarth and I. Cbabay, J. Chem. Phys. 57, 1632 (1972). 2z I. Tinoco, Radiat. Res. 20, 133 (1963).

[13]

INFRARED

CIRCULAR

315

DICHROISM

2.0 x 0.0 < <1 -2"0f I

2440

I

I

I

I

I

I

I

2380 2320 2260 2200 2140 2080 2020 WAVENUMBER

FIG.2. Circulardichroismspectrumof praesodymiumL-tartratecomplexin H20 solution (1.5 M in tartrate, 1 M in Pr3+, and 5 M in NaOH).

valid when any vibrationally generated electronic charge flow occurs only within the local oscillator. The coupling can occur by means of throughspace dipolar interaction or by kinetic or potential energy terms, which allow coupling among the oscillators and introduce a splitting in the energies of the coupled modes. A local oscillator in a chiral environment can generate a monosignate VCD feature characteristic of the local environment.3 Such features, which can be attributed to chiral electronic charge circulation in the vicinity of the oscillator generated by the nuclear motion, serve as empirical markers for configuration and conformation. When oscillators of this type couple, biased VCD couplets can be generated. Application to Metal Complexes and Metalloproteins To illustrate the applications of both the experimental methodology and interpretational techniques of IRCD to biologically relevant metal complexes and metalloproteins, we summarize several pertinent studies. The IRCD arising from low-lying electronic f--~ f or d--~ d transitions at a metal center has been detected indirectly through enhancement, owing to interaction between the vibrational and electronic states, of the VCD intensity in tris[3(trifluoromethylhydroxymethylene)-d-camphorato]europium compared to the corresponding praseodymium complex, ]5 and in

316

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[131

20

% 0 X

--20 2000 z G

ml000

\ 0

I

1700

1650

WAVENUMSER FIG. 3. Carbonyl-stretching absorbance and VCD spectra for L,L,L-tris(N-Boc-leucylamidoethyl)amine, 5 x 10-4 M in C2C14 solution, 4.0 mm path length.

the Co(II) and Ni(II) complexes of (-)-spartein compared to the corresponding Zn(II) complex. 23 Electronic IRCD has been observed directly for a praseodymium L-tartrate complex by Chabay et al. ~ with a dispersive CD instrument and in our laboratory with a PMI-CD instrument (Fig. 2)24 Coupled oscillator VCD effects can be used to determine the solution conformation of the tripodal peptide L,L,L-tris(N-Boc-leucylamidoethyl)amine (1; Boc, t e r t - b u t y l o x y c a r b o n y l ) , a precursor to a synthetic ion carrier that is a biomimetic analog of the siderophore enterobactin. In enterobactin, the A - c i s - F e 3+ complex, but not the A - c i s - F e 3+ complex, interacts with a cell membrane receptor, and it is of interest to determine whether the biomimetic ligands assume an interchain hydrogen-bonded conformation of a specific handedness even in the absence of metal ion. z3 C. J. Barnett, A. F. Drake, R. Kuroda, S. F. Mason, and S. Savage, Chem. Phys. Lett. 70, 8 (1980). 24 N. Ragunathan, Ph.D. Dissertation, Syracuse University, Syracuse, New York (1991).

[13]

INFRARED CIRCULAR DICHROISM Rt

R~'O

I

t

o,." c..

C

/ . \ ~N.,C

N

tt3 :

#01"

317

\ /'*"

......N

I H~

q3 ". "H2

0

F

""taa

l 0 ~c~ I R

0

SCHEME I. L,L,L-Tris(N-Boc-leucylamidoethyl)amine, right-handed C3 propeller.

The carbonyl stretching region of 1, recorded on an F T - V C D instrument, is shown in Fig. 3 for a 5 x 10 -4 M solution in C2C14.25 The absorbance feature and ( - , +) VCD couplet centered at 1699 cm -~ is assigned to the urethane carbonyl group stretches, and the absorbance and (+, - ) VCD couplet centered at 1676 cm -1 is assigned to the leucine carbonyl stretches in the C3 symmetry structure depicted in Scheme I. VCD couplets of the observed senses are consistent with the coupled oscillator model predictions for dipolar coupling among C3-equivalent carbonyl groups, when the interchain hydrogen-bonding network forms a right-handed propeller with the oxygen ends of both sets of carbonyl dipoles directed upward along the C3 axis, as shown in Scheme I. 25 The coupled oscillator model has also been applied to the interpretation of the mid-infrared VCD spectra of bis(acetylacetonato)(L-alaninato)cobalt(III). 26 The NH- and CH-stretching VCD spectra of metal complexes with ethylenediamine, 27 trans- 1,2-diaminocyclohexane, 2s alanine, 26,29glycine,29 proline, 29 threonine, 29 and/3-alanine 3° ligands have been recorded with 25 M. G. Paterlini, T. B. Freedman, L. A. Nafie, Y. Tor, and A. Shanzer, Biopolymers 32, 759 (1992). ~_6D. A. Young, E. D. Lipp, and L. A. Nafie, J. Am. Chem. Soc. 107, 6205 (1985). 27 D. A. Young, T. B. Freedman, E. D. Lipp, and L. A. Nafie, J. Am. Chem. Soc. 108, 7255 (1986). 28 H. Morimoto, I. Kinoshita, M. Mori, Y. Kyogoku, and H. Sugeta, Chem. Lett. 1989, 73 (1989). 29 T. B. Freedman, D. A. Young, M. R. Oboodi, and L. A. Nafie, J. Am. Chem. Soc. 109, 1551 (1987). 3o D. A. Young, T. B. Freedman, and L. A. Nafie, J. Am. Chem. Soc. 109, 7674 (1987).

318

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

t

0-

%

[13]

-1-

x

<~

-2-3-

0.03o t-

Horse Myoglo.__.bin

A [ / / /

Human

A

Mutant Human Myoglobin

0.02 O ..Q

0.01

% x

-1

< -2

0.02-

O

0.01

0

21oo

2dso

2000

19so

Wavenumbers (cm -1) FIG. 4. Absorption and VCD spectra of the antisymmetric N 3- stretch in horse azidometmyoglobin (11.5 raM), mutant human azidometmyoglobin (7.1 raM, asparagine replacing valine at Ell), and human azidomethemoglobin (8.0 raM). Samples were buffered at pH 7, and all path lengths were 26 k~m.

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

319

dispersive VCD instruments. Metal ions studied include Co(III), Cr(III), Cu(II), Pd(II), and Ni(II). The VCD features are characteristic of the configuration of the complex and the ligand conformations. VCD has been employed to examine the environment of the iron ion in heme proteins through a study of the interactions of an azide ligand with the distal heme residues in azidomethemoglobins and azidometmyoglobins obtained from diverse species and from site-directed mutagenesis techniques.31 An example of the VCD assigned to azide antisymmetric stretch is shown in Fig. 4. The spectra were obtained on a dispersive VCD instrument. The azide stretch exhibits very large VCD (g ~ 10-3) for the covalently bound low-spin Fe(III) species at approximately 2020 cm -1 but no VCD intensity for the ionically bound high spin azide at around 2040 cm -1. The anisotropy ratio is not altered on R to T quaternary structural changes, but the band disappears for site-directed mutants with the histidine at E7 replaced by glycine or the valine at E11 replaced by asparagine. The VCD intensity is thus extremely sensitive to the exact nature of the chiral environment of the azide ligand. We also note that a related technique, magnetic vibrational circular dichroism (MVCD), has been employed to investigate metallotetraphenylporphyrins. 32 Acknowledgments Support of this work from a grant from the National Institutes of Health (GM-23567) is gratefully acknowledged. 31 R. W. Bormett, S. A. Asher, P. J. Larkin, W. G. Gustafsom N. Ragunathan, T. B. Freedman, L. A. Nafie, S. Balasubramanian, S. G. Boxer, N.-T. Yu, K. Gersonde, R. W. Nobel, B. A. Springer, and S. G. Sligar, J. Am. Chem. Soc. 114, 6864 (1992). 32 p. V. Croatto and T. A. Keiderling, Chem. Phys. Lett. 144, 455 (1988).

[14] R a m a n a n d R e s o n a n c e R a m a n S p e c t r o s c o p y

By YANG WANG and HAROLD E. VAN WART Introduction Because of its high content of structural information and its applicability to a wide variety of samples, Raman spectroscopy has become an important and widely used method for studying biological macromolecules. Raman spectroscopy is a form of vibrational spectroscopy in which a sample is interrogated with an intense light beam and the spectrum of METHODS IN ENZYMOLOGY,VOL. 226

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