[14] Raman and resonance Raman spectroscopy

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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

Copyright© 1993by AcademicPress, Inc. All rightsof reproductionin any form reserved.

320

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

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R a m a n active vibrational m o d e s obtained through analysis of the inelastically scattered photons. 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 is a specialized form of R a m a n s p e c t r o s c o p y in which the wavelength of the interrogating light b e a m lies under an electronic absorption band of the sample. This produces a r e s o n a n c e e n h a n c e m e n t in the scattering intensity of only those bands that are coupled to the excited electronic transition. This r e s o n a n c e effect minimizes the interference from bands due to nonabsorbing portions of the sample, allowing one to z o o m in on and selectively p r o b e c h r o m o p h o r i c sites. The metal centers of metalloproteins are quite often chromophoric, having electronic transitions that can be selectively excited with nearultraviolet or visible radiation. M o r e o v e r , m a n y of these centers are located at the active site responsible for the function of the metalloprotein. B e c a u s e the frequencies and intensities of r e s o n a n c e R a m a n bands are sensitive to the nature of the chemical bonding and the molecular configuration of the a t o m s participating in the vibrations, the r e s o n a n c e R a m a n technique can be used to monitor structural changes of a c h r o m o p h o r e that are induced or modulated by its environment. Thus, 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 can provide important structural and mechanistic information a b o u t c h r o m o p h o r i c metalloproteins. In this chapter, we describe the R a m a n and resonance R a m a n techniques with an emphasis on their application to the study of metalloproteins. Aspects considered in earlier volumes of this series are not addressed in any detail. 1-3 First, we briefly review the underlying theoretical basis for these techniques in order to provide the reader with an appreciation of the relationship to other forms of s p e c t r o s c o p y and to facilitate an understanding of subsequent sections on spectral interpretation. Next, the instrumentation, p r o c e d u r e s , and methods used to obtain R a m a n and r e s o n a n c e R a m a n spectra are considered in some detail. Last, we illustrate the interpretation of R a m a n spectra by considering several recent examples of studies of metalloproteins. Several specialized R a m a n techniques, including the ultraviolet-excited, 4 time-resolved, 5 surface-enhanced, 6 and circular differential R a m a n methods, 7 as well as applications of the technique to single crystals, 8 and special considerations for obtaining resonance R a m a n spectra of metalloproteins 9 are the subjects of other chapters 1 M. C. Tobin, this series, Vol. 26, [23]. 2 H. E. Van Wart and H. A. Scheraga, this series, Vol. 49, p. 67. 3 N.-T. Yu, this series, Vol. 130, p. 350. 4 j. C. Austin, K. R. Rodgers, and T. G. Spiro, this volume [15]. 5 C. Varotsis and G. T. Babcock, this volume [17]. 6 W. E. Smith, this volume [20]. 7 L. A. Nafie and T. B. Freedman, this volume [19]. 8 G. Smulevich and T. G. Spiro, this volume [16]. 9 T. M. Loehr and J. Sanders-Loehr, this volume [18].

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

321

in this volume. F o r a more complete discussion of other aspects of R a m a n spectroscopy, the reader is referred to several books on the subject.l°-15 Several excellent reviews are also available that specifically consider biological applications of R a m a n and resonance R a m a n spectroscopy. 16-19 Discovery of R a m a n Scattering The essential c o m p o n e n t s o f a R a m a n scattering experiment are a light source and a detection system capable of analyzing the frequency and intensity distributions of the scattered photons. The first experimental observation o f R a m a n scattering was published in 1928 after many years of effort by C. V. R a m a n and associates. 2° Some of the early experiments were c o n d u c t e d using excitation from sunlight that had been directed through a short-pass filter. The light scattered from the sample was directed through a long-pass filter and the Raman shift detected by eye. For example, when blue light was delivered to a sample of neat water or alcohol, R a m a n was able to observe green scattered light. It was subsequently established that the frequency differences between the incident and scattered photons corresponded to the vibrational energies of the sample and that a plot of the intensity of scattering versus frequency shift gave a vibrational spectrum of the sample, similar to that observed by infrared spectroscopy. 21 Theoretical Background

General Description of Raman Scattering The interaction of the electric and magnetic fields of light with the electrons and nuclei of molecules leads to a variety of absorption, emis10T. R. Gilson and P. J. Hendra, "Laser Raman Spectroscopy." Wiley, New York, 1970. 1i M. C. Tobin, "Laser Raman Spectroscopy." Wiley, New York, 1971. lZ S. K. Freeman, "Application of Laser Raman Spectroscopy." Wiley, New York, 1974. 13N. B. Colthup, L. H. Daly, and S. E. Wiberley, "Introduction to Infrared and Raman Spectroscopy," 2rid Ed. Academic Press, New York, 1975. 14D. A. Long, "Raman Spectroscopy." McGraw-Hill, New York, 1977. 15j. A. Konigstein, "Introduction to the Theory of the Raman Effect." D. Reidel, Dordrecht, Holland, 1973. 16A. T. Tu, "Raman Spectroscopy in Biology." Wiley, New York, 1982. 17p. R. Carey, "Biochemical Applications of Raman and Resonance Raman Spectroscopy." Academic Press, New York, 1982. ~8F. S. Parker, "Application of Infrared, Raman and Resonance Raman Spectroscopy in Biochemistry." Plenum, New York, 1983. ~9T. G. Spiro (ed), "Biological Applications of Raman Spectroscopy," Vols. 1-3. Wiley, New York, 1987. 2oC. V. Raman and K. S. Krishnan, Nature (London) 121, 501 and 619 (1928). 2i R. W. Wood, Nature (London) 122, 349 (1928).

322

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

sion, and scattering processes that alter the intensity and frequency distribution of the photons in the radiation field. An analysis of the photon field before and after these processes forms the basis for all optical spectroscopic techniques and yields information about the eigenstates, and hence structures, of the molecule being interrogated by the field. The relationship between Raman spectroscopy and other forms of spectroscopy was described in our earlier contribution to this series 2 and is not repeated here. However, it is appropriate to remind the reader that Raman spectroscopy is a two-photon process that is inherently different from the more familiar one-photon processes (Fig. 1). The single-photon absorption of microwave, infrared, or visible/ultraviolet radiation of frequency v results in rotational, vibrational, and electronic excitation (hE = by), respectively, of the molecule. An infrared spectrum reflects the direct absorption of vibrational quanta by the sample as a function of the frequency of the

IR A b s o r p t i o n - - S

1

Sl

ht~

h~ hv,

hE=h~

So

S1

- -l--F- Virtual I - l - ~ - State

',

by,

E=hv, Stokes R a m a n Band

v , c m -I

Resonance Scattering

Nonresonance Scattering

Rayleigh Band

\

&v ,em -1

anti-Stokes Raman Band

So /

5E=hv,-hu~

So

?~~aman Excitatio

gavelength,nm

FIG. 1. Schematic diagram showing the relationship between infrared absorption, Rayleigh scattering, Stokes and anti-Stokes Raman scattering, and resonance Raman scattering. So and S~ are the ground and excited singlet states of the molecule, respectively. Infrared absorption is a single-photon process, whereas all of the others are two-photon processes. In resonance Raman scattering, the variation in the intensity of a vibrational band with the excitation wavelength is called an excitation profile and often follows the contour of the absorption band.

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

323

exciting light. The equivalent single-photon emission processes also exist, where emission arising from electronic deexcitation (fluorescence or phosphorescence) is the most efficient. Two-photon processes involve the simultaneous interaction of two photons with the molecule and are inherently much weaker phenomena. Three possible types of two-photon processes are the absorption of two photons (hE = h v 1 + hv2) , the emission of two photons (AE = h v 1 + hv2), and the scattering event in which there is the simultaneous absorption of the first and emission of the second photon (hE = h v I - hv2). Rayleigh and Raman scattering correspond to the cases where vl = v2 (elastic scattering) and v~ # v2 (inelastic scattering), respectively. In Raman scattering, the Raman shift, Av = v 1 - v2, corresponds to the energy of a vibrational transition. Stokes transitions are those in which the molecule is excited by the radiation field with Av < 0, whereas anti-Stokes transitions are those in which the molecule is deexcited by the field, Av > 0 (Fig. 1). A Raman spectrum is obtained by plotting the scattering intensity as a function of the Raman shift and yields information similar to that provided by an infrared spectrum. However, because the selection rules for Raman and infrared transitions are different (see below), the two techniques complement one another. Because of the impact of fluorescence on the ability to observe Raman scattering, it is worthwhile to reemphasize the relationship of Raman scattering to fluorescence. The latter consists of two one-photon processes that are separated in time, whereas the former is a single two-photon process. As shown in Fig. 2, these processes can be distinguished by the difference in the lifetime of the "intermediate state" involved. Raman scattering is completed within a time interval comparable to that of a vibrational cycle (-10 -15 to 10 -13 sec), where the two-photon process connects the ground state to a so-called virtual state. On the other hand, fluorescence involves excitation by the first photon to a real intermediate state with a finite lifetime (-10 -~° to 10 .7 sec). Following vibrational relaxation or nonradiative interstate crossing to lower vibrational levels of the excited state, the second photon is emitted. Because the excited state has a finite lifetime, fluorescence can be quenched by collisions between the excited fluorophore and other molecules. No such quenching of Raman scattering is possible. Because fluorescence is a first-order process, it is generally much stronger than Raman scattering (even though quantum yields vary considerably). Both fluorescence and Stokes Raman scattering produce photons with frequencies on the long-wavelength side of the incident light. As a result, fluorescence from a sample, even one with a low quantum yield, can easily obscure the Raman scattering. Thus,

324

[14]

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

Singlet e~ Excited Star

\ Triplet Excited State

Ground S t a t e ~

~luoreseence

Rayleigh \ \ \ ~ i \ ~Rayleigh ~Excitation

Fluorescence Phosphorescence aaman I

u~o,~t,oo

I

v~....

I

[

~ .......... u~h............. A v, c m - 1 FIG.2. Diagramsillustratingthe relativefrequencies,v, ofRayleighandRamanscattering, fluorescence,and phosphorescence.The overlapin PRamanand/)fluorescencecan causethe Raman signal to be obscured.

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

325

interference from fluorescence is the most frequent problem associated with acquiring Raman spectra.

Information Content and Interpretation of Raman Spectra Both Raman and resonance Raman spectroscopy can be applied phenomenologically to some problems without a detailed understanding of the underlying theory, or even a basic knowledge of spectral interpretation. In such studies, one utilizes direct comparisons of spectra without detailed analysis. For example, comparative studies can establish whether one substance is identical to another or spectral changes can be used as a means to monitor a sample in different environments or undergoing a reaction (i.e., by obtaining spectra at different time intervals). The structural information obtained through such comparative studies is, however, quite limited. To extract the most information possible from Raman spectra, detailed analyses of the spectral features are required. The important spectral parameters are the Raman shifts, Av, of the bands and their intensities and depolarization ratios. These parameters are related to the structure and symmetry of the molecule. The depolarization ratio of a Raman band (defined below) is particularly helpful in making vibrational assignments. Some of the underlying theory related to the interpretation of Raman spectra from an analysis of these parameters is reviewed briefly in the subsequent sections.

Normal Coordinates and Vibrational Modes The vibrational motions of atoms in a polyatomic molecule can be described by a series of normal coordinates designated Qi.22 The normal coordinates describe the motions of groups of atoms along eigenvectors that are solutions to Newton's equation of motion for the molecule, where the corresponding eigenvalues correspond to the vibrational frequencies for these modes. Such a calculation is referred to as a normal mode analysis and is extremely helpful in relating the motions of individual atoms to individual Raman bands. A simplified, but nevertheless instructive, way to understand vibrational frequencies is provided by Hooke's law for a diatomic oscillator. This model is conceptually applicable to "local" vibrational modes of a polyatomic molecule that are centered predominantly on a few atoms, such as those involving metal ions and their ligands. These modes can yield important structural information concerning the metal centers of 22 E. B. Wilson, Jr., J. C. Decius, and P. C. Cross, "Molecular Vibrations." Dover, New York, 1955.

326

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

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metalloproteins. According to Hooke's law, the vibrational frequency, v, is given by v (1/2~)(k/tz) 1/2, where k is the force constant (which is proportional to the strength of the restoring force) and/z is the reduced mass of the bonded atoms. Owing to the heavy mass of most metal atoms, metal-ligand stretching modes usually have low (200 to 500 cm-1) vibrational frequencies, although they can occur at higher frequencies for higher order bonds. Isotopic substitutions are quite useful for locating such modes, as a shift in vibrational frequency (Av = v 1 - v2) is expected. Assuming that the force constant, k, is not affected by the isotopic replacement, /)2 = /)l(itL1/~2) 1/2, where/x 1 and/t£ 2 are the reduced masses of the metal and ligand with the two different isotopes, respectively. For metal centers having a trans ligand, the calculation is slightly more complicated.23 =

Classic Mechanical Description of Raman Effect: Polarizability Tensor An understanding of the difference in selection rules between Raman and infrared spectroscopies and of the polarization properties of Raman bands can be gleaned from classic electromagnetic theory. An infraredactive vibrational mode is one that introduces a change in the permanent dipole moment of the molecule. 24 Thus, the intensity of an infrared transition, IrR, is given by ( 0/'z~2

(1)

where t~ is the permanent dipole moment of the molecule and Qi is the normal coordinate for the vibrational mode in question. Thus, symmetric vibrations in a molecule with a center of symmetry are infrared inactive. This includes, for example, the stretching mode of a diatomic molecule (X2), the symmetric stretching mode of a linear triatomic molecule (XYX), and the breathing mode of a benzene ring that involves uniform ring expansion. A Raman-active mode, on the other hand, is one that produces a change in molecular polarizability, o~.24 Thus, the intensity of a Raman band, IRaman, is given by

IRarnan~ P2 = [ (~Qi)o QiE] 2

(2)

23 E. A. Kerr, H. C. Mackin, and N.-T. Yu, Biochemistry 22, 4373 (1983). 24 G. W. Chantry, "Laser Raman Spectroscopy," Vol. 1, Chap. 2. Dekker, New York, 1971.

[14]

RAMAN

AND

RESONANCE

RAMAN

SPECTROSCOPY

327

where P is the induced dipole moment, E is the electric field strength of the exciting radiation, and Qi is the normal coordinate associated with the vibration. It should be noted that the molecular polarizability, a, is described by the following tensor:

t

Olxx OLxy OLxzt

Ol. = ~ O£yx

OI.yy Ol-yz ]

(3)

/

\ O£zx OLzy OI.zz

The polarization of the scattered light with respect to the incident light is determined by the orientation of the scattering molecules and the symmetry of the vibrational mode, as reflected by the tensor elements of a. The parameter used to describe the polarization of Raman scattering for fluid samples produced by a polarized incident beam is the depolarization ratio, p = I±/Ii[, where/It and I± are the intensities of light scattered parallel and perpendicular to the polarization of the incident light (Fig. 3).

.? .--.r.z.~e..:mo ,~' o.o

t~et.O) r ~-L-Ox,

FIG. 3. Measurement of the depolarization ratio in a 90° Raman-scattering experiment. Utilization of a polarization scrambler eliminates the grating bias for different polarizations.

328

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

The relationship between p and the elements of a is 25-27 I j_ 3y 2 + 5y2s O - /IL - 45&2 + 4y2

(4)

where ys2, YZas, and & are the anisotropy, antisymmetry, and trace of the polarizability tensor defined as , ~ = ! Z (~ii -- Oljj)2 %-

2ij

3Z(Ol'ij 4ij

2'

(5) (6)

4;j

1~.

& = -~

°tii

(7)

where i and j represent the axial directions in a Cartesian coordinate system. The depolarization of the incident light is a consequence of the optical anisotropy of the sample. In normal Raman scattering, the polarizability tensor is symmetric, ai~ = a~i, and consequently "/as ---- 0. In this case, p varies between 0 (when & # 0) and 3/4 (when & = 0). Totally symmetric vibrations are those that preserve the symmetry of the molecule and have nonzero diagonal polarizability tensor elements (& # 0); these modes have 0 < p < 3/4 and are said to be polarized. All other modes are nontotally symmetric (& = 0) and give rise to depolarized bands (p = 3/4). When approaching resonance, the scattering tensors for certain modes can be nonsymmetric, (aij # aj;). This results in anomalously polarized bands for which YZas# 0 and p > 3/4. 27-29 In the limiting case where the symmetry is high enough, the scattering tensor may be antisymmetric with o~ij = - a j i , in which case % = 0 and p = oo (the scattering intensity becomes zero for parallel orientation). The depolarization ratio of a Raman band is very helpful in making vibrational assignments. The ranges of values expected for the depolarization ratio for bands arising 25 T. G. Spiro and P. Stein, Annu. Rev. Phys. Chem. 28, 501 (1977). 26 O. S. Mortensen and S. Hassing, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, eds.), Chap. 1, p. 1. Heyden, London, 1980. 27 H. Hamoguchi, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, eds.), Chap. 6, p. 273. Heyden, London, 1985. 28 G. Placzek, in "Rayleigh and Raman Scattering" UCRL Trans. No. 526(L) from "Handbuch der Radiologie" (E. Marx, ed.), Vol. 2, p. 209. Alkademishe Verlagsgesellschaft, Leipzig, 1934. 29 T. G. Spiro and T. C. Strekas, Proc. Natl. Acad. Sci. U.S.A. 69, 2622 (1972).

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

329

TABLE I RANGE OF VALUES OF DEPOLARIZATION RATIO OF RAMAN BANDS OBSERVED IN AND OUT OF RESONANCE

Type of Raman scattering Nonresonance (all cases) and resonance (most cases) Resonance (special cases)

Mode character Polarized (totally symmetric modes) Depolarized (nontotally symmetric modes) Anomalously polarized (nontotally symmetric modes) Inversely polarized (nontotally symmetric modes)

Tensor symmetry Symmetric,

Depolarization ratio 0< p <

aij = aji

Symmetric, aij = aji

p =

Nonsymmetric,

p >

aij ¢= aji

Antisymmetric,

p =

aij = - aji

from modes of various symmetries both in and out of resonance are summarized in Table I.

Quantum Mechanical Description of Raman Scattering: Theory of Resonance Enhancement The classic treatment of the Raman effect provides only limited insight into Raman intensities. The application of quantum theory to the Raman effect shows that the intensity of a Raman band, IRama,, fi associated with a transition from the initial state Ii) to final state If), is proportional to the intensity of the incident light, I0, and the Raman cross section, 0n, for the transition: Ifiaman (1"0, Pfi) = 0fi(/~0, Ufi)Io

(8)

where v0 and v~ are the excitation and vibrational frequencies, respectively. For a given transition, 0fi is related to the elements of the molecular polarizability tensor, af, '~ (p, o- = x, y, z), by 3° 0( 0,

--

c 0( 0 -

I UI 2

(9)

p,lY

where C is a constant and p and cr represent one of the three axes of a Cartesian coordinate system on which the dipole moment operator/z is projected. From second-order perturbation theory, the molecular polarizability can be expressed as a sum of the contributions from the wave 30 A. C. Albrecht, J. C h e m . P h y s . 34, 1476 (1961).

330

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

functions of all excited states, [e), to give the Kramers-Herzberg-Dirac dispersion formula3°:

p,~

2~

((J~ole>(el~.li>

(j~.le>(el~pli>h

(10)

OLfi : X ~e \ ~ e i = ~'0 ~/-~e ~- l"fe -b 120 q- ire/

Here, F e is a damping factor that ensures a finite bandwidth for the excited state. Equation (10) is valid for all Raman scattering, and the summation is carried out over all electronic states. The basis for the resonance Raman effect follows naturally from Eq. (10). As the excitation frequency, v0, is tuned into the vicinity of an electronic transition, Vei, the value of the first term will increase (but not go to infinity owing to the Fe term) because of the reduction in its denominator, and the second (nonresonance) term will become insignificant. When the resonance is with a single electronic transition, the contribution to the first term from other electronic transitions will become insignificant, and Eq. (10) becomes

p,~ aft

27r ~ <~ple> = -~

(11)

Pvei _ VO + iFve

Here, the summation is over all of the vibrational states of a single resonant electronic state. Separation of the electronic and vibrational wave functions using the Born-Oppenheimer approximation followed by a Herzberg-Taylor expansion of the electronic wave functions in terms of the nuclear displacements produced by the normal coordinates, Q, gives 3~ p,o" a n =A +B+C (12) where 9,.-

A = hM~M~ B

~-n (M~M~'Eve

'. ~v~Vvevi - Vo + iFve

(13)

1)s=yOo + MO'M'~ EVeVVev.(vflQlve)PO (velv+i)- irvo/) (14)

C= h M°'M'~' E ~o~i--- <-+

(15)

in which M ° = (e°l/~o[e °) and M o' = M°(alOM°/OQle>/(Va - vs), where [a> is another excited state with the associated transition dipole moment 31 j. Tang, and A. C. Albrecht, in "Raman Spectroscopy" (H. Szymanski, ed.), Vol. 2, p. 33. Plenum, New York, 1970.

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

331

M ° and energy Va, ]Ui) and [vf) are the initial and final vibrational wave functions, respectively, of the ground state and [re) the ones for the excited state, and [el°), Ie e), ° and [el°) are the electronic wave functions for the final, excited, and initial states, respectively, defined at the ground state equilibrium geometry indicated by the superscript 0. Each term described above contains contributions that can be related to the electronic and vibrational wave functions and excitation energy. Because the spacing between vibrational energy levels is small compared with the gap between electronic transitions, the energy denominators (vooo - Vo + iFve) for different ve act as small differential weighting factors. Together with the electronic contribution described by Mo and M ' , they determine the total enhancement of all of the vibrational modes for a given electronic transition and a given laser excitation frequency. The contribution of vibrational wave functions is manifested by the Franck-Condon overlap integrals, (vflve)and (Ve[Vi)in the A- and B-terms, and the normal mode (Q)-dependent integrals, (vf[Q]ve) and (ve]Q[vi) in the B- and C-terms. These integrals relate the symmetry of a vibrational mode to its Raman activity and are the origin of selection rules for Raman scattering. For fully allowed transitions that have large M 0 values, the most common mechanism of resonance enhancement arises from the A-term. The Franck-Condon overlap integrals must vanish by symmetry unless the mode is totally symmetric. Therefore, A-term resonance enhancement leads to polarized Raman bands. When the resonant electronic transition is only weakly allowed and M ' > M 0 , the contribution from the B-term can be dominant. Because B-terms involve the vibronic mixing of two excited electronic states, the B-term contribution is favored when a weak electronic transition is vibronically coupled with an intense one, as is the case for metalloporphyrins. The B-term active vibration may have any symmetry that is contained in the direct product of the group-theory representations of the two electronic states .32The numerator of the C-term contains products of two normal mode (Q)-dependent integrals. Because each of these terms connects vibrational levels differing by one quantum, the final level must differ from the initial level by two quanta. Consequently, only overtones are enhanced by the C-term. The contribution from C-term enhancement can be significant when M ' is equal to or larger than Mo. The three terms described above account for the most important enhancement mechanisms in resonance Raman scattering. Some of the results described above are applied in a later section to the interpretation 32 F. A. Cotton, "Chemical Applications of Group Theory." Wiley, New York, 1963.

332

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

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of resonance Raman spectra of metalloproteins. First, however, some of the experimental aspects of Raman and resonance Raman spectroscopy are considered. Experimental Considerations

Equipment Raman scattering is inherently weak, typically 10 -9 t o 10 -6 of the intensity of Rayleigh scattering. For this reason, it is difficult to observe without an intense monochromatic excitation source and a sensitive detector, and Raman spectroscopy was overshadowed by infrared spectroscopy for 40 years. It was not until the 1960s with the development of commercial lasers that the modern Raman renaissance took place. In recent years, significant progress has been made in all aspects of Raman instrumentation, and the technique has now become routinely used in a wide spectrum of research activities carried out in both academic and industrial laboratories. A modern Raman spectrometer consists of three major spectroscopic components: a monochromatic light source for excitation, a spectrometer for dispersion of the scattered light by frequency, and a detection system for light analysis and data acquisition. In addition, the system must also have sample illumination and collection optics and an assortment of devices to hold samples. A schematic of the Raman spectroscopy system used in the laboratory of the authors is shown in Fig. 4, where three different lasers, two kinds of spectrometers, and two kinds of detectors are integrated to accommodate a variety of diverse applications. The specifications of the individual components available from commercial sources vary significantly. Unfortunately, a single combination of components that satisfies a broad range of Raman applications does not exist. Therefore, whether one plans to assemble a system oneself or purchase a ready-to-run package, there are some important features and potential capabilities that should be kept in mind in order to understand the tradeoffs that are an inherent part of any system. As a general rule, the most important consideration in the choice of components is their suitability to the samples that are to be examined. For resonance Raman studies of metalloproteins, the most critical decisions involve the choice of excitation wavelength and the choice between pulsed versus continuous wave (CW) excitation. These decisions are often dictated by the optical properties of the sample or by the nature (static versus kinetic) of the experiment. The excitation wavelength is important in all forms of Raman spectroscopy because every optical component of a Ra-

[14]

333

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

K r y p t o n Ion L a s e r Argon Ion L a s e r

I ~Collection~llumina Optics

I

.......

I pptics

io~ |

SPEX 1403 Double M o n o e h r o m a t o r

io;rf

Controller Photon Counter & High Voltage

Collection ~ l l u m i n a t i o ~ Optics II . . . . . r ' ~ I 0pries

/

IDA Controler

Nd:YAG L a s e r

SPEX 1877 Triplmate

i =

Dye L a s e r

FIG. 4. Schematic diagram of the Raman spectroscopic system used in the authors' laboratory. It consists of three different types of lasers (a CW argon ion and a CW krypton ion laser, and a pulsed Nd : YAG/dye laser system), two different monochromators (a spectrometer with two gratings and a spectrograph with a single exchangeable grating and a twograting filter stage), and two detection systems [a cooled photomultiplier tube (PMT) with photon counting electronics and an intensified diode array (IDA) controlled by an optical multichannel analyzer (OMA)].

man spectrometer has an efficiency that is wavelength dependent. The excitation wavelength in resonance Raman studies has the additional importance that it must match the absorption of the sample under study. A brief consideration of each of the components of a Raman spectrometer

J

334

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS .<

1000 o.

u'3

800 ,,<

©

600

r-~

r:.

ff3t¢3 ¢'3¢~

©

400

o~. p,. ke3

<

g ,~

un

? 200

v

co

=

©

J

o

go0

400

500 Wavelength,

p-

6(30

700

L 800

nm

FIG. 5. Approximate output of the most commonly used lines from argon (A), krypton (K), H e - N e ( H - N ) , and H e - C d (H-C) lasers.

is provided below, starting with the laser and following the incident and scattered light through to the detec[or. More comprehensive discussions can be found elsewhere. 33-35 Lasers. Because they provide exceedingly monochromatic, coherent, intense, and well-collimated light beams, lasers are the only excitation sources currently used for Raman spectroscopy. Lasers can be divided into CW and pulsed models on the basis of the temporal nature of light output. As the name implies, CW lasers emit photons continuously and are the most widely used lasers for Raman spectroscopy. The helium/neon laser, which has a strong line at 632.8 nm, was the earliest reliable gas laser. However, it has now been largely superseded by the much more powerful argon and krypton ion lasers. These lasers emit a series of lines in the visible region from 406.7 to 676.4 nm and in the near-ultraviolet region from 330 to 370 nm (Fig. 5) that can be selected with an intracavity 33 p. p. Strommen and K. Nakamoto, "Laboratory Raman Spectroscopy." Wiley, New York, 1984. 34 D. J. Gardiner and P. R. Graves (eds.), "Practical Raman Spectroscopy." SpringerVerlag, N e w York, 1989. 35 j. G. Grasselli and B. J. Bulkin, "Analytic Raman Spectroscopy." Wiley, New York, 1991.

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335

or out-of-cavity prism. The weaker lines are available at a useful intensity only from the most powerful models of these lasers. In addition, the helium-cadmium laser offers two relatively weak lines at 325 and 442 nm that can be useful for resonance or preresonance Raman studies of certain chromophoric species. On the whole, the argon ion laser is the most useful for studies requiring blue or green excitation, whereas the krypton ion laser is of greatest value in the violet, yellow, and red regions of the spectrum. The krypton ion laser is attractive in that it provides lines over a broad range of the spectrum. However, this feature is somewhat diminished by the reality that there are different optimal tube pressures and cavity mirrors (at least five sets of mirrors are needed to cover all the frequency windows) for the lines in different spectral regions, a fact that limits facile changes in excitation wavelength. Argon ion lasers can be purchased with higher power ratings (up to 20 W of all-line power centered near 500 nm and 9 W of single-line power at 514.5 rim) than krypton ion lasers (maximum 4.6 W of all-line power anywhere, 3.5 W for the most powerful single line at 647.1 nm, and less than 1.5 W for any single line below 600 nm). In addition, argon ion lasers are less susceptible to instabilities created by changes in plasma tube pressure and give a greater number of lines (from 457.9 to 514.5 nm) without the need for changing laser optics. Krypton ion lasers have a spectral gap from 482.5 to 520.8 nm within which there are four powerful argon lines (488.0, 496.5,501.7, and 514.5 nm). The argon ion laser is the easier of the two for the nonexpert to use and tends to have less downtime. The krypton ion laser, however, has played a key role in resonance Raman studies of heme proteins because its violet output in the 407 and 415 nm regions falls under the Soret band of the heme chromophore. All in all, if there is no specific wavelength requirement, as in the case of nonresonance Raman spectroscopy, the argon ion laser is the more reliable and economic choice, although this is much less true today than several years ago. Another potentially useful alternative offered by laser manufacturers is the mixed-gas ion laser that delivers certain frequencies of both argon and krypton ion lasers. 36 The systems that are currently available do not have violet output,

36 Specifications on the o u t p u t p o w e r and f r e q u e n c y of the m i x e d gas l a s e r from Coherent: Wavelength(nrn): 752.5 647.1 568.2 530.9 520.8 514.5 488.0 457.9 Multiline-UV Multiline-Vis Power (mW): 30 250 150 200 130 250 250 30 50 2500 N o t e that several sets of optics are require d to obtain all of the individual lines in bot h the visible and n e a r - u l t r a v i o l e t regions.

336

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It is advantageous for several reasons to purchase a laser with the highest output power that the budget will allow. This allows one to use the weaker lines as excitation sources and extends the usefulness of the stronger lines, since output power inevitably decreases with tube life. Moreover, a high-power CW laser can be used to pump a tunable dye laser to give access to other desired excitation wavelengths. Finally, a powerful argon ion CW laser allows one to use nonlinear optical crystals to frequency-double or mix the 488.0 and 514.5 nm lines to produce ultraviolet lines at 244.0, 250.5, and 257.3 nm. 37 Although the lines produced are weak, new developments in both spectrometers and detectors are making high laser power unnecessary for many applications. Several examples of Raman applications have been reported in which diode lasers with weak output (1-20 roW) in the red region of the spectrum were used for excitation, 38'39 In purchasing an ion laser, it should be kept in mind that the laser tube is an expendable item that has a relatively short lifetime (normally less than 3-5 years), even when great care is taken to ensure the use of very clean cooling water and to operate at power outputs well below the maximum. The plasma tubes, especially those for more powerful large-frame models, are very expensive to replace. This problem has been diminished somewhat by the development of ceramic tubes that have largely replaced the more costly glass tubes, but it is nevertheless significant. The purchase of a larger laser commits one to greater plasma tube replacement costs somewhere down the road. In contrast to CW lasers, pulsed lasers deliver light in a series of bursts. The duration of the burst (usually defined as the temporal width at half the intensity of the pulse) and the pulse repetition rate (usually measured as the number of bursts in pulses per second) are important parameters of a pulsed laser. Thus, whereas the "intensity" of a CW laser is described by its power, the "average power" of a pulsed laser is 37 Specifications on output p o w e r and f r e q u e n c y of the intracavity frequency doubler for an argon ion laser from L e x e l Laser: F u n d a m e n t a l line

Power

514.5 n m 496.5 n m 488.0 n m

2400 m W 720 m W 1800 m W

H a r m o n i c line 257.3 248.2 244.0 250.5

nm nm nm nm

(doubled 514.5 nm) (doubled 496.5 nm) (doubled 488.0 nm) (mixed 514.5 and 488.0 nm)

Power 30 10 25 30

mW mW mW mW

F o r the h a r m o n i c lines, note that different nonlinear crystals are needed to cover all ultraviolet frequencies. 38 y . W a n g and R. L. M c C r e e r y , Anal. Chem. 61, 2647 (1989). 39 C. D. Allred and R. L. M c C r e e r y , Appl. Spectrosc. 44, 1229 (1990).

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determined by both the energy per pulse and the repetition rate. In principle, there are a number of potential advantages to employing pulsed Raman excitation. First, the laser pulse can be used to initiate a photochemical event so that the sample can be interrogated at later fixed times by other pulses. This allows time-resolved studies to be carried out and is most applicable to reversible photochemical reactions. Second, pulsed lasers are ideal sources for the nonlinear optical processes used to generate new lines with wavelengths that are tunable over a wide spectral range. Most commercially available pulsed lasers are designed to operate with relatively low repetition rates, ranging from 10 to several hundred hertz (Hz). The high peak powers of such lasers are well suited for the generation of visible, near-ultraviolet, and far-ultraviolet lines by nonlinear processes such as pumping dye lasers, inducing stimulated Raman emission from gas media, and frequency doubling or mixing by nonlinear crystals, since the efficiencies of these processes are greater at higher field strengths. On the other hand, the high peak powers of these pulses can damage the sample, and this has tended to limit their utility as excitation sources for routine Raman experiments. Pulsed excitation sources have been most widely used in the study of biological molecules by UV resonance Raman spectroscopy.4°-42 The two most commonly used pulsed lasers are the neodymium-yttrium-alluminum-garnet (Nd : YAG) and excimer lasers. The Nd : YAG laser has a fundamental output at 1064 nm with second to fifth harmonics at 532, 355,266, and 213 nm, respectively, that are generated by various types of nonlinear optical crystals. Excimer lasers produce a series of fundamentals ranging from 193 to 351 nm, depending on the gas being used (ArF, KrC1, KrF, XeC1, and XeF at 193, 222, 248, 308, and 351 nm, respectively). Both lasers can be used in conjunction with dye lasers, Raman shifting devices, and a range of nonlinear optical crystals for additional frequency coverage. As mentioned above, the main drawback with the use of N d : Y A G and excimer lasers for Raman spectroscopy is their low repetition rate and high peak power. A newly introduced alternative is the quasi-CW (80 to 100 MHz) mode-locked Ti : sapphire laser. 43It is an attractive alternative because of its high repetition rate and because its tunability over the visible, near-UV, and UV regions is achieved without the complicated

4o I. Harada and H. Takeuchi, in "Spectroscopy of Biological Systems" (R. J. H. Clark and R. E. Hester, eds.), p. 113. Wiley, New York, 1986. 41 B. Hudson and L. C. Mayne, in "Biological Applications of Raman Spectroscopy" (T. G. Spiro, ed.), Vol. 2, p. 181. Wiley, New York, 1987. 42 S. A. Asher, Annu. Rev. Phys. Chem. 39, 537 (1988). 43 p. M. W. French, J. A. R. Williams, and J. R. Taylor, Opt, Len. 14, 686 (1989).

338

S P E C T R O S C O P IMETHODS C FOR METALLOPROTEINS

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TABLE II PEAK POWER COMPARISON OF THREE TYPES OF LASERS OPERATING AT SAME AVERAGEPOWER

Parameter

Ar ÷ laser (364 nm)

Mode-locked Ti : sapphire laser (360 nm, 100 MHz, 2 psec)

Excimer/ dye laser (410 rim, 400 Hz, 10 nsec)

Nd : YAG laser (355 nm, 10 Hz, 5 nsec)

Average power (W) Peak power (W) Ratio to peak power of Ar + laser

0.01 0.01 1

0.01 50 5000

0.01 2500 250,000

0.01 200,000 20,000,000

a

Typical wavelengths, repetition rates, and pulse durations have been selected for the purposes of illustration.

setups for laser amplification associated with mode-locked argon ion or N d : Y A G laser systems. 44 Frequency doubling produces a second harmonic in the range from 350 to 450 nm, with the third and fourth harmonics having the potential to provide excitation lines in the 210 to 500 nm region. 45,46This could significantly broaden the range of organic and enzymatic chromophores that could be examined by resonance Raman spectroscopy. The peak power of the Ti : sapphire laser (100 MHz, 2 psec pulses) is lower than that of a Nd : YAG laser (10 Hz, nsec pulses) operating at the same average power by a factor of 4000, as illustrated by the comparison shown in Table II. It is, however, still larger than the "peak power" of a CW argon laser by a factor of 5000. The significance of this for photolabile samples can be quite profound. For example, if 5% of the sample within the volume element of the focused laser beam was damaged by the pulses from a Nd : YAG laser, less than 0.00125% of the same sample would be damaged under the same conditions with the Ti : sapphire laser. A 40- to 80-fold reduction in peak laser power has been shown to result in significantly lower photon saturation and sample damage. 47"48 Finally, it should be emphasized that the major safety hazard encountered in operating Raman instruments is the laser radiation, especially for pulsed lasers. When aligning the system and positioning the sample in the laser beam, one is often subjected to reflected beams from the sample cell or other surfaces. To prevent eye damage, protective glasses should be worn. 44 T. L. Gustafson, J. F. Palmer, and D. M. Roberts, Chem. Phys. Lett. 127, 505 (1986). 45 p. F. Curley and A. I. Ferguson, Opt. Lett. 16, 321 (1991). 46 A. Nebel and R. Beigang, Opt. Lett. 16, 1729 (1991). 47 C. M. Jones, V. L. Devito, P. A. Harmon, and S. A. Asher, Appl. Spectrosc. 41, 1268 (1987). 48 C. Su, Y. Wang, and T. G. Spiro, J. Raman Spectrosc. 21, 435 (1990).

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339

Illumination and Collection Optics. The performance o f a Raman spectrometer is markedly influenced by the sample illumination and collection optics. Many research groups prefer to design and install their own optics to maximize flexibility and efficiency, and minimize cost. The design of the illumination and collection optics is influenced by the scattering geometry, which is discussed later under the section on sample illumination techniques. Features independent of the scattering geometry of these optics, however, are discussed here. The purpose of the illumination optics is to deliver and focus a monochromatic laser beam to the sample. The first problem that must be addressed by the illumination optics is the fact that CW lasers emit plasma lines from nonlasing transitions of the gas. Although much weaker than the laser output, they are very close in energy to the lasing line and can be comparable to, or stronger than, typical Raman bands. To avoid their interference in Raman spectra, it is best to eliminate them before they reach the sample. This can be achieved by using either dispersion or filtering optics. In the dispersion method, either a prism or a grating is used to spatially disperse the light according to frequency, and an aperture such as an iris, pinhole, or slit is used to select the desired frequency. Alternatively, one can use a laser interference filter. Depending on the requirement, a 1 to 10 nm bandpass filter can be used for this purpose, with typical throughputs of approximately 25 to 80%, respectively. The cost, however, can become significant if filters for a large number of laser lines are needed. Aside from the filters for removing these lines from the laser beam, the remainder of the illumination optics usually consists of several mirrors to steer the beam by 90 ° reflections and a lens with a short (typically 3-10 cm) focal length to focus the beam on the sample. The purpose of the collection optics is to focus the scattered light into the spectrometer. A minimum of two lenses is required for optimal collection of the scattered light. Optical collection efficiency also requires that the focal length of the last lens be matched to the f number of the first mirror in the monochromator. Under- and overfill of this mirror will result in a decrease in resolution or efficiency, respectively. If the instrument is to be used solely with visible radiation, the first lens can be a camera lens. A Cassegrain mirror is an alternative to the first lens for samples positioned at significant distances (3 to 5 inches) from its first optical surface. Owing to its central reflector, however, it is inefficient at short working distances. Cassegrain mirrors can be readily obtained for use in the ultraviolet regions. 49'5° A schematic showing the three types of light collection systems discussed above is given in Fig. 6. 49 K. Bajdor, Y. Nishimura, and W. L. Peticolas, J. Am. Chem. Soc. 109, 3514 (1987). 50 K. R. Rodgers, C. Su, S. Subramaniam, and T. G. Spiro, J. Am. Chem. Soc. 114, 3697 (1992).

340

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

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To obtain unbiased Raman band intensities, which is particularly important for the measurement of depolarization ratios, a polarization scrambler is recommended (Fig. 3). This is a quartz wedge that scrambles the polarization of the scattered light in order to eliminate the unequal efficiency of the gratings toward the two polarization directions. The quartz wedge is placed between the sample and entrance slit to ensure maximum area or aperture of illumination by the scattered light. Sample Illumination Techniques. Raman spectroscopy is directly applicable to samples in all phases. It is noteworthy that the technique can offer the opportunity to study aqueous biological solutes and to compare samples in the solution and solid states. The handling of samples and the various accessories associated therewith has become an increasingly important aspect of the technique that often determines the success of the experiments. The first important issue related to sample illumination is the excitation geometry (i.e., the angle between the incident laser beam and the axis of the collection optics). This is usually restricted to values between 90° and 180° and is often dictated by the geometry of the experimental setup. The two most popular scattering geometries have incident and collection angles that differ by 90 ° and 180°. The inherent scattering intensity is similar for the two scattering geometries; however, there are some trade-offs associated with the use of each configuration. A 180° scattering geometry minimizes absorption of the incident light, which is important in resonance Raman experiments (see below). Owing to geometric considerations, all Raman microprobe instruments also use a 180° geometry. The disadvantage of this geometry is that it casts an image of the last laser reflecting optic (prism or mirror) onto the slit. Most commercial Raman spectrometers use a 90 ° scattering geometry because it is the most convenient for routine operation. However, the dependence of scattering intensity on the sample concentration for absorbing samples in a 90° scattering geometry is quite severe (see below) and makes certain resonance Raman experiments more difficult. Other scattering geometries between 90° and 180° can also be employed when deemed appropriate. The absorption of the incident and scattered light by the sample in resonance Raman spectroscopy is markedly dependent on the scattering geometry. In ordinary nonresonance Raman measurements on a transparent liquid sample, the intensity of a Raman band will increase with an increase in either the sample concentration or the laser power (10-500 mW). In resonance Raman measurements, however, the laser power at the sample must generally be below 50 mW because light absorption may lead to sample heating and/or photodamage. In addition, the scattering intensity depends on the sample concentration and the excitation geome-

[14]

RAMAN

AND

RESONANCE

RAMAN

SPECTROSCOPY

341

+.a

0

0 , " ,..~ 0 .,a

0 0 ~-~

.~

o

o

0

~

~,.~ 0 ~-~

o

0 ,..-i 0 r..)

o

342

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

try, since photons from both the incoming laser beam and the outgoing Raman scattering can be absorbed by the sample. The equation describing the dependence of Raman scattering intensity (IRs) on sample concentration (c) depends on the excitation geometry. For a 90° scattering geometry

Ias ~ Ioce -(cb/2)(ee+es)

(16)

where b is the sample path length and ee and es are the extinction coefficients of the sample at the exciting and scattering wavelengths, respectively. 5~'52 For a 180° scattering geometry, the appropriate equation is IRs ~ 7r(1 -- l/e) I0 [1 -- e -c%+~s)b] 16 ee + es

(17)

These functions have been plotted in Fig. 7 for typical values of ~ (ee = es) and a capillary diameter of 1 mm. The results agree quantitatively with the experimental data of Hendra and associates. 53 With a 90° scattering geometry, there exists an optimal concentration, c = 2/b(ee + es), at which to observe the Raman scattering. The dependence on sample concentration for the 180° (back-scattering) geometry is much less severe and offers a distinct advantage over the usual 90 ° configuration. The second important issue related to sample illumination is the sample cell. In the most favorable cases, as little as 1 /zl of solution or 1 mg of a solid biological sample can be illuminated in a static capillary tube. In most instances, however, more sophisticated sampling techniques are required to prevent damage to the sample from photochemical events associated with exposure to the laser beam. The primary strategy in sample illumination is to minimize absorption of the exciting photons by the sample. Three popular sample illumination techniques are illustrated in Fig. 8 and include interrogation of a solution sample while flowing through a capillary tube or in a cell stirred from above as well as examination of a frozen sample on a cryogenic tip. There are many variations of each of these sampling techniques that may be more suitable for a given application. For photolabile samples, for example, the continuously flowing sample stream in the capillary tube shown in Fig. 8 could be replaced by a continuous unconfined flowing jet or a discrete stream of microdroplets for very photolabile samples.

51 j. C. Merlin and M. Delhaye, in "Laser Scattering Spectroscopy of Biological Objects" (J. Stepanek, P. Anzenbacher, and P. Sedlacek, eds.), p. 49. Elsevier, Amsterdam, 1987. 52 T. C. Strekas, D. H. Adams, A. Packer, and T. G. Spiro, Appl. Spectrosc. 28, 324 (1974). 53 p. j. Hendra, J. Chem. Soc. A, 1298 (1967).

343

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

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1.0-

Configuration

900

/

/

/ //

//

//

/e=

ixlO*

we=

2xlO*

1 /

/~/ /I/e= 4xlO 4 / ~=

/

~

8xlO* ~e=16x104

~0.0

180 o C o n f i g u r a t i o n

//i

/

I i ///

/~e= /

I

1

2xlO 4

//i / / I / /~= 4xlO 4 ~= 8xlO* 11 I i j

l

~=16xlO*

0.0

2.0

4.0

6.0

8.0

-logic] FIG. 7. Plots of the Raman scattering intensity versus sampie concentration for resonance Raman experiments carried out on samples with different extinction coefficients in a 1 m m diameter capillary tube excited in the 90 ° and 180° scattering geometries.

Alternatively, the capillary flow could be configured to allow an upstream pumping/downstream probing experiment to be carried out. 54 In other setups, the stirred cell could be a spinning nuclear magnetic resonance (NMR) tube, an airtight anaerobic cell, or one with the capability to perform electrochemistry.55 For the study of relatively stable samples by 54 S. Han, Y.-C. Ching, and D. L. Rousseau, Proc. Natl. Acad. Sci. U.S.A. 87, 2491 (1990). 55 p. Hildebrandt, K. A. Macor, and R. S. Czernuszewicz, J. Raman Spectrosc. 19, 65 (1988).

344

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

Flowing

Sample

Stirring

Sample

Stirring Device Pumping Laser

Frozen

Sample

Liquid Nitrogen or Other Coolant

Vacuum

!

UV/Vis Cell

Focusing Lenses ~

< ~

Capillary Tube

Probing Laser

Probing Laser

Probing Laser

FIG. 8. Schematic representation of three popular techniques for illuminating samples in Raman spectroscopic experiments.

resonance Raman spectroscopy, a spinning NMR tube is a convenient sample cell that requires only small sample volumes (e.g., 50 to 100/xl). It is sometimes useful to examine the sample at cryogenic temperatures. This not only allows the effects of temperature changes to be studied, but it also gives enhanced resolution by observation of sharper Raman bands. The lower temperature can also reduce excitation damage through better heat transfer, and the method requires only a small amount of sample. On the other hand, it has the disadvantages that spinning the sample is more difficult and that there may be enhanced luminescence. The photolability of samples is a common cause of failure to obtain the Raman spectra. An example of this has been the difficulties encountered in acquiring the authentic spectrum of horseradish peroxidase (HRP) compound 156 discussed in the last section of this chapter. To overcome photolability, one can try to make the residence time of the sample in the laser beam shorter than that of the photoconversion time. Although the use of a pulsed laser can shorten the temporal width of the light to nanoseconds or picoseconds, many photoconversions are still completed within the 56 H. E. Van Wart and J. Zimmer, J. A m . Chem. Soc. 107, 3379 (1985).

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RAMAN AND RESONANCE RAMAN SPECTROSCOPY

345

durations of these pulses. In such cases, the only alternative is to minimize the total number of photons absorbed by the sample element being interrogated by employing a faster flow rate and using lower probing powers. Another way to minimize sample absorption is through the use of a uniform microdroplet stream generator, 57 which allows a residence time in the laser beam ranging from approximately 1 to 100 /zsec. 58 There is less absorption of light by a sample illuminated in a microdroplet stream than in a simple flowing jet with the equivalent linear flow velocity because there is light piping that illuminates the sample in the direction parallel to the continuous jet stream that is prevented when the stream is chopped into a series of discrete microdroplets. A drawback of this technique is the need for large volumes of sample (e.g., 5 to 10 ml). Another common difficulty in observing the Raman spectrum of a sample is interference from fluorescence. Fluorescence frequently arises from impurities, and the recommended solution is purification of the sample. Alternatively, one can attempt to shift the excitation wavelength to avoid or minimize excitation of the contaminating chromophore that is responsible for the fluorescence. Interference from fluorescence with both ultraviolet and infrared excitation wavelengths is generally less serious owing to the fact that the regions in which the Raman scattering and fluorescence are observed are noncoincident. The use of a pulsed laser for excitation can sometimes circumvent interference from fluorescence by saturating the chromophore responsible. With the use of small shifted excitation wavelengths, a mathematical procedure has been developed to identify genuine Raman bands in the presence of a large fluorescence background. 59 Spectrometers. The choice of the dispersing spectrometer needed to analyze the frequency distribution of the Raman scattering should be based on the type of research carried out. The major obstacle to the analysis of Raman scattering is that the signal is only one-millionth or less in intensity of that of the elastic Rayleigh scattering. As a consequence, Raman scattering is much harder to detect than either Rayleigh scattering or fluorescence. In addition, the resolution required for Raman spectroscopy is approximately two orders of magnitude higher than in electronic emission spectroscopy because of the narrow spacing of vibrational transitions. The weaker signal demands a high throughput spectrometer. This translates into one with a small f number and smallest number of high57 S. F. Simpson, J. R. Kincaid, and F. J. Holler, Anal. Chem. 58, 3163 (1986). 58 W.-J. Chuang, Ph.D. Thesis, Department of Chemistry, Florida State University, Tallahassee 0991). 59 A. P. Shreve, N. J. Cherepy, S. Franzen, S. G. Boxer, and R. A. Mathies, Proc. Natl. Acad. Sci. U.S.A. 88, 11207 (1991).

346

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

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quality optical elements. Unfortunately, the need to eliminate interference from stray light, to achieve the necessary resolution, and to observe small Raman shifts requires the use of a high dispersion spectrometer. This usually translates into one with an increased f number, multiple gratings and optics, and a higher cost. As the criteria for sensitivity and resolution play against each other, an appropriate trade-off must be reached. Therefore, the choice of the appropriate spectrometer is not a simple task and should be made in the light of the considerations discussed below. In modern Raman spectrometers, one or more gratings are used to disperse the light. Both ruled and holographic gratings are available, but holographic gratings are currently the most popular because of their wide frequency coverage and absence of grating ghost lines. They are also available with higher groove densities and are usually more economical. The dispersion of a spectrometer is increased by using more than one grating in an additive dispersion mode, by using gratings with higher groove densities, or by using higher order diffractions. A Raman spectrometer can be used in two fundamentally different modes. It can be used as a scanning instrument in which the grating(s) disperses the entering light, which is ultimately focused through a variable-width exit slit onto a singlechannel detector. This light has a very narrow bandwidth and may b e considered essentially monochromatic. In this mode, the Raman spectrum is acquired by slowly rotating (or stepping) the grating(s) so that all of the photons that are Raman-shifted from the exciting line are eventually presented to the detector. In most cases, the detector is a cooled photomultiplier tube equipped with photon-counting electronics. Scanning the whole spectral range of interest to the Raman spectroscopist (10-4000 cm-1) typically takes 1 hr or more. The Raman spectrometer can also be used as a spectrograph in which the scattered light is dispersed by a coarsely ruled grating and focused in a plane parallel to an exit port. The density of the grating and focal length of the spectrometer are usually chosen so that the scattered light in the region of interest is spatially dispersed over approximately 1 inch of the focal plane onto a multichannel detector (see below). In this mode, the grating(s) are not moved during spectral acquisition, but rather the detector "scans" the spectrum, resulting in a significantly shorter acquisition time. These two modes of usage each have advantages and disadvantages. In general, the scanning instrument is best for studies requiring high resolution, such as identification of isotopic shifts, deconvolution of Raman bands, and the study of small Raman shifts. Data acquisition is much faster in the spectrograph mode, which is advised for studies of photon-sensitive samples and shortlived transient species.

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TABLE III ESTIMATED THROUGHPUTS OF THREE COMMERCIAL SPECTROMETERS Optical component Aperture Slits a Mirrors Gratings Lenses Overall efficiency b

Spex 1296

Spex 1403

Spex 1877

f /9

f/6.9

f/6.3

2 2 1 0 34%

4 5 2 2 10%

4 7 3 2 3.4%

a Number of slits specified is for operation in the scanning mode. For operation in the spectrograph mode, there will be one less slit for each system. b The efficiency is estimated based on the assumptions that the light collection and coupling efficiencies are equal in all the spectrometers and that the mirrors, gratings, and lenses have efficiencies of 92, 40, and 98%, respectively.

The overall throughput of a monochromator is also a significant parameter, particularly for samples with low Raman cross sections and a high susceptibility toward photochemical damage. The throughput of a spectrometer or spectrograph is determined by the efficiency of all of its optical elements. The throughputs of three commercial Raman spectrometers have been estimated by assuming typical efficiencies for their optical components (Table III). A typical single grating system has a throughput of 34%, a two-grating system 10%, and a triple-grating system only 3.4%. In other words, if all other factors were equal, the same Raman signal detected on a triple-grating system would be approximately one-tenth of the intensity of one observed on a single monochromator. To compensate for such a reduction in intensity, one must either open the entrance slit wider, which will cause a reduction in spectral resolution, or increase the laser power, which might cause damage to the sample. The two most popular configurations for Raman spectrometers are a double-grating instrument that uses a photomultiplier tube for detection and a triple-grating system (consisting of a double-grating subtractive dispersion filter stage coupled to a spectrograph stage containing a single grating) that uses a multichannel detector. Spex (Edison, N J), Jobin-Yvon (Edison, NJ), and Dilor (Lille, France) all market triple-grating systems that consist of a double-grating monochromator and a spectrograph. In the Spex 1877 Triplemate and Jobin-Yvon $3000 systems, the two-grating stage operates in a subtractive dispersion mode, which acts as a variable frequency filter that allows a selected frequency window to enter the spectrograph stage. In the new Jobin-Yvon T64000 and Dilor XY-24 sys-

348

S P E C T R O S C O P IMETHODS C FOR METALLOPROTEINS

[14]

terns, the double monochromator can be operated in either the subtractive or additive dispersion modes, where the latter is useful for applications involving higher resolution. In addition, the Jobin-Yvon system has the unmatched capability that it allows one to separate the double monochromator in the filter stage from the single-grating spectrograph stage. Consequently, it can be used as a true 0.64 meter single monochromator for certain types of experiments. A single-grating spectrograph used with various types of Rayleigh light rejection f i l t e r s 6°-62 offers the same or better resolution as the triple-grating system. Its high throughput and ease of grating exchange are advantageous for many biological applications. 54'63 Detectors. Several types of detectors can be used for Raman studies. The cooled photomultiplier tube (PMT) with photon-counting electronics is used on most Raman spectrometers. Specially selected tubes with very low background signals and a high sensitivity from the near-ultraviolet all the way to the infrared are readily available. It should be noted that the photon-counting mode of PMT operations is not compatible with a pulsed laser excitation source. In this case, the PMT should be operated in the analog mode. Because the efficiency of a PMT device is frequency dependent, a careful comparison of intensities of Raman bands with different Raman shifts would require that the observed intensities be corrected for PMT response. When using a PMT, the resolution of the spectrum that is acquired is normally spectrometer limited (in the range of 0.2 cm-1). Thus, this is the detection mode of choice for studies requiring high resolution. Multichannel detectors in use for Raman spectroscopy include intensified diode array (IDA), charge coupled device (CCD), and vidicon (VID) detectors. The high quantum efficiency, relatively low dark current (extremely low in the case of CCDs), and wide dynamic range of such detectors provide sensitivity comparable to, and sometimes surpassing, that of a PMT, especially in the 600 to 900 nm region. Whereas a typical PMT offers 5 to 30% quantum efficiency in most spectral regions, the efficiency can be 10 to 40% for a typical CCD or IDA, and a thinned and backilluminated CCD detector can offer an efficiency of up to 80% in the red region of the spectrum. However, unlike the PMT, where resolution is limited by the spectrometer, the spectral resolution of multichannel detectors can be limited by the detector itself because of the nonzero width of 6o S. A. Asher, P. L. Flaugh, and G. Washinger, Spectroscopy 1, 26 (1986). 61 M. M. Carrabba, K. M. Spencer, C. Rich, and D. Rauh, Appl. Spectrosc. 44, 1558 (1990). 62 H. Horinaka, N. Yamamoto, and H. Hamaguchi, Appl. Spectrosc. 46, 379 (1992). 63 V. Palaniappan and J. Terner, J. Biol. Chem. 264, 16046 (1989).

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

349

the individual detector elements. As a consequence, there exists a point where one cannot gain resolution by reducing the width of the entrance slit. A CCD offers the best resolution in the sense that it is limited only by the physical dimensions of the pixels, which usually have a width of approximately 10 to 25 ~m. An IDA, on the other hand, has a resolution that is limited by both the pixel width and a "blooming" or "cross talk" effect which partially delocalizes the signal incident on a single pixel (25 /~m in width) onto the two adjacent pixels. Resolution is also compromised for both CCD and IDA detectors by the intensifier tube installed at the front of the sensors. These resolution issues limit one's ability to study small changes in vibrational frequencies induced by weak interactions or isotopic substitutions. Therefore, resolution can be a critical factor that needs to be considered in the choice of a multichannel detector for Raman spectroscopy. The relatively high cost of commercial photodiode array detectors can be overcome by constructing one from commercially available components. 64Homebuilt multichannel detectors offer similar performance but at a fraction of the cost of comparable commercial systems. 65

Obtaining Raman Spectra Choice of Excitation Wavelength. In nonresonance Raman spectroscopy, the choice of excitation wavelength is not critical and is made on the basis of purely practical considerations including instrumental sensitivity, available laser power, and sample fluorescence. The choice of excitation wavelength in resonance Raman spectroscopy is much more critical and must be made with reference to the absorption bands of the sample. Because the intensity of resonance Raman scattering is, to a first approximation, proportional to the square of the absorbance of the sample at the excitation wavelength, a laser line near the absorption maximum is often chosen. To minimize reabsorption of Raman scattering, excitation on the long-wavelength, descending part of the absorption band is usually advantageous. The choice of exciting line should also be one that provides minimum interference from sample fluorescence and minimal photodamage, where the presence and severity of these problems must be determined empirically. Quite often, the choice of excitation wavelength for a sample can only be made by trial and error. As pointed out in the theoretical section, excitation of allowed electronic transitions enhances totally 64 T. P. Carter, H. K. Baek, L. Bonninghausen, R. J. Morris, and H. E. Van Wart, Anal. Biochem. 134, 134 (1990). 65 y . Wang, T. P. Carter, L. van de Burgt, L. Bonninghausen, and H. E. Van Wart, manuscript in preparation (1990).

350

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

symmetric modes, whereas excitation of partially allowed or forbidden transitions is required to observe nontotally symmetric modes and overtones. Therefore, to observe the maximum number of resonance-enhanced bands, it is advantageous to probe all of the known accessible absorption bands of the sample. The choice of an excitation wavelength suitable for observing metalligand vibrational bands in heme proteins is somewhat more complex and deserves separate discussion. Unlike the case of iron-ligand modes from nonheme iron chromophores, a variety of resonance enhancement mechanisms are possible for the modes involving the axial ligands. Any mode that modulates the porphyrin ~r-~r* transitions, either through Fe(d~)-porphyrin(n'*) back bonding or mixing of the antibonding ironligand orbitals with the porphyrin ~'* orbitals via tilting of the proximal ligands, can be enhanced via Soret band e x c i t a t i o n . 66'67As a consequence, excitation profiles (plots of Raman intensity relative to an internal standard and corrected for the spectral response of the spectrometer and the v 4 dependence of the scattering; see below) for these bands exhibit maxima with Sorer band excitation. Excitation profiles can provide valuable information about the enhancement mechanism and vibronic nature of excited electronic states. Another resonance enhancement mechanism for metal-axial ligand modes is through excitation within charge transfer transitions. Because iron has partially filled d orbitals, a variety of charge transfer transitions from filled 7r orbitals of the axial ligands to vacancies in the dr or dz2 orbitals of the iron, or from filled d orbitals of the iron to a 7r* orbital of the ligand, are possible. All of these transitions are allowed, but their intensities depend on the extent of orbital overlap. Charge transfer transitions can also take place from the filled porphyrin alu or azu orbitals to vacancies in the d= or dz2 orbitals of the iron, or from filled d orbitals of the iron to eg* orbitals of the porphyrin. Excitation within any of these transitions can give enhancement of iron-ligand modes. The location of charge transfer bands is often dependent on the type of ligands as well as the mechanism of charge transfer. Because the charge transfer bands are weak and often obscured by nearby porphyrin bands, the optimal excitation wavelength must often be determined by trial and error. Frequency Calibration. The frequency response of a Raman spectrometer can best be calibrated using known standards. One source of calibration lines for assigning absolute positions is the plasma emission from an 66 A. Desbois and M. Lutz, Biochim. Biophys. Acta 671, 168 (1981). 67 0 . Bangcharoenpaurpong, K. T. Schomacker, and P. M. Champion, J. Am. Chem. Soc. 106, 5688 (1984).

[14]

351

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

¢q h,3

c5

= =

i h'3 J

oo

eq

400

sdo

'

12bo

'

lgoo

Raman S h i f t , c m -1 FIG. 9. R a m a n s p e c t r u m of indene, w h i c h is frequently u s e d as a standard for freq u e n c y calibration.

ion laser. Low-pressure discharge lamps such as krypton, argon, neon, and mercury are suitable alternative sources of calibration lines. They are easy to use, readily available, and have well-documented frequencies. 6a The best method of calibrating a cosecant-driven Raman spectrometer is to perform a linear least-squares fit of peak position against absolute line frequency. For a sine-driven monochromator (linear in wavelength) used with a multichannel detector, a linear least-squares calibration is not applicable. In this case, calibration involves at least a third-order polynomial curve-fitting procedure based on four or more internal or external calibration frequencies that are evenly distributed within the window of interest. 69 For Raman spectroscopy, the absolute frequency of a band (v) is not as important as the Raman shift (Av) from the exciting frequency. Thus, the most often used approach for assigning Raman shifts in an acquired spectrum is to use the Raman shifts from a standard sample for calibration. The two most widely used liquid reference materials are indene (Fig. 9)69 and carbon tetrachloride. 33 The 1 cm -1 accuracy obtained with these calibration samples is usually good enough for applications with condensed phase samples. 68 K. B u r n s , K. B. A d a m s , and J. Longwell, J. Am. Chem. Soc. 40, 339 (1950). 69 H.-O. H a m a g u c h i , Appl. Spectrosc. Rev. 24, 137 (1988).

352

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

Intensity Calibration. To compare the intensities of Raman bands acquired in different regions of the spectrum in a scan on the same instrument, or to compare spectra obtained from different Raman instruments, it is necessary to correct the intensities of the bands for the unequal wavelength sensitivities of the detector, gratings, and other optical components. This procedure is crucial for the correct acquisition of excitation profiles in resonance Raman experiments. The fluorescence from a laserexcited dye standard or the output of a calibrated white light source such as a deuterium or tungsten lamp are suitable calibration standards. Calibration methods using the pure rotational Raman spectrum of D 2 and the fluorescence spectrum of quinine have been described in detail. 69 The quantification of the intensity of spectral bands is usually made from the peak heights or peak areas. In principle, the peak area is the proper measure of the band cross section, since it takes into account variations in the bandwidths, which may be substantial. For practical reasons, however, the peak height is often a more reliable gauge of intensity in a complex spectrum because of ambiguities in the multiparameter spectral fitting procedures used to deconvolute the individual bands. Therefore, the peak height is the parameter most often used to describe the intensity of a Raman band. Raman band intensities should be measured relative to an internal standard and converted to molar scattering intensities via Eq. (18). 70

IR CS (eRX "~-eE)

OR = kOs ~-~R \esX + -~z

(18)

where

, = s ,Ecos,(O

cos ~

)]

(19)

and ORand 0s are the absolute Raman cross sections of the measured band and of the internal standard, respectively, I R and Is are the measured Raman band intensities, and CR and Cs are the molar concentrations of sample and standard, respectively. Here, k is a factor equal to (v0 - ~s)/ (v0 - VR) that corrects for the dependence of scattering on wavelength; k varies from 1.0 to 1.2. The quotient given in parentheses is the selfabsorption correction for front-scattering geometry, and eE, eR, and es are the extinction coefficients of the sample at the excitation wavelength and the wavelengths of the internal standard and sample bands, respec7o S. P. A. Fodor, R. P. Rava, T. R. H a y s , and T. G. Spiro, 1520 (1985).

J. Am. Chem. Soc. 101,

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

353

tively. These can be determined directly from absorption spectra. Last, nl and n2 are the refractive indices of air and the sample, respectively, and a is the angle between the laser beam and the sample surface. The most commonly used internal standard bands are those arising from inorganic salts such as the sulfate band at 981 cm- 1, the nitrate band at 1050 cm -1, the perchlorate band at 930 cm-1, and the phosphate bands at 880, 990, and 1070 cm -1. Some of the Raman cross sections of these standards have been determined throughout the visible, near-UV, and farUV (184 nm) regions. 33,71'72 Because the Raman bands of the internal standards are not resonance enhanced, the typical concentrations required are high and lie in the range from 0.1 to 0.5 M. The choice of the internal standard is governed by its chemical inactivity toward the biological sample under study. Spectral Processing. Spectral processing of the raw data can greatly enhance the quality of the resultant spectra and allow the maximum interpretability. Procedures commonly carried out include smoothing spectra to improve the signal-to-noise ratio (S/N), correcting the baseline to eliminate sloping backgrounds arising from either stray light or sample fluorescence, removing Raman line broadening caused by instrumental functions, and deconvoluting congested spectra into individual components for quantitative analysis. 73-78 There are many modern analysis techniques and computer software programs available to facilitate these procedures. Their success usually depends on how much is already known about the source of data corruption (noise and distortion) and the algorithm on which the software is based. Caution should be paid to the limitations of each spectral processing technique so that unreasonable interpretations are not applied to overprocessed experimental data.

Interpretation of Raman Spectra: Selected Examples In this section, three examples have been chosen to illustrate how the Raman and resonance Raman techniques have been successfully applied 71 j. M. Dudik, C. R. Johnson, and S. A. Asher, J. Phys. Chem. 89, 3805 (1985). 72 S. A. Fodors, R. A. Copeland. C. A. Grygon, and T. G. Spiro, J. Am. Chem. Soc. U l , 5509 (1989). 73 A. Savitsky and M. J. E. Golay, Anal. Chem. 36, 1627 (1964). 74 j. Steinier, Y. Termonia, and G. S. Deltour, Anal. Chem. 44, 1906 (1972). 75 S. Kawata and S. Minami, Appl. Spectrosc. 38, 49 (1984). 76 S. A. Dyer and D. S. Hardin, Appl. Spectrosc. 39, 655 (1985). 77 S. F. Gull and G. G. Daniell, Nature (London) 272, 686 (1978). 78 R. K. Bryan, M. Bansal, W. Folkard, C. Nave, and D. A. Marvin, Proc. Natl. Acad. Sci. U.S.A. 80, 4728 (1980).

354

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

p

+

o =

~o .......

O z

0

I 0 i

+ 0

--•/+[,•1•

(D

°

.......0 ~ ( . ~ _ _ 0 _ _

I

....... O ~

/ '0 O

¢~

[-.

o

I o

_g~

0

I

I

b o o

__i

\

°

~(~ ~

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

355

to metalloproteins. Rather than focus on the scientific issues relevant to the study of each metalloprotein, we emphasize interpretation of the Raman spectra to obtain structural information.

Preresonance Raman Spectroscopy of Carboxypeptidase A Carboxypeptidase A (CPA) is a zinc hydrolase that cleaves the peptide bond to hydrophobic side chains of carboxyl-terminal residues of peptides and proteins. 79'8° The enzyme will also hydrolyze the structurally analogous ester substrates. The active site of the enzyme contains the zinc atom, which acts as a Lewis acid by coordinating the carbonyl group of the substrate and activating it toward nucleophilic attack. Active site residues include Arg-145, which serves as a cationic recognition site for the a-carboxyl group of the substrate, and Tyr-248, whose OH group is in close enough proximity to the scissile amide bond of the substrate to donate a proton to the amine fragment formed during catalysis. The other critical residue is Glu-270, which has been postulated to have two different roles in catalysis (Fig. 10). 81 In mechanism I, Glu-270 acts as a general base to assist the attack of the substrate carbonyl group by water. In mechanism II, Glu-270 directly attacks the carbonyl group to form a mixed acid anhydride intermediate that is subsequently hydrolyzed. Britt and Peticolas have used a Raman microscope to obtain Raman spectra from the interior of a single crystal of CPA while reacting with the chromophoric ester substrate L-/3-phenyllactic acid p-dimethylaminobenzoate (DABPLA). 82 There is a preresonance enhancement of the substrate scattering with 514.5 nm excitation that enables certain of its bands to be distinguished from those of enzyme, particularly in the 1700-1800 c m - 1region. Spectra obtained during the reverse reaction [i.e., on addition of/3-phenyllactic acid (PLA) and p-dimethylaminobenzoic acid (DABA)] and of the enzyme in the absence of products are shown in Fig. 11, together with a difference spectrum. The four bands observed in the difference spectrum between 1750 and 1800 cm-1 have been assigned to an anhydride 79 B. L. Vallee, A. Galdes, D. S. Auld, and J. F. Riordan, in "Zinc Enzymes" (T. G. Spiro, ed.), p. 26. Wiley, New York, 1983. 8o j. E. Coleman and B. L. Vallee, J. Biol. Chem. 235, 390 (1960). 81 W. N. Lipscomb, Chem. Soc. Rev. 1, 319 (1972). 82 B. M. Britt and W. L. Peticolas, J. Am. Chem. Soc. 114, 5295 (1992).

FIG. 10. Alternate mechanisms of reaction for carboxypeptidase A in which Glu-270 acts as a general base to facilitate the nucleophilic attack of water (mechanism I) or as a nucleophile to attack the substrate directly to form an anhydride intermediate (mechanism II).

356

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

C

N

1200

1400

1600

1800

R a m a n Shift, cm 1

FIG. 11. Preresonance Raman spectra of carboxypeptidase A crystals soaked for 48 hr in a 20 mM buffered solution of p-dimethylaminobenzoic acid (DABA) and B-phenyllactic acid (PLA) (spectrum B) and of the same enzyme crystals in mother liquor (spectrum A). Spectrum C is a difference spectrum (B - A). The excitmion wavelength was 514.5 nm. (Adapted, with permission, from Ref. 82. Copyright 1992 American Chemical Society.)

intermediate, since only organic acid anhydrides (and not the esters or acids) give carbonyl R a m a n bands with frequencies in this range. The o b s e r v a t i o n of this anhydride intermediate constitutes evidence that DABP L A is h y d r o l y z e d b y CPA according to m e c h a n i s m II of Fig. 10. The 1769 and 1796 cm-~ bands h a v e b e e n attributed to the u n c o m p l e x e d anhydride intermediate and the 1753 and 1784 cm-1 bands to the Zn(II)-complexed anhydride intermediate. Support for these assignments c o m e s f r o m the data in Fig. 12, which show the p r e r e s o n a n c e R a m a n spectra of the mixed anhydride of D A B A and acetic acid in the presence and absence of zinc. 82 The u n c o m p l e x e d anhydride exhibits bands at 1758 and 1795 cm i, whereas the zinc c o m p l e x has bands at 1765 and 1787 cm -1. This study exemplifies the ability of the R a m a n technique to provide insights into m e t a l l o e n z y m e reaction mechanisms.

Resonance Raman Studies of Metalloproteins In 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 , one sacrifices scope for selectivity and studies vibrational m o d e s arising from the motions of only those atoms that are located in the immediate vicinity of the excited c h r o m o p h o r e .

[141

357

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

B

.=

800

10'00

12{)0

14f00

1600

'

1800

Raman Shift, cm -1 FIG. 12. Raman spectra of the mixed anhydride between p-dimethylaminobenzoic acid and acetic acid (0.1 M) in CDC13 in the presence (B) and absence (A) of ZnC12 obtained with 514.5 nm excitation. (Adapted, with permission, from Ref. 82. Copyright 1992 American Chemical Society.)

Following a brief introduction to heme frequency-structure correlations, the use of resonance Raman spectroscopy to obtain structural information on several heme-containing hydroperoxidases is presented. This is followed by a discussion of the application of the resonance Raman technique to the nonheme iron protein hemerythrin. Heine Group Frequency-Structure Correlations. The structure of the heme group is shown at the top of Fig. 13, where the C a, Cb, and C m atoms of the porphyrin are defined. Also shown is the absorption spectrum of cytochrome c,S3 which is representative of the spectra of a wide variety of heme enzymes. The spectrum is dominated by ¢r-Tr* transitions of the porphyrin ring with an intense Soret or B band near 400 nm and a weaker Q0 or a band near 500 nm. There is also a vibronic side band referred to as the Q1 or/3 band. The heme group contains an iron atom coordinated to the four pyrrole nitrogen atoms of protoporphyrin IX. The iron atom can also bind one axial ligand on each side of the porphyrin ring. The porphyrin macrocycle is a highly symmetric (D4hif the peripheral groups are considered as point masses) and conjugated aromatic system. The porphyrin core consists of 37 atoms, for which there are 105 normal 83 R. E. Dickerson and R. Timkovich, in "The Enzymes" (P. D. Boyer, ed.), 2nd ed. Vol. 11, p. 397. Academic Press, New York, 1975.

358

SPECTROSCOPIC METHODS FOR METALLOPROTEINS Ligand

/

[141

\\

B O~

150'

_.100

ferri-cyt c

ferro-cyt c

/1!t

° Oo

50

0 200

300

400

500

600

Wavelength, nm FIG. 13. Structure of the heme group shown complexed to two axial ligands (top). Typical ultraviolet/visible absorption spectra of a heme protein are illustrated (bottom) by cytochrome c in its reduced (dashed line) and oxidized (solid line) forms. (The absorption spectra were adapted, with permission from Ref. 83.)

modes of vibrations, of which 71 are in-plane and 34 are out-of-plane modes. There are 51 Raman-active and 54 infrared-active modes, and, owing to the center of symmetry of the heme group, their activities are mutually exclusive. The most intense bands in the resonance Raman spectra of metalloporphyrins are the skeletal stretching bands of the porphyrin. Excitation within the B band gives rise to resonance enhancement of totally symmetric modes, such as b,2,/-~3, and v4 (nomenclature of Abe and co-workers 84) that produce polarized bands via A-term enhancement [Eq. (13)]. Excitation within the Q band gives rise to nontotally symmetric modes, such as vl0, vn, and v~9, that produce depolarized or anomalously polarized bands via B-term resonance enhancement [Eq. (14)]. 84 M. Abe, T. Kitagawa, and Y. Kyogoku, J. Chem. Phys. 69, 4526 (1978).

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

359

T A B L E IV HEME SKELETAL STRETCHING MODES AND SENSITIVITIES TO HEME STRUCTURAL AND ELECTRONIC PERTURBATIONS a

Mode composition (predominant coordinates)

Response to porphyrin oxidation

Response to core expansion

Response to iron oxidation

2Aau

2AZu

~ --

---

Mode

Polarization

Vl0 1'19

dp ap

v(Ca-Cm) ~'(Ca-Cm)

~ ~

---

~2

p

v(Cb-Cb)

$

--

1'

$

Vll

dp

U3

P

P4

P

V(Cb-Cb) ~'(Ca-Cm) v(Ca-N) + v(Ca-Cb)

$ ~, --

--i'

1" ~ i

--$

a U p and down arrows represent the directions of frequency shift in response to perturbation.

The major internal coordinate contributions to each of these six modes are listed in Table IV. The frequencies of these and other vibrational modes of the heme group are sensitive to the electronic structure and ligation state of the central metal ion and the oxidation state of the porphyfin ring. The coordination number, spin, and oxidation state of the iron atom affect the core size of the porphyrin, which is defined by the distance between nonadjacent pyrrole nitrogen atoms.85,86 Increasing the size of the core weakens the porphyrin bonds, with the most striking and systematic effects observed for the porphyrin skeletal stretching modes that lie above 1350 cm -~. The frequencies of these modes have been found to show an inverse linear dependence on the core size of porphyrin, where the order of core sizes is 87'88 [6c, Is, Fe 3+ ] < [6c, ls, Fe z+ ] < [5c, hs, Fe 3+] < [6c, hs, Fe 3+ and 5c, hs, Fe z+ ] < [6c, hs, Fe z+ ] Here, c indicates coordination, and ls and hs represent low and high spin, respectively. Superimposed on this core size effect are perturbations arising from doming of the porphyrin in five-coordinate heroes, back donation of d= electrons from the iron atom to the porphyrin in low-spin ferrous 85 T. Kitagawa, M. Abe, and H. Ogoshi, J. Chem. Phys. 69, 4516 (1978). 86 S. Choi, T. G. Spiro, K. C. L a n g r y , K. M. Smith, L. D. Budd, and G. N. L a Mar, J. Am. Chem. Soc. 104, 4345 (1982). 87 T. Kitagawa, Y. Ozaki, and Y. K y o g o k u , Adv. Biophys. U , 153 (1978). 88 L. D. Spaulding, C. C. Chang, N.-T. Yu, and R. H. Felton, J. Am. Chem. Soc. 97, 2517 (1975).

360

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

hemes, and effects caused by oxidation of the heme ring. When these complicating effects are not present, the frequencies of the core size marker bands can be used to assess the coordination number and spin state of the heme species in a straightforward manner. Another informative band is the so-called oxidation state marker band 0'4) that is observed near 1370 cm- 1. Its frequency is sensitive to forward ~r donation from the porphyrin to the metal, giving rise to a correlation with the oxidation state of the iron a t o m . 89'90 It is found near 1380, 1375, and 1360 cm-1 for ferryl, ferric, and ferrous hemes, respectively. For lowspin ferrous heme complexes, this band shifts up toward the ferric value when the axial ligands are good ~- acceptors (e.g., O2, CO, NO). 91 The frequencies of the heme group are also markedly perturbed by oxidation of the porphyrin ring. One-electron oxidation produces a ~--cation radical formed formally by removal of an electron from either of the nearly degenerate aau or azu orbitals of the porphyrin to give the 2Alu or 2A2uground state configurations, respectively. To a first approximation, the effects of removal of an electron from an alu versus a2u orbital of a metalloporphyrin on a particular skeletal stretching mode can be understood by considering the molecular orbital phasing diagrams for the two orbitals in conjunction with the internal coordinates that are the major contributors to each mode. The directions of the shifts in vibrational frequencies predicted on formation of 2Alu and 2A2u ~r-cation radicals agree well with the values actually observed for a variety of four- and fivecoordinate metalloporphyrins (Table IV) and are diagnostic toward distinguishing between the formation of a predominantly 2Alu v e r s u s 2Azu radical. In particular, mode v2 shows large opposite shifts for these two species. Important structural information can also be obtained from the analysis of metal-ligand bands. 92'93 Many metal-ligand stretching, bending, and internal ligand modes have been observed for heine proteins. One can obtain force constants, bond lengths, bond angles, and bond strengths from an analysis of these modes. Owing to the heavy mass of the iron atom, most of these modes for the heme group are located in the lowfrequency region from about 180 cm-~ for large and heavy ligands, such as Fe(II)(Im)2 (Im, imidazole), to as high as 800 cm-1 for doubly bonded 89 p. V. Huong and J.-G. Pommier, C.R. Acad. Sci. Ser. C285, 519 (1977). 90 T. Yamamoto, G. Palmer, D. Gill, I. T. Salmeen, and L. Ramai, J. Biol. Chem. 248, 5211 (1973). 91 W. R. Scheidt and C. A. Reed, Chem. Rev. 81, 543 (1981). 92 S. Han, Y. Ching, and D. L. Rousseau, Nature (London) 348, 89 (1990). 93 N.-T. Yu and E. A. Kerr, in "Biological Applications of Raman Spectroscopy" (T. G. Spiro, ed.), Vol. 3, p. 39. Wiley, New York, 1988.

[14]

RAMAN AND RESONANCERAMAN SPECTROSCOPY

361

Peroxidase Cycles Hz0 z

[(HRP)Fe(III)__]

L

H20

/

[(HRP)Fe(IV)=O] '+

=

Ferri-HRP, 5c, hs

Compound I, 6c, ls

/<

~ ~"AH[(HRP)Fe(IV)=O] / ~ AH.

H20~

Compound 1I, 6C, ls AH2

20 [(CCP)Fe(IV)=O] P r + Compound I (ES), 6e, Is Ferro-eyt e

[(CCP)Fe(III)__] Ferri-CCP, 5c, hs

~

Ferri-Cyt e Ferro-Cyt e

Ferri-Cyt e

[(CCP)Fe(IV)=O] Compound II, 6e, ls

Catalytic Cycle l

~02+H20 Hz0e

[(BLC)Fe(III)__] Ferri-CAT, 5c, hs

L

He0

/

=-

' ~---H eOa- + [(BLC)Fe(IV)=O] Compound I, 6c, Is

Fro. 14. Catalytic cycles for horseradish peroxidase (HRP), cytochrome-c peroxidase (CCP), and bovine liver catalase (BLC). [(XXX)Fe(III)__] represents a five-coordinate resting enzyme with a vacant sixth site. Pr" ÷ represents a protein radical.

362

SPECTROSCOPIC METHODS FOR METALLOPROTEINS V4

H4]

V~

o

~

1300

1400

15'00

m~

N

16~00

1700

R a m a n Shift, c m 1 FIG. 15. Comparison of the resonance Raman spectra of resting HRP, HRP compound I, and HRP compound II species obtained with 413.1 nm excitation. (Adapted, with permission, from Ref. 96.)

ligands such as Fe(IV)=O. These bands are usually weakly enhanced through the various mechanisms discussed previously and are often obscured by the numerous in-plane and out-of-plane porphyrin modes. Nevertheless, it has proved possible to assign a number of them through the use of isotopic substitutions. 9z'93 Studies of Specific Hydroperoxidases. The hydroperoxidases are a class of ferric hemoproteins that catalyze the oxidation of various substrates by H202. Members of this family include peroxidases such as horseradish peroxidase (HRP), which oxidizes a variety of organic substrates, and cytochrome-c peroxidase (CCP), which oxidizes ferrocytochrome c. Also included in this family are catalases, such as bovine liver catalase (BLC), where the substrate oxidized is a second molecule of H 2 0 2 94,95 All three of these enzymes are five-coordinate high spin in the 94 G. R. Schonbaum and B. Chance, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed. Vol. 13, p. 363. Academic Press, New York, 1976. 9s I. Yamazaki, in "Molecular Mechanisms of Oxygen Activation" (O. Hayaishi, ed.), p. 535. Academic Press, New York, 1974.

[14]

363

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

~w~2 V3 Resting Slate

J

-r,

~

~

I

L__, A

C°mp°und I

II

o

:

~

,,'v'-

.=

i

Compound II

1300

1400

:

t

t

i

!

i

[

1500 Raman

1600

1700

Shift, cml

FIG. 16. Comparison of the resonance Raman spectra of resting BLC, BLC compound I, and BLC compound II species obtained with 413.1 nm excitation. (Adapted, with permission, from Ref. 96.)

resting state. HRP and CCP both have proximal histidine ligands (His170 in HRP and His-175 in CCP), whereas BLC has a proximal tyrosine ligand (Tyr-357). The peroxidases and catalases have similar catalytic cycles, as shown in Fig. 14. Ferri-HRP is oxidized by H202 to form a species called compound I, which is two oxidizing equivalents above the resting enzyme. One electron is removed from the iron atom and one oxygen atom of H202 is captured to form an oxoferryl species, F e ( I V ) = O , while the second electron is abstracted from the porphyrin ring to form a porphyrin 7r-cation

364

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

~ F e l

I

H ASh

I 2 0 5 c m "~

~Fe

I

~

I 233 em a

~Fe

I

~

I 246 cm "~

250

450 Raman

Shift,

650 c m -1

FIG. 17. R e s o n a n c e R a m a n spectra of the low-frequency region of native ferro-CCP (bottom) and the Ash-235 m u t a n t (top), showing the v ( F e - H i s ) band that is sensitive to the extent of h y d r o g e n bonding of the ligated His to a residue (Asp-235) on the protein (left). (Adapted, with permission, from Ref. 98).

radical that is thought to have the 2A2uground state. The one-electron reduction of compound I by substrate (AH2) produces a species called compound II. It has a normal porphyrin spectrum, implying that the added electron has gone into the porphyrin orbital that gave rise to the radical. Finally, compound II is reduced back to ferri-HRP by reaction with a second molecule of substrate. Ferri-CCP also reacts with H202to form a compound I species (sometimes called compound ES) that is isoelectronic with compound I of HRP but has different characteristics. Like HRP, the iron atom is oxidized by one electron to form an Fe(IV)~---O species. However, the second electron is removed from an amino acid side chain on the protein (Pr) to form a protein radical (Pr'+). Thus, CCP compound I has an optical spectrum like that of HRP compound II. It reacts with one molecule of ferrocytochrome

[141

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

365

H

\ H

i

779

crn -i

0

_.!!v t

His

.=

H

788 c m -x 0

_!!v 6 Raman Shift, cm-1 FZG. 18. Resonance Raman spectra of the low-frequency region of compound II of HRP lsoenzymes B and C excited at 406.7 nm. The changes in frequency of the v(FelV=O) band indicate that there is hydrogen bonding to a distal His (left). (Adapted, with permission, from Ref. 102).

c to form its own compound II and then with a second molecule of ferrocytochrome c to regenerate ferri-CCP. The reaction of ferri-BLC with H20 2 also produces a compound I species that is two oxidizing equivalents above the resting enzyme. As with HRP, BLC compound I is thought to be a heme 7r-cation radical species containing an oxoferryl group, but the porphyrin 7r-radical was thought to have the 2Alu ground state configuration. In the normal catalactic cycle, compound I oxidizes a second molecule of H 2 0 2 to form molecular oxygen (with both oxygen atoms of dioxygen coming from the second peroxide) and is itself reduced back to the native ferric enzyme. The resonance Raman spectra of the resting forms of HRP and BLC excited at 413.1 nm are shown as the top curves in Figs. 15 and 16, 96 96 W.-J. Chuang and H. E. Van Wart, J. Biol. Chem. 267, 13293 (1992).

366

[14]

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

~

..~1376

e~

o~

1300

1400

1500

1600

1700

Raman Shift, cm-' Fro. 19. R e s o n a n c e R a m a n spectra of H R P c o m p o u n d I excited with 406.7 n m in a microdroplet stream with a residence time of 1.5 /~sec at powers of 1, 3, and 20 roW. T h e b o t t o m curve is the s p e c t r u m of c o m p o u n d II. (Adapted, with permission, from Ref. 96.)

respectively. The oxidation marker band,/24, is observed near 1374 cm-1 for both enzymes, confirming that they exist in the ferric oxidation state. The frequencies of the core size marker bands/22 and v3 both fall into the range expected for five-coordinate, high-spin s p e c i e s . 97 97 N. Parthasarathi, C. H a n s o n , S. Yamaguchi, and T. G. Spiro, J. Am. Chem. Soc. 109, 3865 (1987).

[14]

367

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

~a~'l'5

?~

AsPI06~ O?~xsqq~O-H t

%

\o /oI A

02

/

'o,

\ /

0

OH

B

C

D

FIG. 20. Structure of the binuclear iron center in deoxy- and oxyhemerythrin as suggested by the X-ray structure of azidomethemerythrin 107(top). (A-D) Four possible binding configurations of a peroxide ligand to the hemerythrin binuclear iron center (bottom).

The nature of the His ligation and proximal interactions in CCP have been revealed by resonance Raman studies of native and mutant CCP species. The peroxidases have a stronger Fe(III)-proximal His bond than found in myoglobin or hemoglobin owing to a modulation of the proximal His-electron distribution via strong hydrogen bonding to another protein residue. Thus, u(Fe-His) bands are observed at 233 and 246 cm -l for ferro-CCP (Fig. 17) compared with 220 cm-1 for deoxyhemoglobin. The bands at 233 and 246 cm -1 are assigned to protein species in which the proximal His-8 proton is strongly hydrogen bonded to and transferred completely to the adjacent Asp-235 residue, respectively. These interactions make the proximal His residue a stronger ligand and increase the frequencies of the v(Fe-His) band. No analogous proton-accepting residue is present in deoxyhemoglobin. This interpretation is supported by the resonance Raman spectrum of the Asn-235 mutant, for which the u(Fe-His) band shifts down to 205 cm -1. A similar dependence of v(Fe-His) on the hydrogen-bonding state of a His residue has been ob-

368

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

I

[14]

t

80001

6000

t-Hr

4000'

2000-

0

300

~,,.Doo~-.r ~

400

"~

560

600

Wavelength, nm Fro. 21. Optical spectra of apo-, deoxy-, oxy-, and methemerythrin. (Adapted, with permission, from Ref. 110.)

served for the model Fe(II) protoheme 2-methylimidazole complex. Here, the ~,(Fe-His) frequency shifts from 205 cm-1 in benzene to 220 cm-1 in water and to 239 cm i when the imidazole is deprotonated. 9s Resonance Raman spectra of HRP and BLC compound II species have been well studied 99-~°j and are shown in Figs. 15 and 16, respectively. The upshifts in the oxidation state marker band P4 by 2 and 5 cm-~ for BLC and HRP, respectively, indicate that the iron atom has been oxidized to Fe(IV). The core size marker bands v2 and v3 are both up-shifted into the range expected for six-coordinate low-spin heme species. 1°2"1°3The observation of an oxoferryl band, u(Fe~-O), near 779 cm-1 establishes that the sixth ligand is an oxo atom (Fig. 18). Interestingly, the frequency 98 T. G. Spiro, G. Smulevich, and C. Su, Biochemistry 29, 4497 (1990). 99 W. H. Woodruff and T. G. Spiro, Appl. Spectrosc. 28, 576 (1974). 100j. Terner and D. E. Reed, Biochim. Biophys. Acta 789, 80 (1984). i01 S. Hashimoto, H. Teraoka, T. Inubushi, T. Yonetani, and T. Kitagawa, Y. Biol. Chem. 261, 11110 (1986). I02 A. J. Sitter, C. M. Reczek, and J. Terner, J. Biol. Chem. 260, 7515 (1985). I03 W.-J. Chuang, J. Heldt, and H. E. Van Wart, J. Biol. Chem. 264, 14209 (1989).

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

369

8

(~178

300

400

500

O2-IIr

600

~

700

~

800

900

Raman Shift, cm -1 FIG. 22. Resonance Raman spectra of oxyhemerythrin obtained with 1602 and tsO2 using 488.0 nm excitation. Plasma lines from the laser are identified by the letter p. (Adapted, with permission, from Ref. 110.)

of the v ( F e : O ) band in both enzymes is pH dependent because of a hydrogen bond formed between the oxo atom and a proton of a distal His residue with a pK near 8. When the distal His is protonated, this hydrogen bond lowers the v(Fem-O) frequency. These data show that the structure and distal environment of the heme group in the compound II species of BLC and HRP are very similar. 1°2,1°3 The observation of the authentic resonance Raman spectra of HRP and BLC compound I species has been a challenging task because of their extreme photolability.56,1°4-1°6 The key to observing the correct spectra of these species has been to use low laser power, to reduce the residence time in the laser beam, and to examine the samples in a microdroplet stream. 5s'96'1°6 A set of resonance Raman spectra of HRP compound I measured with a 1.5 t~sec residence time with variable laser powers is shown in Fig. 19. Only when the power is reduced to 1 mW is the true spectrum of compound I acquired. 96,1°6 The same holds true for BLC compound I. The best resonance Raman spectra of the HRP and BLC 104j. Teraoka, T. Ogura, and T. Kitagawa, J. A m . Chem. Soc. 104, 7354 (1982). t05 W. A. Oertling and G. T. Babcock, J. A m . Chem. Soc. 107, 3379 (1985). 106 K.-J. Paeng and J. R. Kincaid, J. A m . Chem. Soc. 110, 7913 (1988).

370

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

I

780

I,

I

8OO

82O

I ~ 840

860

Raman Shift, cm1 FIG. 23. Resonance Raman spectra of oxyhemerythrin obtained with a mixture of 1602, 1802, and ~60180 using 514.5 nm excitation. (Adapted, with permission, from Ref. 114. Copyright 1976 American Chemical Society.)

compound I species are shown together with that of the native enzymes and compound II species in Figs. 15 and 16, respectively. 96,1°6The shifts in porphyrin skeletal modes ~'2or v3 compared to compound II are negative and establish that both HRP and BLC compound I species have predominant 2A2u ground state character (Table IV). Hemerythrin. Resonance Raman spectroscopy can also provide detailed structural information on nonheme iron-containing proteins such as hemerythrin (Hr). Myoglobin and hemoglobin are vertebrate proteins that utilize a heme group to bind, transport, and store oxygen. Hemerythrin is an invertebrate nonheme respiratory protein that uses a binuclear iron center (Fig. 20) 1°7 to carry out these same functions. 1°8 The absorption 107R. E. Stenkamp, L. C. Sieker, and L. H. Jensen, J. A m . Chem. Soc. 106, 618 (1984). ~08N. B. Terwilliger, R. C. TerwiUiger, and R. Schabtach, in "Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology" (W. D. Cohen, ed.), p. 193. Alan R. Liss, New York, 1985.

[14]

371

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

486 981 n~'o 753 475-

;0

I

410

5~0

730

890

1050

R a m a n Shift, cm a Fro. 24. Resonance Raman spectra of oxyhemerythrin obtained in H2160 (top) and H2180 (bottom) using 363.8 nm excitation. (Adapted, with permission, from Ref. 116. Copyright 1984 American Chemical Society.)

spectra of four forms of hemerythrin are shown in Fig. 21. ApoHr and deoxyHr do not have any distinct absorption bands above 300 nm. However, oxyHr and metHr (an oxidized and inactive form 1°9) exhibit several distinct bands in the 300-600 nm region that are due to iron-ligand charge transfer. 11° For oxyHr, excitation within these bands gives rise to resonance Raman spectra that are valuable in elucidating the nature of the bonding between oxygen and the binuclear iron center. The resonance Raman spectra of 16OzHr and 1802Hr obtained with 488.0 nm excitation are shown in Fig. 22. The spectrum of 16OzHr exhibits bands at 500 and 844 cm-1 that are shifted to 478 and 798 cm -1, respectively, for 1802Hr. These bands are due to the u(Fe-O2) and u(O-O) stretching modes, respectively, of the oxygen-binuclear iron corn1o9I. M. Klotz and D. M. Kurtz, Jr., Acc. Chem. Res. 17, 16 (1984). 11oj. B. R. Dunn, D. F. Shriver, and I. M. Klotz, Proc. Natl. Acad. Sci. U.S.A. 70, 2582 (1973).

372

SPECTROSCOPIC METHODS FOR METALLOPROTEINS

[14]

7.0

5.o ~ 3.0 X 1.0

I

I

/',,,

2.0 a

I

I

• 4 8 6 cm'l

I

.-rr~,fla'e-O-~-)sym

~

1.0

m '~

I

0.0 2.0

~

I

" l l l - ~

•

844 cm

(~o-o)

•

503

(VFe.Oz)

cm

--

--

I

1.0

0.0

I

400

500

Wavelength,

600

nm

FtG. 25. Optical spectrum (top) and Raman excitation profiles of selected modes of oxyhemerythrin (middle and bottom). The uI band of sulfate was used as an internal standard for the intensity measurements. (Adapted, with permission, from Ref. 116. Copyright 1984 American Chemical Society.)

The frequency of 844 cm 1 for the v(O-O) band indicates that the bound dioxygen has an electron distribution similar to that of a peroxide. This establishes that, although oxyHr is formally a ferrous-oxygen complex, it can be regarded more accurately as a ferric-peroxide complex. The peroxide ligand can be bonded to the binuclear iron center in several possible geometries (Fig. 20, bottom). Configuration A has the two oxygen atoms bridging the two iron atoms, structure B has a single p l e x . 109-113

111 L. Vaska, Acc. Chem. Res. 9, 175 (1976). 112 j. B. R. Dunn, D. F. Shriver, and I. M. Klotz, Biochemistry 14, 2689 (1975). 113 S. M. Freier, L. L. Duff, D. F. Shriver, and I. M. Klotz, Arch. Biochem. Biophys. 205, 449 (1980).

[14]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

373

oxygen atom bridging the two iron atoms, structure C has the peroxy group bonded to a single iron atom through a single oxygen atom, and structure D has both peroxy oxygen atoms bonded to a single iron. One can discriminate between the possible structures by examining the resonance Raman spectrum of oxyHr prepared from unsymmetrically labeled oxygen gas (160180). 114New v(O-O) bands are observed at 825 and 818 cm-1 (Fig. 23). This establishes that the two oxygen atoms of the peroxide in oxyHr are inequivalent. Thus, only structures B and C are consistent with the resonance Raman data. X-Ray crystal studies of oxyHr rule out structure B, H5 leading to the conclusion that oxyHr has unsymmetrical configuration C. In addition to the modes associated with the bound peroxide ligand in oxyHr, bands associated with the oxybridge can be observed at 486 and 753 cm -1 with near-ultraviolet excitation (363.8 nm) (Fig. 24). ll6 These two bands are assigned to the symmetric and asymmetric stretching modes of the F e - O - F e unit and are shifted to 475 and 720 cm -~, respectively, on 180 substitution. Excitation profiles for the v(Fe-O2), v(O-O), and Vsym(Fe-O-Fe) bands have been acquired 116 and demonstrate different enhancement patterns (Fig. 25). The v(O-O) mode is enhanced only by excitation in the green, the Vsym(Fe-O-Fe) mode only by excitation in the near-ultraviolet, and the v(Fe-O2) mode by excitation in both regions. Although these differences in the coupling of individual vibrational modes to the different electronic transitions of the oxo-bridged binuclear iron-peroxo center are not yet fully understood, they provide a potential means to sort out these interactions that is not offered by the optical spectra. Acknowledgments The authors thank Professor Warner L. Peticolas for providinga preprint of the work cited above. This work was supported by a research grant from the National Institutes of Health (GM 27276).

114 D. M. Kurtz, Jr., D. F. Shriver, and I. M. Klotz, J. A m . Chem. Soc. 98, 5033 (1976). 115R. E. Stenkamp, L. C. Sieker, L. H. Jensen, J. D. McCallum, and J. Sanders-Loehr, Proc. Natl. Acad. Sci. U.S.A. 82, 713 (1985). 116 A. K. Shiemke, T. M. Loehr, and J. Sanders-Loehr, J. A m . Chem. Soc. 106, 4951 (1984).