Metal Clusters in Proteins - American Chemical Society

Chapter 3

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Resonance Raman Spectroscopy of Iron—Oxo and Iron—Sulfur Clusters in Proteins Joann Sanders-Loehr Department of Chemical and Biological Sciences, Oregon Graduate Center, Beaverton, OR 97006-1999 Resonance Raman studies of Fe- and Cu-containing proteins have led to the identification of tyro sine, h i s t i d i n e , cysteine, and hydroxide ligands as well as Fe-O and Fe-S c l u s t e r s . For the Fe-O c l u s ters, the frequency and oxygen isotope dependence of the Fe-O-Fe symmetric stretch relates to Fe-O-Fe bond angle, while the peak intensity relates to the disposition of the other ligands i n the c l u s t e r . For the Fe-S proteins, the frequencies and sulfur isotope dependence of the Fe-S v i b r a t i o n a l modes can be used to d i s t i n g u i s h mononuclear, binuclear, and tetranuclear c l u s t e r s . Hydrogen bonding of both Fe-O and Fe-S clusters can be detected by frequency shifts i n deuterium-substituted proteins. In resonance Raman spectroscopy, excitation within a metal-ligand charge transfer band leads to enhanced i n t e n s i t i e s of M-L and Internal ligand v i b r a t i o n a l modes, thereby allowing these modes to be s e l e c t i v e l y observed i n metalloproteins. Because v i b r a t i o n a l frequencies are sensitive to bond strength and coordination envi ronment, this technique provides information which Is complementary to that obtained by x-ray crystallography or x-ray absorption spec troscopy. This chapter w i l l discuss the general p r i n c i p l e s of resonance Raman spectroscopy as they apply to metal centers In proteins and then focus s p e c i f i c a l l y on binuclear and tetranuclear iron clusters with oxo or sulfido bridging groups. Raman Spectroscopy Raman spectroscopy i s a form of v i b r a t i o n a l spectroscopy which, l i k e infrared spectroscopy, i s sensitive to transitions between different v i b r a t i o n a l energy l e v e l s i n a molecule (1). It differs from infrared spectroscopy In that information i s derived from a l i g h t scattering rather than a d i r e c t absorption process. Further more, different selection rules govern the Intensity of the respec tive v i b r a t i o n a l modes. Infrared absorptions are observed for v i b r a t i o n a l modes which change the permanent dipole moment of the 0097-6156/88/0372-0049$06.00/0 • 1988 American Chemical Society

In Metal Clusters in Proteins; Que, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

METAL CLUSTERS IN PROTEINS


50

molecule while Raman scattering i s associated with normal modes which produce a change in the p o l a r i z a b i l i t y (or induced dipole moment) of the molecule. Thus, symmetric stretching modes tend to be the most intense features i n Raman spectra, whereas asymmetric stretches and deformation modes tend to be more intense in infrared spectra. This d i f f e r e n t i a l character makes Raman spectroscopy more favorable for the study of b i o l o g i c a l materials since there i s considerably less spectral interference from the Intra- and intermolecular deformation modes of water molecules; these are dominant features in the Infrared spectra of aqueous samples. The predominant type of l i g h t scattering which occurs when radiation impinges upon atoms i n a sample i s known as Rayleigh scattering. The scattered l i g h t appears equally i n a l l directions and i s of the same frequency as the incident l i g h t . The Raman effect relates to the fact that a small f r a c t i o n of the scattered radiation may have undergone a change i n frequency (2). This can be explained as the r e s u l t of an i n e l a s t i c c o l l i s i o n with an incoming photon (hv ) such that some of i t s energy (hv ) i s trans ferred to a v i b r a t i o n a l mode of the molecule. The energy of the Stokes Raman scattered photon (TZVR) i s , thus, equal to hv - hv (Figure l a ) . Since Raman scattering Is 1000-fold weaker than Rayleigh scattering, i t s detection requires the use of an Intense monochromatic l i g h t source, a high-resolution monochromator, and a highly e f f i c i e n t photodetector (4). In practice, v i s i b l e or uv laser l i n e s provide the best input radiation and the Instrument Is calibrated for the p a r t i c u l a r v being used. The scattered photons of varying frequencies (VR) are resolved by a scanning monochroma tor (Figure 2) or a diode array instrument, and their i n t e n s i t i e s are plotted as a function of frequency, v = v - v . Use of computer-related c a p a b i l i t i e s such as multiple scanning, signal averaging, background subtraction, and data smoothing leads to s i g n i f i c a n t enhancements In spectral quality (5). Raman spectra obtained on concentrated protein solutions (ca. 20 mM) are dominated by contributions from the repeating amide group of the polypeptide backbone and from the sidechains of aromatic amino acids (6). Phenylalanine and tryptophan exhibit p a r t i c u l a r l y intense ring breathing modes at 1006 and 1014 cm" , respectively. An additional strong peak due to tryptophan i s t y p i c a l l y observed a t 760 cm" . Q

v

Q

v

Q

v

Q

R

1

1

Resonance Raman Spectroscopy Resonance-enhanced Raman scattering occurs when the energy of the Incident r a d i a t i o n , hv i s close to or within an electronic absorption band of the sample (7,8). In this case, vibronic coupling with the e l e c t r o n i c a l l y excited state increases the probability of observing Raman scattering (TZVRR) from v i b r a t i o n a l transitions In the electronic ground state (Figure l b ) . The intensity of such resonance-enhanced v i b r a t i o n a l transitions can be described i n simplified terms as: Q9

2

(1)


3.

SANDERS-LOEHR

Resonance Raman Spectroscopy

51


V

q

q

(a)

(b)

Figure 1. Diagrams of potential energy, V, versus Internuclear separation, q, for a molecule undergoing v i b r a t i o n a l excitation by (a) the Raman effect or (b) a resonance Raman e f f e c t (hv *h\> ) or a pre-resonance e f f e c t (hv approaches v , the frequency of the m to e electronic t r a n s i tion; the damping term, V prevents the denominator from becoming zero. Thus, while v i b r a t i o n a l frequencies i n resonance Raman spectroscopy are s t i l l a function of the electronic ground state, v i b r a t i o n a l i n t e n s i t i e s are determined by the properties of the electronic excited state (8,9). The exact features of molecular and electronic structure which give r i s e to the resonance Raman effect are not well understood. I t i s believed that those vibrations whose normal modes most c l o s e l y approximate the nuclear displacements In the electronic excited state are the ones which are the most susceptible to resonance enhancement (10). Hence, the nature of the electronic t r a n s i t i o n becomes an Important factor i n determining the extent of Franck-Condon overlap between the v i b r a t i o n a l wave functions i n the ground and excited electronic states. In considering metal-based chromophores, the two most common electronic transitions are metalcentered ligand f i e l d (LF) transitions and metal-ligand charge transfer (CT) t r a n s i t i o n s . Since the vibrations associated with the metal center generally involve either M-L or Internal ligand modes, they are more l i k e l y to e x h i b i t resonance-enhanced Intensi ties i n conjunction with CT transitions which r e d i s t r i b u t e electron density along M-L coordinates. Q

v

e


e>

Sample Requirements. While ordinary Raman spectroscopy requires a scatterer concentration of 100 mM or higher, metalloprotein resonance Raman spectra are t y p i c a l l y obtained i n the 1 mM concen tration range. For metal-ligand CT transitions having extinction coefficients of 2,000-4,000 M~ cm" , this corresponds to sample absorbances of 2 to 4 ( I . e . , samples which are strongly colored). For frozen samples In which the laser l i g h t is scattered only off the surface of the sample ( ~ 1 5 0 ° backscattering geometry), two to four times higher concentrations are needed. The sharply focused nature of the Incident laser beam means that a volume of 10-20 y l i t e r s is s u f f i c i e n t for s t a t i c samples, but larger volumes must be used for rotating or flowing samples. Since metalloprotein samples are highly absorbing (as well as highly scattering), spectral contributions from amide and sidechain vibrations are suppressed. However, the strongly scattering modes of tryptophan and phenylalanine near 750 and 1000 cm" , respectively, can often s t i l l be detected i n addition to the vibrations of the metal chromophore. The use of an Intense laser l i g h t source with b i o l o g i c a l materials i s accompanied by the concomitant problems of l o c a l i z e d sample heating and the p o s s i b i l i t y of protein denaturation. A further complication introduced by resonance Raman spectroscopy is the Increased potential for photochemical destruction of chromophoric metal centers as a r e s u l t of the absorption of large amounts of incident r a d i a t i o n . Both of these situations may be ameliorated by freezing samples to l i q u i d nitrogen temperature (~90 K ) , while the even lower temperatures made possible with a closed-cycle 1

1

1


3. SANDERS-LOEHR

53


helium r e f r i g e r a t o r (10-20 K) y i e l d even greater sample s t a b i l i t y . Lowered sample temperatures have the additional advantage of offering s i g n i f i c a n t Improvements in spectral resolution and signal to noise r a t i o s (11). Various designs for low temperature work include cold-finger Dewars capable of cooling samples i n c a p i l l a r i e s or devices i n which the sample i s frozen d i r e c t l y onto a cold finger (3,4). Since i n the l a t t e r case the sample is gen e r a l l y farther removed from any glass windows, such devices can r e s u l t i n less interference from the broad glass band in the 430500 cm" region. However, frozen samples do e x h i b i t a strong peak at ~230 cm" and a weaker feature at ~310 cm" which are due to ice translational vibrations and must be c a r e f u l l y distinguished from sample vibrations (12). Concentrated buffer solutions can also contribute Raman l i n e s , and i t i s wise to run a dialysate or u l t r a f i l t r a t e from the protein sample as a c o n t r o l . Preparation of the sample as a frozen glass through the addition of glycerol or ethylene glycol does not improve spectral quality and introduces excessive d e t a i l from the solvent. 1


1

1

I d e n t i f i c a t i o n of Vibrational Modes. Ligands containing C=C and C=N bonds exhibit Raman peaks In the 1000-1600 cm" region. These are often present i n a c h a r a c t e r i s t i c pattern of frequency and Intensity which can be i d e n t i f i e d by reference to the Raman spectra of model compounds. Metal-ligand stretching vibrations occur In a narrower range of frequencies, 200-600 cm" , and are considerably harder to assign because d i f f e r e n t ligands can give r i s e to M-L modes of similar energy and because internal ligand vibrations can occur in this energy range, as w e l l . Where possible, i t i s wise to try to v e r i f y an assignment by atom or Isotope substitution. Vibrational frequencies, v, are dependent on the masses of the v i b r a t i n g atoms and the strength of the bond force constant, k> according the harmonic o s c i l l a t o r relationship: 1

1

(2)

where u is the reduced mass: m m2/(m + m ). The inverse depen dence on mass predicts a decrease i n frequency for an increase In mass. Atom substitutions ( e . g . , Se for S; Cu for Fe) a l t e r force constants and coordination geometry as well as mass, while isotopic substitutions ( e . g . , 0-18 for 0-16; Cu-65 for Cu-63) mainly a f f e c t the reduced mass without s i g n i f i c a n t l y a l t e r i n g the force constant or the coordination environment. Thus, although isotope-dependent s h i f t s are more d i f f i c u l t to detect, they are considerably easier to interpret p a r t i c u l a r l y when used in conjunction with normal coordinate analysis c a l c u l a t i o n s . The most common ligands found at metal centers i n metalloproteins are the imidazole group of h i s t i d i n e , the thiolate group of cysteine, the phenolate group of tyrosine, the carboxylate groups of aspartic and glutamic acids, solvent-derived or exchange able groups ( e . g . , aquo, hydroxo, oxo, s u l f i d o ) , and the tetrapyrrole macrocycle of porphyrins. The porphyrin chromophores, by virtue of their extended conjugated TT systems, have very d i s t i n c tive o p t i c a l and resonance Raman spectra (13). The only amino acid moiety which has a s u f f i c i e n t l y c h a r a c t e r i s t i c resonance Raman 1

1

2


METAL CLUSTERS IN PROTEINS

54

spectrum to be immediately i d e n t i f i e d as a metal ligand i s tyrosine. E x c i t a t i o n within the phenolate Fe CT band results in the selective enhancement of phenolate ring vibrations (Figure 3, Table I ) . Although imidazole also has c h a r a c t e r i s t i c ring modes i n the 1000-1400 cm" region, they are only weakly resonance-enhanced and have not yet been detected in metalloprotein spectra. The location of the imidazole -> Cu CT bands i n the uv region, which i s more d i f f i c u l t to probe experimentally, may be partly responsible for this d i f f i c u l t y . However, resonance-enhanced metal-histidine stretching modes have been observed i n the 220-290 cm" region. These undergo 1 to 2 cm" s h i f t s to lower energy when the proteins are incubated i n D20. Although a pure M-N v i b r a t i o n should occur closer to 400 cm" (1), the Imidazole ring behaves as a r i g i d moiety with an effective mass of 67 (including hydrogens) and, thus, the M-N(imidazole) vibrations are found at lower energy. Substitution of the Nl and, less frequently, the C2 hydrogens of imidazole by deuterium lowers the effective mass to 66 or 65, thereby accounting for the observed s h i f t s (15). I t should be noted, however, that hydrogen bonding of l i g a t i n g oxygen or sulfur atoms to the polypeptide backbone can cause s i m i l a r shifts of 1 to 2 cm" i n D20 (21). Thus, deuterium isotope effects do not unequivocally signify h i s t i d i n e l i g a t i o n . Hydroxo ligands produce resonance-enhanced Fe-OH stretching vibrations in the 550-600 cm" region (Table I ) . These have been v e r i f i e d by observing a ~25 cm" s h i f t to lower energy in 18-0labeled water and a somewhat lesser s h i f t in D20 (22). The aquo group, another potential source of an M-0 stretch between 300 and 500 cm" (1), has not yet been detected i n resonance Raman spectra, even in such examples as Cu-substituted alcohol dehydrogenase where a water molecule i s believed to be metal-coordinated (12). The M-0 stretch of metal carboxylates which occurs i n the 300-400 cm" region (Table I) has also not yet been observed in metalloprotein spectra. However, a peak at 530 cm" i n methemerythrin (Table I) has been tentatively assigned to a carboxylate internal deformation which undergoes "intensity borrowing" by virtue of i t s proximity to a strongly enhanced Fe-L mode a t 510 cm" . The cysteine-ligated metal centers y i e l d more complicated resonance Raman spectra, i n part due to the coupling of different v i b r a t i o n s . The Fe-4Cys protein, rubredoxin, has a series of four Fe-S stretching modes between 314 and 376 cm" (Table I) a r i s i n g from symmetric and asymmetric vibrations of the four sulfur ligands, with contributions from S-C-C (cysteine) deformations (19). S i m i l a r l y , the 2Fe-2S-4Cys and 4Fe-4S-4Cys ferredoxins exhibit multiple Fe-S stretching modes of predominately cysteinate character from 310-370 cm" and from 360-390 cm" , respectively (23, 24). In rubredoxin the C-S stretch of the cysteinate ligand i s assigned at 653 cm" , while Fe-S-Fe and S-Fe-S deformation modes account for the weaker peaks i n the region between 130 and 200 cm" (19). The blue copper proteins such as azurin (Table I) with their unusually short Cu-S(Cys) bonds of ~2.1 A offer a s t r i k i n g con t r a s t . The Cu-cysteinate chromophore gives r i s e to a number of intense features i n the 370-430 cm" range whose higher frequency i s commensurate with a stronger Cu-S bond, but whose multiple bands are surprising i n view of the presence of only a single cysteinate ligand. I t i s l i k e l y that these additional peaks represent admix tures of internal ligand motions (such as the S-C-C bend) with the 1

1


1

1

1

1

1

1

1

1

1

1

1

1

1

1

1


3.

SANDERS-LOEHR

55


C-S stretch, a coupling phenomenon f a c i l i t a t e d by the distorted geometry of the blue copper s i t e (20, 25). A v i b r a t i o n near 750 cm" i s assigned to the Cu-S stretch of cysteine. Both i r o n - s u l f u r and the blue copper proteins exhibit an additional set of bands above 600 cm" which are due to overtones and combinations of the fundamentals In the 300-450 cm" region. 1

1

1


Raman Spectra of Metal Clusters The p r i n c i p a l types of metal clusters which have been characterized in metalloproteins are binuclear copper, Iron-oxo, and Iron-sulfur clusters. In each case the multiple metal ions appear to be con nected by one or more bridging ligands which mediate strong a n t i ferromagnetic Interactions between the metal centers. The clusters containing oxo (0 ") or sulfido (S ") bridges also have intense absorption bands a r i s i n g from 0 -> Fe or S Fe charge transfer which f a c i l i t a t e the observation of resonance-enhanced M-0 and M-S vibrations. Conversion of an oxo bridge to a hydroxo bridge by protonation leads to a weaker chromophore which i s less l i k e l y to produce a resonance Raman spectrum. Although the bridging ligand In a binuclear copper s i t e has yet to be i d e n t i f i e d , x-ray c r y s t a l lographic studies of deoxyhemocyanin indicate that i t i s unlikely to be an endogenous amino acid group. An a t t r a c t i v e candidate would be a bridging hydroxide which would account for both the strong antiferromagnetic coupling in oxyhemocyanin (-J > 550 cm" ) and the f a i l u r e to observe any resonance Raman modes attributable to a Cu-L-Cu moiety (26). The structures of typical binuclear iron-oxo and i r o n - s u l f u r clusters are shown In Figure 4. The Iron-oxo c l u s t e r , as exempli fied by the respiratory protein hemerythrin (26, 29), contains octahedral iron atoms bridged by two protein carboxylates and a single, solvent-derived oxo group. The s i t e denoted L accepts exogenous ligands and i s the place where dioxygen binds in the b i o l o g i c a l l y active form of the protein. In contrast, Ironsulfur clusters have tetrahedral iron atoms with multiple sulfido bridging groups and tend to be coordinatively saturated, in keeping with their role as redox-active, electron transfer proteins (30). Common features of the Iron-oxo and i r o n - s u l f u r clusters are that they exhibit vibrational modes encompassing the entire Fe-O-Fe or Fe-S-Fe moiety and that these assignments can be r e a d i l y v e r i f i e d by substitution of i s o t o p i c a l l y labeled oxo or sulfido groups. 2

2

1

Iron-Oxo Clusters. The Fe-O-Fe moiety has three normal modes of vibration: a symmetric stretch ( v ) , Fe-Fe, an asymmetric stretch ( v ) , Fe->0->Fe, and a bending mode (6), Fe-O-Fe. A l l three have been observed In resonance Raman spectra, but the intense symmetric mode i s by far the easiest to detect (26, 33). A typical spectrum Is shown i n Figure 5 for oxyhemerythrin where the only three c l e a r l y v i s i b l e peaks are v a t 486 cm" , v at 753 cm" , and v of sulfate at 981 cm" (an internal frequency and intensity standard). The bridging oxo group in oxyhemerythrin does not exchange with solvent when the protein Is equilibrated in water containing 18-0, but w i l l exchange when the protein Is reduced to Its deoxygenated state. The l a t t e r , which Is believed to contain an Fe(II)-OH-Fe(II) cluster and i s c o l o r l e s s , cannot be probed by resonance Raman spectroscopy. However, after reconversion to s

a s

1

s

1

a

s

1

x



(373,

-CH -S---Cu

1

401, 409,

363,

428)

376

1140, 1330,

753

653

(530)

662

n.o.

1168, 1426, n.o.

1256, 1490

803, 869, 1164, 1281, 1497, 1597

v(Ligand)

2

2

Azurin

2,

Rubredoxin^

2

Methemerythrin^

2

3

Fe 0(Ac) (HBpz )

2

/

Methemerythrin(OH)

Oxyhemocyanin^

A

Cu(ImH) Cl

e

Purple acid phosphatase

Example

Vibrational Modes in Metalloproteins

d

e

V i b r a t i o n a l frequencies in cm"" . v(M-L) = metal-ligand stretching mode, v(Hgand) • internal ligand mode, n.o. - not observed. Values in parentheses are tentative assignments. ^Coupled v(Cu-S) +

Metal Clusters in Proteins - American Chemical Society

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