Near-infrared excitation of Raman scattering by chromophoric proteins

SpectrochimicaACM, Vol. 47A, No. 9/10, pp. 1413-1421.1991 F’rinted in Great Britain

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Near-infrared excitation of Raman scattering by cemophoric

proteins

CAREY K. JOHNSON* Department of Chemistry, University of Kansas, Lawrence, KS 66045, U.S.A.

and RONALD

RUBINOVITZ-~

Bruker Instruments, Inc., Manning Park, 19 Fortune Dr., Biller&

MA 01821, U.S.A.

(Received 18 February 1991; in revised form and accepted 3 April 1991) Ah&act-Fourier-transformed Raman spectra of bacteriorhodopsin, the photosynthetic reaction center, and myoglobin in aqueous solution excited at 1064 nm are presented. These proteins are representative of three important classes of chromophoric proteins. The observed vibrational modes are assigned and discussed based on the known resonance Raman spectra of these proteins. In each case, chromophore vibrations dominate the Raman scattering, with little or no contribution from other protein vibrations. However, the limitations encountered in resonance Raman studies of chromophoric proteins due to sample fluorescence or sample photolability are circumvented. The relative intensities in the bacteriorhodopsin Raman spectrum excited at 1064 nm are nearly identical to the relative intensities previously observed by resonance excitation. The Raman spectrum of the reaction center of the photosynthetic bacterium Rhodobacter sphaeroides excited at 1064 mn contains contributions from both bacteriochlorophyll and bacteriopheophytin pigments, with possible preresonance enhancement of bacteriochlorophyll modes. The lWnm-excited Raman spectrum of myoglobin displays several marker bands that have been characterized previously in resonance Raman investigations with excitation in both the Soret and Q-band regions.

INTR~DUC~ON THE CLASS of proteins containing visible pigments comprises some of the most fascinat-

ing of biological molecules: proteins responsible for trapping light energy, for converting electronic excitation into chemical potential, and proteins with specialized prosthetic groups. Although proteins pose a stiff challenge to the vibrational spectroscopist, for reasons that include the large number of vibrational modes, sample availability, and the need in many cases to work with aqueous samples, chromophoric proteins have been particularly susceptible to Raman vibrational studies by excitation in resonance with the visible absorption bands of the pigments [l, 21. This method has been highly successful because it specifically enhances vibrations of the chromophores, often precisely the region of the protein in which one is most interested. Nevertheless, the application of resonance Raman spectroscopy to chromophoric proteins is often limited by two constraints: sample fluorescence and sample photolability. Fluorescence-either intrinsic fluorescence of the protein, or, in cases where the fluorescence quantum efficiency of the chromophores is low due to efficient ultrafast processes,7 fluorescence from impurities or breakdown products-may overwhelm the Raman signal and preclude successful generation of resonance Raman spectra. Photolability, a consequence of the high photochemical quantum efficiency typically encountered in photoactive proteins, places constraints on the laser power (cw or pulsed) that can be employed for Raman excitation. This limitation is particularly severe with I pulsed lasers, for example in time-resolved applications. In recent years the realization has grown that these limitations can be avoided with excitation of Raman scattering by a near-infrared (NIR) laser beam [3-51. Combined with Fourier transform (FT) spectroscopic methods, NIR excitation has proven to be a * Author to whom correspondence should be addressed. t Current address: Spectra-Tech, Inc., Applied Systems Division, 200 Harry S. Truman Parkway, Annapolis, MD 21401, U.S.A. Editor’s note: further work on bacteriochlorophyll will be found elsewhere in this Special Issue (Ref. [41]). 1413

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K. JOHNSON and R. RUBINOVIT~

very powerful technique indeed [6]. Several recent papers have emphasized in particular

the application of FT-Raman spectroscopy to biological materials [5,7-91. However, several potential difficulties and limitations must be addressed. Biological samples are often most easily handled in aqueous solution, where overtone vibrations of water absorb in the NIR [8]. Another concern regards the importance of contributions of nonresonant Raman scattering from the polypeptide chain and amino-acid side chains relative to Raman scattering from visible chromophores. Recently, we reported the FT-Raman spectra of two chromophoric proteins, bacteriorhodopsin (BR) and the photosynthetic reaction center (RC), both in aqueous solution excited at 1064 nm [5]. Interestingly, the FT-Raman spectra are dominated in both cases by non-resonant Raman scattering by the protein-bound pigments retinal (in BR) and bacteriopheophytin (BPh) and bacteriochlorophyll (BCh) (in RCs). Hence, we concluded that FT-Raman spectroscopy with NIR excitation represents a potent probe of vibrational modes of chromophores in proteins, circumventing the photolability and fluorescence drawbacks of resonant excitation. In this paper, we present a more complete analysis of the FT-Raman spectra of BR and the photosynthetic RC, and we report the ET-Raman spectrum of the heme protein myoglobin (Mb).

EXPERIMENTAL Fourier-transformed Raman spectra were measured with a Bruker Instruments IFS 66 FMR spectrometer coupled to an FRA-106 FT-Raman accessory. Raman scattering was excited by OS1.3 W of multimode cw 1064 nm radiation, and collected in a backscattering geometry. Double-sided interferograms were acquired at lOcm-’ resolution, and were transformed to the corresponding power spectrum to eliminate phase error. The FT-Raman spectrum of water was also measured and was subtracted from each protein Raman spectrum with a subtraction coefficient adjusted to null out the water bending mode at 1645 cm-i. Bacteriorhodopsin was prepared from cultures of Halobacterium halobium according to standard techniques [lo]. The sample was light-adapted by light from a 14W lamp for 45 min before Raman analysis. Samples of the photosynthetic RCs were generously donated by Prof. Robert Blankenship or were isolated from Rhodobacter sphaeroides at the University of Kansas and prepared according to published procedures [ 111. Myoglobin (from horse skeletal muscle) was purchased from Sigma Chemical Co. and purified by preparative isoelectric focusing on a flat bed of Sephadex gel inoculated with a pH gradient of 7-9. After isoelectric focusing, the darkest band was cut from the gel and eluted with buffer. The eluent was dialyzed against buffer solution to remove the ampholines and subsequently dialyzed against distilled water. Before use, a small amount of sodium dithionite was added to reduce Mb to its deoxygenated form.

RESULTS AND DISCUSSION Bacteriorhodopsin

The FT-Raman spectrum of BR is shown in Fig. 1. This spectrum was discussed previously in some detail [5]; the most striking feature is its strong resemblance to the resonance Raman spectrum of BR [12]. Almost without exception, every band observed between 700 cm-’ and 1700 cm-’ was observed under resonant excitation, and has been assigned to vibrational modes of the retinal Schiff base chromophore [ 131. Furthermore, the relative intensities of the bands with 1064 nm excitation match closely those observed with resonant excitation [12] or 752 nm pre-resonant excitation [14]. One exception, the 798 cm-’ band, was noted [5]. A strong band at 800 cm-’ is observed in dark-adapted BR [12]. Hence, the intensity in this mode, as well as a shoulder at 1179 cm-‘, may result from incomplete light adaptation in this sample. A similar resemblance between resonance Raman and 1064 nm PI’-Raman spectra has been noted for all-trans retinal [7]. FT-Raman spectra of BR (both light- and dark-adapted) excited at 1064 nm, as well those of rhodopsin and phycocyanin, were reported recently by SAWATZKI et al. [15]. Their results are essentially in agreement with ours. A comparison of the spectra of light-

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Fig. 1. FT-Raman spectrum of bacteriorhodopsin, excited with 1.3 W at 1064 nm with 10 cm-’ resolution. A total of 1500 scans was co-added and the Fl’-Raman spectrum of water was subtracted. (A) 100-1000 cm-’ region; (B) MO-1700 cm-’ region.

and dark-adapted BR supports our assignment of the 798 cm-’ band to dark-adapted BR. We attributed [S] the dominance of the 1064nm IT-Raman spectrum by pigment vibrational modes and the strong similarity between the non-resonance PT-Raman spectrum and resonance Raman spectra to pre-resonance Raman enhancement [16]. In this effect, enhancement is related to the leading terms in an expansion of the Condon and vibronic contributions (ALBRECHT’S A and B + C terms [ 171, respectively) in powers of the detuning from resonance [16]. This enhancement is evidently strong enough at 1064 nm to completely dominate the PI-Raman spectrum. An examination of the leading term in the expansion for the Condon and vibronic contributions shows that whereas the first term in the expansion for the Condon contribution depends inversely on the square of the detuning, the first term in the expansion for the vibronic contribution is inversely proportional to the detuning. This suggests dominance of the vibronic (B term) contribution with 1064 nm excitation. A similar argument was put forward by SAWATZKI and coworkers 1151, who noted that the A and B term relative intensities may be similar in BR because of the large geometry change in the excited state. Photosynthetic reaction centers Since the determination of the crystallographic structure of RCs of the photosynthetic bacteria R. viridis [18] and R. sphaeroides [19,20] interest in the spectroscopic properties of these proteins has intensified [21-231. The RC contains several chromophores: two bacteriochlorophyll (BCh) molecules form a dimer known as the primary donor or special pair (P860 in R. sphaeroides); two monomeric BCh pigments are adjacent to the special pair; and two bacteriopheophytin (BPh) molecules serve as intermediate electron

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acceptors. A carotenoid pigment is also present. While transient absorption experiments are capable of sensitive measurements of the electron transfer rates involved, several outstanding questions relating to structure and electron transfer mechanism in RCs require vibrational information to be answered. A number of resonance Raman studies of bacterial RCs have been reported with resonant excitation in the Soret and QXbands of BCh or BPh [24]. Resonance Raman excitation in the QYbands is a tantalizing alternative [25], since the NIR QYabsorption bands are about four times as intense as the QX bands. However, fluorescence has hampered Q,-band excitation, with the notable exception of the work of BOCIAN and co-workers [25,26]. Non- or pre-resonant excitation at longer wavelengths thus represents a potentially attractive alternative. Our report of the FT-Raman spectrum of RCs with 1064 nm excitation showed that detailed Raman spectra can be generated with nonresonant excitation [5]. Hence, the FI method with 1064 nm excitation represents a new approach to the Raman spectroscopy of RCs, complementing resonance Raman methods. The FT-Raman spectra of photosynthetic RCs is shown in Fig. 2. This spectrum appears to be dominated by Raman scattering by the visible chromophores BPh and BCh, although a shoulder at about 1650 cm-’ may be attributable to the amide I vibration of the protein. The prominent bands observed in the FT-Raman spectrum are presented in Table 1. As a result of the continuing efforts of a number of researchers [2428] the Raman spectra of RC pigments are increasingly well understood. The assignments in Table 1 are based on this body of work, and are intended to demonstrate the

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Fig. 2. FT-Raman spectrum of photosynthetic reaction centers of R. sphueroidv, excited with 1.0 W at 1064 nm with 10 cm-’ resolution. A total of 1500 scans was co-added and the FT-Raman spectrum of water was subtracted. (A) 20O-llOOc11-~ region; (B) 900_1800cm-’ region.

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Table 1. Prominent vibrational modes of the reaction center of R. sphaeroides observed by FT-Raman spectroscopy with excitation at 1064 nm Excitation*

Mode (cn-‘) 251 334 361 474 563 582 617 669 727 893 922 945 1013 1133 1309 1348 1407 1497 1522 1571 1597

BCh, BPh BCh, BPh BCh BPh BCh, BPh BCh, BPh BPh BPh BCh, BPh BCh BCh, BPh BcH, BPh BCh BCh, BPh BPh BPh BPh BPh BCh, BPh BPh BPh

S, Qz S, Q.

s,ci QX

S,Q,, S, Qx

IR

Qxv S,Qx,

IR IR

ii,

IR IR

Q,, s,

IR Q, IR

EQY

* Modes were previously observed with excitation in band indicated (Refs [25, 27291). S=Soret; IR denotes excitation at 1064 nm.

dominance of the FT-Raman spectrum by vibrational modes of BCh and BPh by comparing the vibrational frequencies observed here with those observed previously by resonance Raman excitation. Since the frequencies of BPh and BCh modes are typically quite similar [27], it is not possible in many cases to distinguish between BPh and BCh contributions to the observed modes. Modes which involve the Mg in BCh are an exception. Several of these have been identified by LUTZ [27] in the low-frequency region of the spectrum, including modes observed in Fig. 2 at 214 and 291 cm-‘. A number of low-frequency modes are clearly discernible in the 200-500 cm-’ region. These modes are potentially of interest because low-frequency vibrations are likely to be involved in any intramolecular structural reorganization accompanying electron transfer. Unlike the BR spectrum, the NIR-excited non-resonant Raman spectrum of RCs in Fig. 2 does not closely resemble the resonance Raman spectra of RCs. Notably absent, for example, are strong bands observed with Soret excitation [27] at 1612, 1287 and 1068 cm-‘. Similarly, modes observed under 753 nm Q,, excitation of BPh [25] at 1726, 1690,1054 and 984 cm-’ are either weak or absent. Recently, the FT-Raman spectrum of BCh a with 1064 mu excitation was reported [29]. This spectrum includes strong modes at 1518, 1358, 1137, 1014, 892 and 73Ocmy’, each of which agrees within a few cm-’ with frequencies observed in Fig. 2. MA~IOLI et al. [29] also report having observed 730,900, 1010, 1130 and 1350cm-’ Raman bands from RCs, although the spectrum was not published. These are among the stronger bands observed in our spectrum. Hence, it may be that pre-resonance enhancement of BCh modes of the special pair generates some of the stronger contributions to the FT spectrum. However, other modes (e.g. at 1571,1497 and 1309 cm-‘) are not accounted for by BCh scattering. One of the most striking features of the FT-Raman spectrum of RCs is the broad band

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Fig. 3. FT-Raman spectrum of deoxy-myglobin excited with 0.5 W at 1064nm with lOcmresolution. A total of 2160 scans was co-added and the FT-Raman spectrum of water was subtracted. (A) 200-1100 cm-’ region; (B) !900-1800 cm-’ region.

covering the 1300-1700 cm-’ region. It may be that this band is caused by congestion due to contributions from the six pigments in the RC. In support of this possibility is the broad feature observed by BOCIAN and co-workers [25] between 1500 and 1750 cm-’ by resonance excitation of BPh at 753 nm. Although that spectrum shows less congestion, perhaps due in part to the higher resolution employed (3 cm-‘), the additional scattering due to the four BCh pigments in the RC may lead to the severe spectral congestion in Fig. 2. Another possibility is that this band may represent luminescence from an as-yet-uncharacterized absorbing state present in the RC. Although no absorption has been observed at 1064 nm, luminescence from quinone-depleted RCs of R. sphaeroides has been observed in the 7500-7800 cm-’ region following excitation of the RC at 532 nm [30]. (Q uinone depletion allows efficient population of 3P860 by visible excitation of the RC.) The emission was peaked at 7600 cm-‘, and assigned to the triplet state of P860. Phosphorescence from BCh a was also observed following excitation at 605 nm, with peak emission at about 8200 cm-’ [30]. It may be that irradiation excites the Ti state of P860 or BCh a directly, albeit with extremely low efficiency, resulting in phosphorescence in the 7700-8100 cm-’ region corresponding to Raman shifts of 1300-1700 cm-‘. We also cannot exclude the possibility of emission from P860+, which may be present in our sample. Myoglobin The heme proteins have been among the most thoroughly studied chromophoric proteins by resonance Raman spectroscopy [31-331. This is due in part to the extremely

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Table 2. Vibrational modes of deoxy-myoglobin observed with 1064 nm excitation Mode (cm-‘) 225 247 341 464 530 578 628 674 750 940 loo0 1114 1126 1209 1317 1353 1385 1448 1521 1544 1558 1616 1652

Assignment* Fe-histidine stretch Pyrrole tilt vs Pyrrole fold (?) Pyrrole fold (?) v4lI v1 VI6 v46 v45 v44 v6+% lrg + V18

v,,IG(CH=) v, (oxidation state marker) vm (?) vB (core size marker) vB (core size marker) vI1 (core size marker) v&, (core size marker) v&inyl C=C (vIO:core size marker) Protein amide I

*Modes are labeled following ABE et al. [37]. Assignments are based on the assignments of SPIROand co-workers [34,38].

low fluorescence quantum yield of the heme group. Resonance Raman studies have been particularly useful in identifying marker modes which have been shown to be related to specific structural parameters. These include the oxidation state marker, v~, which dominates the resonance Raman spectrum excited in the Soret region. The frequency of this band is sensitive to the oxidation state of Fe [34]. A series of bands from 1400 to 1650 Cm-‘, including Ye, y3, v3s, vll, v2, v19, v37, and vlo have been found to be sensitive to the porphyrin core size [35]. Some of these modes are enhanced by Soret (B-band) excitation (e.g. v2, v3), while others are enhanced by Q-band excitation (vii, vi9, via). The latter have been highly useful in steady-state and time-resolved studies of heme structure [31,36]. Other modes are sensitive to heme-protein interactions. Especially significant among these has been the iron-histidine stretching mode, vF&s [36]. Mode v8 has been found to be sensitive to peripheral groups of the heme [37]. Both are enhanced by Soret excitation. The FT-Raman spectrum of deoxy-myoglobin excited at 1064 nm is shown in Fig. 3. The major modes observed are listed in Table 2. Assignments follow the extensive analyses reported by CHOI and SPIRO [38] and the normal coordinate labels of ABE et al. [37]. As in the spectra of bacteriorhodopsin and photosynthetic RCs, the visible chromophore clearly dominates the NIR ET-Raman spectrum. Nearly all of the marker modes mentioned above are present in the FT spectrum. In particular, the iron-histidine stretch at 225 cm-‘, the oxidation-state marker at 1353 cm-‘, and the core-size marker bands vu at 1544 cm-’ and vul at 1448 cm-’ are prominent. In order to study these by resonance Raman, experiments with excitation in both the Soret and in the Q-band regions would be necessary. The band at 1652 cm-’ may be the amide I vibration. (A similar assignment was made for the FT-Raman spectrum of cytochrome c [39].) Hence, ET-Raman spectroscopy with NIR excitation is potentially important in its ability to monitor these important modes with a single excitation wavelength,

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CONCLUDINGREMARKS The three examples discussed in this paper demonstrate the potential of the FT-Raman method with NIR excitation for the study of chromophoric proteins in aqueous solution. In each case, vibrational modes of the visible chromophore dominate the Raman scattering. Although PI’-Raman scattering by modes of the polypeptide backbone has been observed [9,40], such modes are not abundant in the spectra reported here. In one protein, BR, we find a pre-resonant Raman spectrum nearly identical to the resonance Raman spectrum, while avoiding the constraints due to sample photolability which can severely complicate resonance Raman studies of BR and similar systems. In another case, the photosynthetic RC, Raman spectra of electron-transfer donor and acceptor pigments are obtained without background fluorescence due to sample impurities or breakdown products. Finally, in myoglobin, several important structural marker bands are present in the same Raman spectrum. The chromophoric proteins reported here represent a large group of proteins. Other related chromophoric proteins, such as rhodopsin, chlorophyll antenna complexes, and cytochromes, will almost certainly be profitably studied by IT-Raman spectroscopy. We conclude that NIR excitation of Raman scattering offers significant advantages for structural and perhaps time-resolved studies of chromophoric proteins. Note added in proof: Since submission of this manuscript, MA’ITIOLI et al. have published the NIR FT-Raman spectrum of oxidized and reduced reaction centers [T. A. Mattioli, A. Hoffmann, B. Robert, B. Schrader, and M. Lutz, Biochemistry 30, 4648 (1991)]. Bands at 669, 1306, 1348, 1407, 1500, and 16OOcm-’ appear in the FI-Raman spectrum of oxidized reaction centers. The modes at or near these frequencies in Fig. 2 may therefore be due to P860+. Acknowledgements-We

thank SARAH MOUNTER and the staff of the Biochemical Research Service Laboratory at the University of Kansas for sample preparation. C.K.J. acknowledges helpful discussions with R. E. BLANKENSHIP,J. NORRIS, and G. J. SMALL.C.K.J. gratefully acknowledges support for this research from the USDA (88-37234-3404) and NIH (GM 40071).

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