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[17] Nanosecond time-resolved resonance Raman spectroscopy
[17]
TIME-RESOLVED
RESONANCE RAMAN SPECTROSCOPY
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[17] N a n o s e c o n d T i m e - R e s o l v e d R e s o n a n c e Raman Spectroscopy B y CONSTANTINOS VAROTSIS a n d GERALD T . BABCOCK
Introduction Resonance Raman (RR) spectroscopy is a powerful technique to probe molecular vibrations that are coupled to electronic transitions. Monochromatic light, now universally obtained from continuous wave (CW) or pulsed lasers, is used to illuminate a sample, and the spectrum of scattered radiation is analyzed to determine vibrational information on molecular species within the sample. By bringing the laser frequency into resonance with an electronic transition of a species of interest, dramatic enhancements in scattered intensity result. The individual vibrational frequencies observed in a Raman spectrum arise from normal modes in the ground electronic state. The intensities of the Raman lines, however, reflect the character of the electronic excited states. Owing to the high selectivity and sensitivity in the enhancement of vibrational modes, resonance Raman spectroscopy offers the opportunity to probe chemical species such as reaction intermediates, excited electronic states, and chromophoric site(s) of biological systems. Biological chromophores such as heroes, flavins, chlorophylls, and a number of different types of metal-containing proteins have been investigated by resonance Raman spectroscopy. The static resonance Raman effect and biological applications of Raman spectroscopy have been the subject of numerous reports and reviews. 1-2° I A. C. Albrecht, J. Chem. Phys. 34, 1476 (1961). 2 S. Y. Lee and E. J. Heller, J. Chem. Phys. 71, 4777 (1979). 3 D. T. Tannor and E. J. Heller, J. Chem. Phys. 77, 202 (1982). 4 B. B. Johnson and W. L. Peticolas, Annu. Rev. Phys. Chem. 27, 465 (1976). 5 T. G. Spiro and P. Stein, Annu. Rev. Phys. Chem. 28, 501 (1977). 6 A. Warshel, Annu. Rev. Biophys. Bioeng. 6, 273 (1977). 7 W. Siebrand and M. Zgierski, in "Excited States" (E. C. Lim, ed.), p. 1. Academic Press, New York, 1979. s j. Tang and A. C. Albrecht, in "Raman Spectroscopy" (H. Szymanski, ed.), p. 2. Plenum, New York, 1970. 9 G. T. Babcock, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 295. Wiley, New York, 1988. s0 T. G. Spiro and X.-Y. Li, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 1. Wiley, New York, 1988. 1~ N.-T. Yu and E. A. Kerr, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 39. Wiley, New York, 1988.
METHODS IN ENZYMOLOGY, VOL. 226
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
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SPECTROSCOPIC METHODS FOR METALLOPROTEINS
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Time-resolved resonance Raman (TR 3) spectroscopy has been applied to measure conformational and structural changes as well as kinetic properties of transient species. Moreover, data from these studies can be used to identify the mechanistic routes of rapid photochemical and photobiological processes. Application of the TR 3 technique to chemical and biological processes of interest can be implemented either with CW lasers or with the use of a single laser pulse or of two laser pulses (Fig. 1). Various pulsed laser systems provide temporal resolution ranging from subpicosecond to several nanoseconds; CW lasers can be applied so as to approach 1/zsec time resolution. Continuous-wave lasers require high flow rates and tight beam focusing in order to achieve high time resolution. The residence time that the sample spends in the probe beam ultimately determines the shortest time range that can be explored. With a pulsed laser source in a one-pulse experiment, both the time delay and the time resolution are determined by the laser pulse duration. This is a fundamental limitation of the technique. In such a single-pulse experiment, photons at the leading edge of the pulse initiate the photochemistry that generates the photoproduct; photons nearer the trailing edge of the same pulse scatter from the newly created species to generate its Raman spectrum. The laser beam is usually tightly focused, so that a high photon density on the sample scattering volume, which is necessary to create the photoproduct and record its Raman spectrum, is produced. However, if high photon densities are required to initiate the reaction,
n T. Kitagawa, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 97. Wiley, New York, 1988. 13 D. L. Rousseau and J. M. Friedman, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 133. Wiley, New York, 1988. 14 p. M. Champion, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 249. Wiley, New York, 1988. 15 B. Cartling, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 217. Wiley, New York, 1988. 16 M. Lutz and B. Robert, in "Biological Applications of Raman Scattering" (T. G, Spiro, ed.), Vol. 3, p. 347. Wiley, New York, 1988. 17 W. H. Woodruff, R. B. Dyer, and J. R. Schoonover, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 413. Wiley, New York, 1988. 18T. M. Loehr and A. K. Shiemke, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 439. Wiley, New York, 1988. ~9L. Que, Jr., in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 491. Wiley, New York, 1988. 20 T. G. Spiro, R. S. Czernuszewics, and S. Han, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 523. Wiley, New York, 1988.
TIME-RESOLVED RESONANCERAMANSPECTROSCOPY
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1 Pulsed Excitation
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Fro. 1. Block diagram used to obtain time-resolved resonance Raman (TR)3 spectra.
multiple excitations of the sample that produce nonlinear processes may occur and contribute complex and artifactual spectral features. A two-pulse, pump-probe, time-resolved Raman approach allows not only the detection of transient intermediates involved in a dynamic process but also the measurement of their formation and decay kinetics. The first pulse, the pump, initiates the reaction or generates the excited state, and the second pulse, the probe, which is usually of a different frequency, produces the Raman scattering. The two-color, pump-probe configuration has been used extensively, since it offers the opportunity to record realtime kinetic measurements as well as to record the time evolution of transient species. In this review of experimental methods, we limit our discussion to time-resolved Raman techniques that provide time resolution in the nanosecond to second time range. Considerable activity is occurring with pico-
412
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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second and subpicosecond lasers that provide finer time resolution. The reader is referred to several reviews that cover these developments. 21-25 Methods and Equipment For time-resolved Raman spectroscopy, low repetition rate, highenergy pulsed excimer and neodymium-yttrium-aluminum-garnet (Nd : YAG) lasers are commonly used, often in conjunction with dye lasers or Raman shifters to extend the accessible wavelength range. In most time-resolved experiments, two pulsed lasers with pulse widths in the 1-10 nsec range and variable repetition rates are coupled together electronically to produce two pulses of different frequency. The pulsed technique is particularly useful, since the time resolution is determined by the programmable time delay between the short (10 nsec) pump and probe laser flashes (Fig. 1). Useful time-resolved work can also be done with CW laser systems. The continuous-wave approach requires one or two laser systems in conjunction with a continuous-flow apparatus (Fig. 1). In the single-beam (one-laser) experiment, the flow rate is varied so that the sample residence time in the laser-irradiated region is varied, allowing time-evolution studies to be made. This approach becomes ambiguous kinetically as the residence time increases. Furthermore, with a single beam at a low flow rate, the sample residence time in the laser is relatively long, and, therefore, artifacts induced by the incident laser beam may occur. In the double-beam CW experiment, the time resolution is determined by the flow rate, the cross-sectional area of the irradiated volume, and the distance between the two beam foci. Lasers
Resonance Raman enhancement of vibrational modes is observed when the f r e q u e n c y of the excitation laser beam is close to the frequency of an electric dipole-allowed transition of the molecule. Therefore, by appropriate choice of excitation frequency, selective enhancement of the zl G. H. Atkinson, in "Time-Resolved Vibrational Spectroscopy" (G. H. Atkinson, ed.), p. 179. Academic Press, New York, 1983. 22C. L. Hsieh, M. Nagumo, M. Nicol, and M. A. E1-Sayed,J. Phys. Chem. 85, 2714 (1981). 23C. L. Hsieh, M. A. E1-Sayed, M. Nagumo, and J.-H. Lee, Photoehem. Photobiol. 38, 83 (1983). .~4M. A. E1-Sayed,Pure Appl. Chem. 57, 187 (1985). 25G. H. Atkinson, T. L. Brack, D. Blanchard, G. Rumbles, and L. Siemankowski, in "'Ultrafast Phenomena V" (G. R. Fleming and A. E. Siegman, eds.), p. 409. (Springer Series, Chem. Phys. 46). Springer-Verlag, Berlin and Heidelberg, 1986.
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TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
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TABLE I CHARACTERISTICS OF WIDELY USED LASERS IN TIMERESOLVED RAMAN SPECTROSCOPY Lasing medium
Output
Wavelength (nm)
Nd:YAG Excimer
Pulsed, CW Pulsed
N2 Ar + Kr + He-Cd
Pulsed CW CW CW
1064, 532, 355,266 193 (ArF), 222 (KrC1), 248 (KrF), 308 (XeC1), 337 (Nz), 351 (XeF) 337 351,364, 488,514.5 406.7, 413,531,676 441,325
molecules that give rise to the electronic transition can be obtained. This resonance aspect of the Raman scattering process adds unique potential to the technique in resolving multichromophoric systems; flexibility in the choice of laser excitation frequency is essential in realizing this potential. Nanosecond, pulsed, and continuous wave lasers provide a unique tool for TR 3 spectroscopy. Available laser systems and suitable wavelengths that can be created to fulfill the demand of frequency selectivity and tunability have expanded the experimental capabilities in this field. Table I summarizes the common frequencies available from pulsed and continuous-wave lasers for time-resolved studies. Excimer and Nd : YAG lasers provide pulses of 7-10 nsec duration and millijoules of energy per pulse, with repetition rates of 10-100 Hz. The excimer wavelength range varies from 190 to 380 rim, depending on the gases used. The more commonly used excimer gases and their laser wavelengths are summarized in Table I. The direction laser light output from an excimer, however, is generally not suitable for Raman scattering. Consequently, this laser is used almost always as a pump source for a dye laser. Such an arrangement provides high-quality monochromatic light that can be tuned over a broad wavelength range that extends from the near-ultraviolet region through the visible and into the near-infrared. The N d : Y A G laser is considerably more versatile in time-resolved Raman experiments, as its 1064 nm fundamental can be used directly to excite a Raman spectrum. Moreover, because of its high power characteristics, frequency doubling and tripling crystals can be used to convert the YAG fundamental to 532 and 355 nm light, respectively; the fourth (266 nm) and the fifth (213 nm) harmonics can be used to generate light in the deep ultraviolet region of the spectrum. Wavelength shifting of the fundamental or the harmonics of the Nd : YAG laser can be achieved by
414
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
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TABLE II PRACTICAL EXCITATION WAVELENGTHS FROM H2, D 2, AND CH4 RAMAN SHIFTERS FOR TIME-RESOLVED RAMAN SPECTROSCOPY 1064 nm
Stokes (Sn) or Anti-Stokes (AS,) line
H2a
D2b
AS4 AS3 AS2 ASs
384.3 457.4 564.7 737.8
468.5 544.7 650.5 807.4
$1 $2 $3 $4
532 nm CH4c
656.8 812.2
355 nm
H2
D2
CH4
H2
D2
CH4
282.3 319.9 368.9 435.7 683.0
325.3 360.3 403.7 459.0 632.5 779.8
406.1 460.6 629.6 771.1
223.1 245.9 273.9 309.1 416.0 502.9 635.7 863.9
249.1 269.1 292.7 320.7 396.7 450.0 519.9 615.5
294.1 321.7 396.0 447.6 514.8 605.6
a 0Jvib, 4 1 5 5 c m -1 f o r H 2. b COvib, 2 9 8 7 c m - 1 f o r D 2 . c r.~vib' 2 9 1 4 c m -1 f o r C H 4.
focusing into a Raman shifter filled with different gases ( H 2 , D 2 , C H 4 ) to produce a wide range of wavelengths. Table II summarizes the wavelengths available from H 2 , D 2 , and C H 4 Raman shifters. Alternatively, the second or the third harmonic can be used to pump a pulsed-dye laser. The dye laser can generate tunable light between 380 and 800 nm. Finally, harmonic generation and nonlinear mixing techniques can be applied to the dye-laser output. For example, the doubled dye-laser output can be mixed with the 1064 nm Nd : YAG fundamental to produce tunable light in the UV spectral region. The doubling and mixing are accomplished by using angle-tuned crystals in either homebuilt or commercially available arrangements. As an example of how various pulsed-laser sources and wavelength-shifting devices can be teamed with a detection system, Fig. 2 shows the experimental arrangement at the shared LASER Laboratory, Michigan State University (East Lansing, MI), for TR 3 studies of biological molecules and metalloporphyrins. There are a variety of continuous-wave lasers. In time-resolved Raman experiments, H e - C d , argon, and krypton lasers are the most widely used sources. The argon and the krypton lasers are used extensively for pumping CW dye lasers and mode-locked dye lasers. A Ti:sapphire laser pumped by an Ar + laser has been used for Raman experiments in the deep-red spectral region. Thus, with the CW technique, as with the pulsed systems described above, there is now the ability to obtain relatively intense, monochromatic radiation throughout the UV, visible, and nearinfrared spectral regions.
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
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FIG. 2. I n s t r u m e n t a l configuration u s e d for pulsed T R 3 m e a s u r e m e n t s .
Monochromators
In TR 3 as well as in normal resonance Raman spectroscopy, the signal intensity is weak, compared to the intensity of the exciting laser beam (a typical ratio of the intensity of elastically scattered light to inelastically scattered light is on the order of 10 4 t o l 0 8 for most chromophores). Moreover, high spectral resolution is necessary to extract vibrationalfrequency information from scattered light in the visible region of the spectrum. Consequently, the requirements for a useful Raman monochromator are several, and considerable effort has gone into the design of the monochromator in commercially available Raman spectrometers. Primary considerations include the spectral dispersion and optical path length of the monochromator, which together determine wavelength resolution and stray-light rejection. In addition, owing to the low Raman signal intensities, the signal throughput of the monochromator is usually of major importance. In practice, single, double, and triple monochromators have all been used in both normal and time-resolved Raman measurements. In double and triple monochromators, two and three gratings, respectively, are used
416
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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sequentially to disperse the scattered light. High-efficiency rejection of the incident laser frequency can be achieved by these monochromators. For the double monochromator, the two gratings are arranged to provide additive dispersion of the radiation; by using a long optical path (typically 1 m), a suitable double monochromator can record useful Raman spectra to within 50 cm- 1of the excitation frequency. For a triple monochromator, the first two gratings are arranged in subtractive dispersion to provide an extremely effective filter for the incident laser beam. All of the dispersion results from the third grating, which is usually used with a 0.5-I m optical path. Triple monochromators generally allow measurements to be made closer to the Rayleigh wavelength than the double monochromators, and they are the most satisfactory choice when information on low-frequency modes is required. Unfortunately, double and triple monochromators suffer significant loss of signal as the scattered radiation passes through the successive optical elements involved in the dispersion process. Triple monochromators typically have a throughput of 3%, whereas the corresponding throughput is less than 10% for a double monochromator. For high dispersion, a single 0.75 m or 1 m single monochromator with throughput efficiency of more than 30% is ideal for TR 3 experiments. However, the single monochromator does not offer sufficient rejection of the Rayleigh scattering to allow effective measurement at frequencies less than 500 cm- 1, even when a long-pass filter is placed between the sample and the monochromator to increase the discrimination against Rayleigh scattering. A single monochromator, in conjunction with a notch filter and ruled holographic grating, can be used to increase the sensitivity of Raman spectral measurements. The efficiency of a ruled holographic grating is generally lower but flatter than a ruled grating. However, its efficiency is much higher than that of a simple holographic grating (JobinYvon, Metuchen, N J). Holographic notch filters provide spectral bandwidth and sharper edges without the unwanted secondary reflection bands associated with dielectric filters. These filters allow the detection of Stokes and anti-Stokes Raman spectra within 100 cm-1 of the laser line, without the need to readjust the filter angle. More recently, supernotch filters have been constructed that provide detection of Raman lines within 50 cmof the excitation frequency. Detectors
Traditionally, Raman spectra have been recorded by using a photomultiplier tube and a scanning monochromator for wavelength dispersion.
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TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
417
For time-resolved resonance Raman spectroscopy, however, optical multichannel analyzers (OMAs) are routinely used as part of the detection system. The major advantage of the OMA detection system is that the entire spectrum is monitored simultaneously, so snapshots of the vibrational Raman spectrum can be recorded during each pair of pump-probe pulses. The detection sensitivity necessary for time-resolved spectroscopy is obtained by the application of intensifiers in conjunction with the diode array. Typical quantum efficiency for a modern, intensified diode-array based OMA is 20% at 400 nm. Moreover, the intensifier on the OMA detector can be controlled by a high-voltage pulser, so as to be synchronized with the laser pulses. For example, an open-gate pulse can be applied when the probe laser fires, but a close-gate situation can be maintained when the pump laser fires. This gating arrangement is useful in minimizing interference from scattered light and fluorescence that is generated by the pump pulse. Charge-coupled device (CCD), two-dimensional, optical array detectors that are based on silicon metal oxide semiconductor technology, have been applied in time-resolved Raman spectroscopy. The major advantages of CCDs over other multichannel detectors are the high quantum efficiency and the low readout noise. The peak quantum efficiencies of the CCDs exceed 70% at 650 nm, and the spectral window can range from 120 to 1000 nm. Peak sensitivity is centered in the red or near-IR spectral region, making the CCD applicable to Raman spectroscopy. The readout noise of a modem CCD is less than 10 photoelectrons, when operated at a readout rate of 20 t~sec/pixel. This is roughly two orders of magnitude less than other multichannel detectors, such as a photodiode array. However, there are some limitations to these detectors. The CCDs are sensitive to cosmic rays and other high-energy photons. These events, which show up as very narrow spikes in the spectrum, can be removed with available software from the manufacturers.
Sample Cells The choice of sample cells in time-resolved Raman spectroscopy is of importance. Spinning cells, circulating sample cells, and jet streams have been successfully applied to avoid damage to photolabile samples by the exciting radiation, a general problem in biological applications of resonance Raman spectroscopy. The application of flowing sample cells, under conditions in which sample recycling is feasible, has the advantage that long acquisition times can be achieved with fairly minimal sample consumption.
418
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
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The most extensively used apparatus for probing transient species and catalytic reactions is the continuous-flow technique. This technique has been applied to the study of fast reactions. Mathies e t al. 26 and Terner e t al. 27 used the jet-stream sampling system to obtain the resonance Raman spectra of rhodopsin, isorhodopsin, and bacteriorhodopsin. The essence of this experimental technique is that the sample is rapidly flowed through the laser beam so that the residence time of the sample in the scattering volume is short, with respect to the time it takes the prepared state to evolve into subsequent products and the time required for significant photodamage of the sample to occur. Woodruff and Spiro 28used an alternative method to avoid photodamage of the sample. This technique involved a circulating sample cell in an ordinary capillary and a constant temperature bath to control the temperature of the sample. Woodruff and Spiro extended the continuous-flow technique and, by introducing rapid-mixing methods, measured the Raman spectra of transient intermediates during the horseradish peroxidase/H202 reaction. 29 Common techniques for achieving rapid mixing are based on Gibson-type mixers 3°or on adapting commercially available stopped-flow accessories.31 Kincaid and co-workers 32 have extended this rapid-mix/flow approach by developing an elegant microdrop technique. In the experimental setup (Fig. 3), which has been used to study the peroxidase reaction, two regularly spaced, high-velocity streams form uniformly sized droplets that are oriented so that the individual droplets from one stream collide and coalesce with droplets from the second stream. The resulting stream is made of uniformly sized droplets approximately 100/zm in diameter that then pass through the focused laser beam. The mixing apparatus can be moved, relative to the focused laser beam, so as to probe the stream at definite distances; thus, precise times after mixing can be determined. The emerging stream of droplets has a linear velocity of approximately 20 m/sec, which corresponds to a 3/zsec residence time in the CW laser. The time lapse between initial mixing of the two solutions and Raman detection is typically 10-50/~sec. For systems that are amenable to photoinitiation, a two-beam approach is feasible, and both CW and pulsed experiments can be carried out (Fig. 26 R. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, 1 (1976). 27 j. Terner, C.-L. Hsieh, A. R. Burns, and M. A. E1-Sayed, Proc. Natl. Acad. Sci. U.S.A. 76, 3046 (1979). 28 W. H. Woodruff and T. G. Spiro, Appl. Spectrosc. 28, 74 (1974). 29 W. H. Woodruff and T. G. Spiro, Appl. Spectrosc. 28, 576 (1974). 3o Q. H. Gibson and L. Milnes, Biochem. J. 91, 161 (1964). 31 S. Han, Y.-C. Ching, and D. L. Rousseau, J. Am. Chem. Soc. 112, 9445 (1990). 32 S. F. Simpson, J. R. Kincaid, and J. F. Holler, J. Am. Chem. Soc. 108, 3136 (1986).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
A
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FIG. 3. (A) System schematic of the microdroplet experiment. (B) Apparatus for droplet generation and collision. The horizontal linear translator adjusts the relative phase of the droplet streams, whereas the vertical translator is used to collide the streams. (From Simpson
et al? 2)
420
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
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1). Han e t al. 3~ used rapid-mix/flow/CW laser methodology to study the reaction between 02 and cytochrome oxidase, which can be initiated by photodissociating CO from the reduced enzyme in the presence of 02. Three time regimes are critical in the flow apparatus. The first one is the t i m e , t l , that it takes for the sample to pass from the mixer to the photolysis beam. The second time, t2, is the time required for the sample to flow through the pump or the probe beam. The third time, t3, involves how long it takes for the sample to flow from the pump beam to the probe beam. In the experimental arrangement, Han e t al. set t 1 at 0.15 sec and t 2 at 25/zsec. The probe-pump delay time, t3, c a n be varied from zero to several milliseconds. For the corresponding two-beam, pulsed experiment, we developed a rapid-mixing/jet apparatus that allows light scattering from a sample jet in air 33 (Fig. 4). The cell is designed to minimize sample consumption and is well suited to pulse-laser excitation and multichannel detection. The mixer/jet cell provides a continuous flow of sample in air in the scattering volume at flow rates as low as 0.3 ml/min, from which high-quality resonance Raman spectra at laser pulse energies as low as 0.1 mJ can be obtained. At this flow rate and with a repetition rate of 10 Hz, the total sample volume per laser shot is 0.5/zl. The photon flux on the sample molecules depends directly on pulse energy. Therefore, an increase in flow rate above 0.2 ml/min will not decrease photon flux, as is the case for continuous-wave excitation, and the high flow rates necessary in other jet systems do not offer any advantage to the pulse application. To illustrate some of the features of the cell design, and particularly the advantages of the jet in overcoming the difficulty of detecting lowfrequency vibrations of large molecules like cytochrome oxidase, we show in Fig. 5 the low-frequency resonance Raman spectra of cytochrome oxidase in its oxidized (inset) and 10 nsec photoproduct states. The mode observed at 220 cm -1 has been assigned to the v(FeE+-His) stretching vibration. The relaxation pathways of many heme proteins including cytochrome oxidase predict that changes in the heme core site depend on the iron motion out of the heme plane and, hence, on the position of the histidine in a direct way. The application of our mixer/flow cell has allowed us to monitor the position of the v(Fe-His) band as a function of time, which provides insight into the mechanisms involved in heine-protein interactions. Applications Nanosecond TR 3 spectroscopy is adaptable for use in monitoring dynamic interactions between protein active sites and their local en33 C. Varotsis, W. A. Oertling, and G. T. Babcock,
Appl. Spectrosc. 44, 742 (1990).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
421
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FIG. 4. Apparatus for room temperature resonance Raman spectra of rapidly mixed/flowing samples. (From Varotsis eta/. 33)
vironment and in the elucidation of the excited state properties of a variety of chromophores. Examples of data from both pioneering and current applications of TR 3 spectroscopy to study photophysical and chemical intermediates formed in the nanosecond to microsecond time range are presented here to demonstrate the versatility of the technique.
422
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
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CO
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Fro. 5. Low-frequencyresonanceRaman spectra of cytochromeoxidasein its oxidized (inset) and 10 nsec photoproductstates.
Excited State Dynamics Transient absorption studies of Zn(TPP) (TPP, tetraphenylporphyrin) have shown that excitation into either singlet states S 1 or $2 is followed by intersystem crossing to the lowest triplet state, TI. However, neither the singlet nor the triplet state were characterized vibrationally. Figure 6 shows the two-pulse, pump-probe TR 3 spectrum from the triplet (T]) state of Zn(TPP) in tetrahydrofuran (THR) and methylcyclohexane obtained with a low-temperature ( - 80°) backscattering apparatus. 34 The first conclusion from this work is that the greater number of vibrational modes observed in the lowest ~r-~r * triplet state of Zn(TPP), relative to the ground state, indicates lowering of the point group symmetry of the porphyrin core from the D4h symmetry of the ground electronic state. The symmetrylowering phenomenon was attributed to the Jahn-Teller effect in the excited triplet state. The second conclusion is that the electronic excitation 34 V, A. Walters, J. C. de Paula, G. T. Babcock, and G. E. Leroi, J. Am. Chem. Soc. 111, 83OO (1989).
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FIG. 6. Time-resolved resonance Raman spectrum of the lowest triplet state of Zn(TPP) in (a) tetrahydrofuran and (b) methylcyclohexane. Spectrum C is the ground-state species. For the T 1 spectrum in T H F (Alfa, ultrapure 99%), 100 m W of the pump pulse (532 nm) and 40 m W of the probe pulse (460 nm) were used. The delay between the p u m p and probe beams was 50 nsec. (From Walters eta/. 34)
424
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
appears to be localized on the porphyrin ring alone and does not extend to the phenyl rings. Reed et al. 35 used the same technique to characterize the anion radical and the photoexcited triplet state of Zn(TPP). Sato et al. 36 have reported the T R 3 of the free base octaethylporphyrin (OEPH2) in its lowest excited singlet and triplet states. Relaxation Pathways in Hemoglobin and Myoglobin Hemoglobin (Hb) and myoglobin (Mb), oxygen transport and storage proteins, respectively, have been studied extensively by nanosecond timeresolved resonance Raman spectroscopy in an effort to understand the molecular and electronic foundations for the function of these proteins. A useful approach is to bind an exogenous ligand, such as CO, photodissociate the heme-bound ligand with pulsed laser radiation, and subsequently monitor relaxation to equilibrium. This provides a means by which to generate transient species and monitor nonequilibrium protein dynamics and recombination processes. Friedman and Lyons 37 used TR 3 spectroscopy to study the properties of the photodissociated Hb and Mb and the recombination processes. The two proteins exhibit very different properties. Photodissociation of HbCO produced a near-unity quantum yield of deoxyHb, followed by recombination of CO within 100 nsec. MbCO, however, shows no significant recombination for times less than 100/xsec. In addition, when pumped in the visible region, MbCO undergoes 97% photolysis, whereas HbCO undergoes 45-47%. The difference in the quantum yield for photodissociation was suggested to originate from differences in geminate recombination rates. Scott and Friedman 38 extended the TR 3 experiments to monitor the time evolution of the Raman band associated with the v(Fe-His) of deoxyHb*. The behavior of the Fe-His bond reflects the relaxation processes within the proximal heine pocket. Their work demonstrated that, in the transition from deoxyHbA to deoxyHb*, the v(Fe-His) frequency increases from 215 to 230 cm -l. This observation was attributed to the heme-histidine tilt, opening the way to the elucidation of the mechanisms involved in the protein control of ligand binding. In addition to these nanosecond dynamics, picosecond studies of both Mb and Hb have been c a r r i e d o u t . 39,40 35 R. A. Reed, R. Purrello, K. Prendergast, and T. Spiro, J. Phys. Chem. 97, 9720 (1991). 36 S.-I. Sato, M. Asano-Someda, and T. Kitagawa, Chem. Phys. Lett. 189, 443 (1992). 37 j. M. Friedman and K. B. Lyons, Nature (London) 284, 570 (1980). 38 T. W. Scott and J. M. Friedman, J. Am. Chem. Soc. 106, 5677 (1984). 39 R. G. Alden, M. D. Chavez, M. R. Ondrias, S. H. Courtney, and J. M. Friedman, J. Am. Chem. Soc. 112, 3241 (1990). 40 R. G. Alden, M. R. Ondrias, S. Courtney, E. W. Findsen, and J. M. Friedman, J. Phys. Chem. 94, 85 (1990).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
425
T o
E ,e4
~J C -r4
@ 0 @ L 0 C
180
230
280
Raman
330
ShiFt
380
(cm
430
-1
480
)
FI~. 7. Low-frequency, time-resolved spectra of cytochrome oxidase subsequent to CO photolysis, in ascending order: (a) equilibrium reduced oxidase, (b) 10 nsec, (c) 125 nsec, (d) 1 /~sec, (e) 150/~sec, (0 750/zsec, (g) 1 msec, and (h) 5 msec. Spectra were obtained with 532 and 440 nm pump-probe beams, respectively, as described in the text. Spectra are the unsmoothed sums of three to five scans at 10 cm-[/min. Spectral band-pass was less than or equal to 7 cm -1 in all cases. (From Findsen et al. 42)
Cytochrome Oxidase Cytochrome oxidase, the terminal enzyme complex of the mitochondrial respiratory chain, has been the subject of intense study because of its electron transfer, proton-pumping function and Oz-reducing catalytic functions. 41 The complex intramolecular electron transfers that occur between cytochrome a/CuA and the binuclear center cytochrome a3/CuB, where the four-electron reduction of molecular oxygen to water occurs, 41 M. Wikstr6m, K. Krab, and M. Saraste, in "Cytochrome Oxidase--A Synthesis." Academic Press, New York and London, 1981.
426
[17]
SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S
427 nm EXCITATION T = 298 K td : 10 ~ s
o"o~,~¢o Absorp,,o.
l
400
500
X,nm leO=- L.P.
~I
~
B
180=. L.P.
C
180=. H.P.
D
180=- H.P.
1 II
~"
I T
•-
I
10 ns
WAVE NUMBERS
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
427
have been studied by various spectroscopic techniques. Two types of TR 3 experiments have been used to study the enzyme. The first experiment involves monitoring the evolution of the CO photolysis product of the cytochrome a3 transient species. The second has been undertaken to characterize intermediates that occur in dioxygen reduction, which is essential for elucidating the chemical mechanisms of the redox processes catalyzed by the enzyme. Cytochrome a3 Hemopocket Relaxation. In a series of pump-probe pulsed TR 3 measurements, Findsen et al. 42 monitored the full time evolution of cytochrome a 3 hemopocket relaxation subsequent to carbon monoxide photolysis from fully reduced cytochrome oxidase. In this work, a scanning monochromator and photomultiplier tube (PMT) detection was used. Figure 7 shows the low-frequency TR 3 spectra of cytochrome oxidase obtained at various delay times subsequent to CO photolysis from cytochrome a3. The band at 214 cm-1 in reduced cytochrome oxidase has been assigned to the F e - H i s stretching mode. The increased frequency of this vibration in the transient species was explained as a result of a strengthened bond. However, other degrees of freedom, such as histidine rotation relative to the heme plane, were postulated. The TR 3 spectra obtained from 10 to 125 nsec are indicative of protein relaxation prior to heme relaxation. At later times (t > 200 nsec), the v(Fe-His) mode decreases, owing to the rearrangement of the proximal heme pocket. Finally, at t = 1 msec, the frequency of the proximal Fe-His appears at 222 cm- 1, which indicates recombination of CO with cytochrome a 3 and subsequent photodissociation of CO by the probe pulse, which produces again the 10 nsec unligated, transient species. The first conclusion from the work of Findsen et al. 42 is that the correlation between the F e - H i s bond and the frequency of/)4 observed in hemoglobins is absent in cytochrome a3 transients. Moreover, geminate CO recombination is also absent in cytochrome a 3. The second conclusion is 42 E. W. Findsen, J. Centeno, G. T. Babcock, and M. R. Ondrias, J. A m . Chem. Soc. 109, 5356 (1987).
FIG. 8. Time-resolved, resonance Raman spectra of cytochrome oxidase, following initiation of the reaction with oxygen at room temperature. The energy of the 532 nm photolysis pump pulse was 1.3 mJ, sufficient to photolyze the enzyme-CO complex and initiate the Oz-reduction reaction. The energy of the probe beam was 0.3 mJ for spectra A and B, 26 and 1.0 mJ for spectra C - E . The repetition rate for both the pump and probe pulses (10 nsec duration) was 10 Hz. The pump-probe delay was 10/~sec for spectra A - D and 10 nsec for transient spectrum E. The accumulation time was 110 min for spectrum A, 70 min for spectrum B, 5 min for spectra C and D, and 15 rain for spectrum E. (From Varotsis eta/. 48)
428
[171
SPECTROSCOPIC METHODS 'FOR METALLOPROTEINS
427 n m EXCITATION T = 298 K
" cO Ou3
[=3 ~D v-|
CD
.-
2
A©
~=© F Resting
,
~
,-~
E. 5 0 0 ~ s
t-(Jr) Z IJJ I-Z
~/~,>~.j ~
//~o
o.
,0o~s
Z
,..
O
C. 5 0 ~ s
¢O O4
B
2~s
cO
A
WAVE
NUMBERS
10ns
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
429
that heme pocket relaxation is biphasic, with no change in Fe-His frequency during the first 200 nsec subsequent to CO photolysis. In recent TR 3 and time-resolved IR measurements, Woodruff et al. 43 have found transient CO binding to Cu B following its photodissociation from cytochrome a 3 and rapid binding (< 10 psec) of a photolabile endogenous ligand to cytochrome a 3 . They suggested that the photolabile ligand is transferred from Cu Bto cytochrome a 3 , when CO binds to Cu B, forming a cytochrome a 3 species having axial ligation that differs from the reduced unligated enzyme. The implications of this work for 0 2 binding pathways have attracted considerable recent attention. 44 Dioxygen Intermediates. Although the reaction between cytochrome oxidase and 0 2 occurs too quickly (tl/2 1 msec) to be studied by conventional stopped-flow techniques, Gibson and Greenwood45 used photolysis of the cytochrome a32+-CO complex of the enzyme in the presence of 0 2 to circumvent this limitation. Babcock et al. 9'46'47 adapted the Gibson-Greenwood technique and used time-resolved resonance Raman spectroscopy to study the reaction of fully and partially reduced oxidase with 02. We have extended the flow/flash time-resolved approach so as to be able to observe the low-frequency region of the Raman spectrum. Figure 8 shows the time-resolved resonance Raman spectra of cytochrome oxidase at 10/xsec subsequent to carbon monoxide photolysis in the presence of 0 2 .48'49 Spectrum E (Fig. 8) is that of the photodissociation product of the 43 W. H. Woodruff, O. Einarsdottir, R. B. Dyer, K. A. Bagley, G. Palmer, S. J. Atherton, R. A. Goldbeck, T. D. Dawes, and D. S. Kliger, Proc. Natl. Acad. ScL U.S.A. 88, 2588 (1991). 44 G. T. Babcock and M. Wikstr6m, Nature (London) 356, 301 (1992). 45 Q. Gibson and C. Greenwood, Biochemistry 86, 541 (1963). 46 G. T. Babcock, J. M. Jean, L. N. Johnston, G. Palmer, and W. H. Woodruff, J. Am. Chem. Soc. 106, 8305 (1984). 47 G. T. Babcock, J. M. Jean, L. N. Johnston, W. H. Woodruff, and G. Palmer, J. lnorg. Biochem. 23, 243 (1985). 48 C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Am. Chem. Soe. 111, 6439 (1989); C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Am. Chem. Soc. 112, 1297 (1990). 49 C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Biol. Chem. 265, 11131 (1990).
FIG. 9. Time-resolved resonance Raman spectra of fully reduced cytochrome oxidase at the indicated times. The energy of the 532 nm photolysis pump/pulse was 1.3 m J, sufficient to photolyze the enzyme-CO complex and initiate the O2-reduction reaction. The energy of the 427 nm probe beam was 0.8 mJ for spectra A and F and 0.3 mJ for spectra B - E . The repetition rate for both the pump and probe pulses (10 nsec duration) was 10 Hz. The accumulation time was 15 rain for both spectra A and F and 50 rain for spectra B - E . The enzyme concentration was 50/~M after mixing, pH 7.4. (From Varotsis and Babcock. 55)
430
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
reduced carbonmonoxy enzyme (pump-probe delay 10 nsec). Spectrum A (Fig. 8) obtained with a low-energy, defocused beam (0.3 mJ), is similar to the 10 nsec spectrum with the exception that a new mode appears at 571 cm -1. Figure 8B shows that the 571 cm -1 mode in the 160z spectrum is downshifted to 546 cm-1 when the experiment is repeated with 1802 . This allowed us to assign it as the iron-oxygen stretching motion in the cytochrome a32+-O2 complex, as the 24 cm -1 shift is in agreement with that expected from the two-body harmonic oscillator approximation for Fe2÷-O2 . The v(Fe2+-O2) in the oxycytochrome a32+ species is very close to the v(FeZ+-O2) frequency of an imidazole-heme a2+-dioxygen complex model compound. Spectra C and D were obtained with relatively high energies (1 mJ), and the absence of modes located at 571 and 546 cm-1 indicates photodissociation of the oxy ligand, as was observed in the high-frequency experiments. Han e t al. 31'5°'51 and Ogura et al., 52-54 using time-resolved CW techniques, also observed the oxy species. Fig. 9 shows the high-frequency resonance Raman spectra of fully reduced t,,2+., ~- -3 2+~j cytochrome oxidase at various delay times subsequent to carbon monoxide photolysis in the presence of O2.55 In the I0 nsec photoproduct spectrum, the oxidation state marker, v4, is at 1355 cm -1, establishing that both cytochrome a and cytochrome a 3 are in the ferrous state. The core expansion regions shows two vibrations at 1570 and 1586 cm -1. The 1622 cm -1 mode arises from the C~---C stretching vibration of cytochrome a 2+ and a32+. Spectrum B (Fig. 9) shows that no significant changes are detected at 2/,sec in the reaction. As the reaction proceeds, oxidation of cytochrome a 3 and a occurs. At 50/,sec, the oxidation state marker has shifted to 1371 cm- 1, and the shoulder at 1358 cm- 1is substantially decreased, indicating that the oxy adduct of cytochrome a 3 is formed in this reaction. The decrease in intensity of the 1613 cm -1 mode and the concomitant increase in scattering at 1650 cm -1 indicate that partial oxidation of cytochrome a has also occurred at 50 ~sec. Oxidation of cytochrome a remains partial, as indicated by the 1358 cm-1 shoulder in the v4 region in the 500/~sec spectrum. The second phase of cytochrome a oxidation occurs in the 500 >sec to 5 msec time range to produce the oxidized enzyme. Insights into 02 activation and energy conservation in 50 S. Han, Y.-C. Ching, and D. L. Rousseau, Proc. Natl. Acad. Sci. U.S.A. 87, 8408 (1990). 51 S. Han, Y.-C. Ching, and D. L. Rousseau, Nature (London) 348, 89 (1990). 52 T. Ogura, S. Yoshikawa, and T. Kitagawa, J. Am. Chem. Soc. 112, 5630 (1990). s3 T. Ogura, S. Takahashi, K. Shinzawa-Itoh, S. Yoshikawa, and T. Kitagawa, J. Biol. Chem. 265, 14721 (1990). 54 T. Ogura, S. Takahashi, K. Shinzawa-Itoh, S. Yoshikawa, and T. Kitagawa, Bull. Chem. Soc. Jpn. 64, 2901 (1991). s5 C. Varotsis and G. T. Babcock, Biochemistry 29, 7357 (1990).
[18]
R E S O N A N C E R A M A N SPECTRA OF M E T A L L O P R O T E I N S
431
cell respiration that can be obtained by combining time-resolved techniques with biochemical approaches have been discussed in detail by Babcock and Wikstr6m. 44 Conclusions In summary, T R 3 spectroscopy is a technique that can be used to probe structural and conformational as well as kinetic properties of transient species. It appears that the two-pulse, pump-probe, time-resolved Raman approach, in conjunction with a single monochromator and a CCD detector, provides the most reliable configuration to record the time evolution of transient species.
[18] T e c h n i q u e s for O b t a i n i n g R e s o n a n c e R a m a n S p e c t r a of Metalloproteins
By THOMAS M. LOEHR and JOANN SANDERS-LOEHR Introduction The focus of this chapter is pragmatic. It provides the reader with tested laboratory procedures that illustrate how resonance Raman spectra of metalloproteins are obtained and verified, how active-site structural and/or mechanistic information is probed by isotopic labeling, and how spectral data interpretation is initiated. The methodology described here, including the majority of illustrative examples, is based on the investigation of copper- and iron-containing proteins carried out in the authors' laboratory. This specific-methods approach discusses sample preparation and temperature control, choice of scattering geometries, data collection, data quality, and the initiation of data analysis. It describes common precautions and pitfalls, as well as criteria for judging sample integrity. It shows how one confirms the resonance Raman phenomenon and also discusses the utility of excitation profiles. This chapter is not intended to review the resonance Raman literature on copper and iron l~roteins. The general theory of Raman spectroscopy, and resonance Raman spectroscopy in particular, including a description of Raman instrumentation, laser sources, and general applications of the technique, is presented in [14] in this volume. The special area of UV resonance Raman spectroscopy, probing ultraviolet-absorbing amino acids and nucleic acids, is covered in [15]. The use of micro (resonance) METHODS IN ENZYMOLOGY, VOL. 226
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
TIME-RESOLVED
RESONANCE RAMAN SPECTROSCOPY
409
[17] N a n o s e c o n d T i m e - R e s o l v e d R e s o n a n c e Raman Spectroscopy B y CONSTANTINOS VAROTSIS a n d GERALD T . BABCOCK
Introduction Resonance Raman (RR) spectroscopy is a powerful technique to probe molecular vibrations that are coupled to electronic transitions. Monochromatic light, now universally obtained from continuous wave (CW) or pulsed lasers, is used to illuminate a sample, and the spectrum of scattered radiation is analyzed to determine vibrational information on molecular species within the sample. By bringing the laser frequency into resonance with an electronic transition of a species of interest, dramatic enhancements in scattered intensity result. The individual vibrational frequencies observed in a Raman spectrum arise from normal modes in the ground electronic state. The intensities of the Raman lines, however, reflect the character of the electronic excited states. Owing to the high selectivity and sensitivity in the enhancement of vibrational modes, resonance Raman spectroscopy offers the opportunity to probe chemical species such as reaction intermediates, excited electronic states, and chromophoric site(s) of biological systems. Biological chromophores such as heroes, flavins, chlorophylls, and a number of different types of metal-containing proteins have been investigated by resonance Raman spectroscopy. The static resonance Raman effect and biological applications of Raman spectroscopy have been the subject of numerous reports and reviews. 1-2° I A. C. Albrecht, J. Chem. Phys. 34, 1476 (1961). 2 S. Y. Lee and E. J. Heller, J. Chem. Phys. 71, 4777 (1979). 3 D. T. Tannor and E. J. Heller, J. Chem. Phys. 77, 202 (1982). 4 B. B. Johnson and W. L. Peticolas, Annu. Rev. Phys. Chem. 27, 465 (1976). 5 T. G. Spiro and P. Stein, Annu. Rev. Phys. Chem. 28, 501 (1977). 6 A. Warshel, Annu. Rev. Biophys. Bioeng. 6, 273 (1977). 7 W. Siebrand and M. Zgierski, in "Excited States" (E. C. Lim, ed.), p. 1. Academic Press, New York, 1979. s j. Tang and A. C. Albrecht, in "Raman Spectroscopy" (H. Szymanski, ed.), p. 2. Plenum, New York, 1970. 9 G. T. Babcock, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 295. Wiley, New York, 1988. s0 T. G. Spiro and X.-Y. Li, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 1. Wiley, New York, 1988. 1~ N.-T. Yu and E. A. Kerr, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 39. Wiley, New York, 1988.
METHODS IN ENZYMOLOGY, VOL. 226
Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
410
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
Time-resolved resonance Raman (TR 3) spectroscopy has been applied to measure conformational and structural changes as well as kinetic properties of transient species. Moreover, data from these studies can be used to identify the mechanistic routes of rapid photochemical and photobiological processes. Application of the TR 3 technique to chemical and biological processes of interest can be implemented either with CW lasers or with the use of a single laser pulse or of two laser pulses (Fig. 1). Various pulsed laser systems provide temporal resolution ranging from subpicosecond to several nanoseconds; CW lasers can be applied so as to approach 1/zsec time resolution. Continuous-wave lasers require high flow rates and tight beam focusing in order to achieve high time resolution. The residence time that the sample spends in the probe beam ultimately determines the shortest time range that can be explored. With a pulsed laser source in a one-pulse experiment, both the time delay and the time resolution are determined by the laser pulse duration. This is a fundamental limitation of the technique. In such a single-pulse experiment, photons at the leading edge of the pulse initiate the photochemistry that generates the photoproduct; photons nearer the trailing edge of the same pulse scatter from the newly created species to generate its Raman spectrum. The laser beam is usually tightly focused, so that a high photon density on the sample scattering volume, which is necessary to create the photoproduct and record its Raman spectrum, is produced. However, if high photon densities are required to initiate the reaction,
n T. Kitagawa, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 97. Wiley, New York, 1988. 13 D. L. Rousseau and J. M. Friedman, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 133. Wiley, New York, 1988. 14 p. M. Champion, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 249. Wiley, New York, 1988. 15 B. Cartling, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 217. Wiley, New York, 1988. 16 M. Lutz and B. Robert, in "Biological Applications of Raman Scattering" (T. G, Spiro, ed.), Vol. 3, p. 347. Wiley, New York, 1988. 17 W. H. Woodruff, R. B. Dyer, and J. R. Schoonover, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 413. Wiley, New York, 1988. 18T. M. Loehr and A. K. Shiemke, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 439. Wiley, New York, 1988. ~9L. Que, Jr., in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 491. Wiley, New York, 1988. 20 T. G. Spiro, R. S. Czernuszewics, and S. Han, in "Biological Applications of Raman Scattering" (T. G. Spiro, ed.), Vol. 3, p. 523. Wiley, New York, 1988.
TIME-RESOLVED RESONANCERAMANSPECTROSCOPY
[17]
1 Pulsed Excitation
/
I1
II Pump Probe,hv
]i'~
b
~< hv2=probe
td
>~'~
411
-F,ow Ce,,
Flow Cell Slow Flow
hv1= pump
2 Continuous wave- excitation
hv1 = pump
"- ~ ii¢ ...
Flow Cell Rapid Flow
hv2= probe
Fro. 1. Block diagram used to obtain time-resolved resonance Raman (TR)3 spectra.
multiple excitations of the sample that produce nonlinear processes may occur and contribute complex and artifactual spectral features. A two-pulse, pump-probe, time-resolved Raman approach allows not only the detection of transient intermediates involved in a dynamic process but also the measurement of their formation and decay kinetics. The first pulse, the pump, initiates the reaction or generates the excited state, and the second pulse, the probe, which is usually of a different frequency, produces the Raman scattering. The two-color, pump-probe configuration has been used extensively, since it offers the opportunity to record realtime kinetic measurements as well as to record the time evolution of transient species. In this review of experimental methods, we limit our discussion to time-resolved Raman techniques that provide time resolution in the nanosecond to second time range. Considerable activity is occurring with pico-
412
SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S
[17]
second and subpicosecond lasers that provide finer time resolution. The reader is referred to several reviews that cover these developments. 21-25 Methods and Equipment For time-resolved Raman spectroscopy, low repetition rate, highenergy pulsed excimer and neodymium-yttrium-aluminum-garnet (Nd : YAG) lasers are commonly used, often in conjunction with dye lasers or Raman shifters to extend the accessible wavelength range. In most time-resolved experiments, two pulsed lasers with pulse widths in the 1-10 nsec range and variable repetition rates are coupled together electronically to produce two pulses of different frequency. The pulsed technique is particularly useful, since the time resolution is determined by the programmable time delay between the short (10 nsec) pump and probe laser flashes (Fig. 1). Useful time-resolved work can also be done with CW laser systems. The continuous-wave approach requires one or two laser systems in conjunction with a continuous-flow apparatus (Fig. 1). In the single-beam (one-laser) experiment, the flow rate is varied so that the sample residence time in the laser-irradiated region is varied, allowing time-evolution studies to be made. This approach becomes ambiguous kinetically as the residence time increases. Furthermore, with a single beam at a low flow rate, the sample residence time in the laser is relatively long, and, therefore, artifacts induced by the incident laser beam may occur. In the double-beam CW experiment, the time resolution is determined by the flow rate, the cross-sectional area of the irradiated volume, and the distance between the two beam foci. Lasers
Resonance Raman enhancement of vibrational modes is observed when the f r e q u e n c y of the excitation laser beam is close to the frequency of an electric dipole-allowed transition of the molecule. Therefore, by appropriate choice of excitation frequency, selective enhancement of the zl G. H. Atkinson, in "Time-Resolved Vibrational Spectroscopy" (G. H. Atkinson, ed.), p. 179. Academic Press, New York, 1983. 22C. L. Hsieh, M. Nagumo, M. Nicol, and M. A. E1-Sayed,J. Phys. Chem. 85, 2714 (1981). 23C. L. Hsieh, M. A. E1-Sayed, M. Nagumo, and J.-H. Lee, Photoehem. Photobiol. 38, 83 (1983). .~4M. A. E1-Sayed,Pure Appl. Chem. 57, 187 (1985). 25G. H. Atkinson, T. L. Brack, D. Blanchard, G. Rumbles, and L. Siemankowski, in "'Ultrafast Phenomena V" (G. R. Fleming and A. E. Siegman, eds.), p. 409. (Springer Series, Chem. Phys. 46). Springer-Verlag, Berlin and Heidelberg, 1986.
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
413
TABLE I CHARACTERISTICS OF WIDELY USED LASERS IN TIMERESOLVED RAMAN SPECTROSCOPY Lasing medium
Output
Wavelength (nm)
Nd:YAG Excimer
Pulsed, CW Pulsed
N2 Ar + Kr + He-Cd
Pulsed CW CW CW
1064, 532, 355,266 193 (ArF), 222 (KrC1), 248 (KrF), 308 (XeC1), 337 (Nz), 351 (XeF) 337 351,364, 488,514.5 406.7, 413,531,676 441,325
molecules that give rise to the electronic transition can be obtained. This resonance aspect of the Raman scattering process adds unique potential to the technique in resolving multichromophoric systems; flexibility in the choice of laser excitation frequency is essential in realizing this potential. Nanosecond, pulsed, and continuous wave lasers provide a unique tool for TR 3 spectroscopy. Available laser systems and suitable wavelengths that can be created to fulfill the demand of frequency selectivity and tunability have expanded the experimental capabilities in this field. Table I summarizes the common frequencies available from pulsed and continuous-wave lasers for time-resolved studies. Excimer and Nd : YAG lasers provide pulses of 7-10 nsec duration and millijoules of energy per pulse, with repetition rates of 10-100 Hz. The excimer wavelength range varies from 190 to 380 rim, depending on the gases used. The more commonly used excimer gases and their laser wavelengths are summarized in Table I. The direction laser light output from an excimer, however, is generally not suitable for Raman scattering. Consequently, this laser is used almost always as a pump source for a dye laser. Such an arrangement provides high-quality monochromatic light that can be tuned over a broad wavelength range that extends from the near-ultraviolet region through the visible and into the near-infrared. The N d : Y A G laser is considerably more versatile in time-resolved Raman experiments, as its 1064 nm fundamental can be used directly to excite a Raman spectrum. Moreover, because of its high power characteristics, frequency doubling and tripling crystals can be used to convert the YAG fundamental to 532 and 355 nm light, respectively; the fourth (266 nm) and the fifth (213 nm) harmonics can be used to generate light in the deep ultraviolet region of the spectrum. Wavelength shifting of the fundamental or the harmonics of the Nd : YAG laser can be achieved by
414
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
TABLE II PRACTICAL EXCITATION WAVELENGTHS FROM H2, D 2, AND CH4 RAMAN SHIFTERS FOR TIME-RESOLVED RAMAN SPECTROSCOPY 1064 nm
Stokes (Sn) or Anti-Stokes (AS,) line
H2a
D2b
AS4 AS3 AS2 ASs
384.3 457.4 564.7 737.8
468.5 544.7 650.5 807.4
$1 $2 $3 $4
532 nm CH4c
656.8 812.2
355 nm
H2
D2
CH4
H2
D2
CH4
282.3 319.9 368.9 435.7 683.0
325.3 360.3 403.7 459.0 632.5 779.8
406.1 460.6 629.6 771.1
223.1 245.9 273.9 309.1 416.0 502.9 635.7 863.9
249.1 269.1 292.7 320.7 396.7 450.0 519.9 615.5
294.1 321.7 396.0 447.6 514.8 605.6
a 0Jvib, 4 1 5 5 c m -1 f o r H 2. b COvib, 2 9 8 7 c m - 1 f o r D 2 . c r.~vib' 2 9 1 4 c m -1 f o r C H 4.
focusing into a Raman shifter filled with different gases ( H 2 , D 2 , C H 4 ) to produce a wide range of wavelengths. Table II summarizes the wavelengths available from H 2 , D 2 , and C H 4 Raman shifters. Alternatively, the second or the third harmonic can be used to pump a pulsed-dye laser. The dye laser can generate tunable light between 380 and 800 nm. Finally, harmonic generation and nonlinear mixing techniques can be applied to the dye-laser output. For example, the doubled dye-laser output can be mixed with the 1064 nm Nd : YAG fundamental to produce tunable light in the UV spectral region. The doubling and mixing are accomplished by using angle-tuned crystals in either homebuilt or commercially available arrangements. As an example of how various pulsed-laser sources and wavelength-shifting devices can be teamed with a detection system, Fig. 2 shows the experimental arrangement at the shared LASER Laboratory, Michigan State University (East Lansing, MI), for TR 3 studies of biological molecules and metalloporphyrins. There are a variety of continuous-wave lasers. In time-resolved Raman experiments, H e - C d , argon, and krypton lasers are the most widely used sources. The argon and the krypton lasers are used extensively for pumping CW dye lasers and mode-locked dye lasers. A Ti:sapphire laser pumped by an Ar + laser has been used for Raman experiments in the deep-red spectral region. Thus, with the CW technique, as with the pulsed systems described above, there is now the ability to obtain relatively intense, monochromatic radiation throughout the UV, visible, and nearinfrared spectral regions.
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
I
EG&G PAR OMAII
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FIG. 2. I n s t r u m e n t a l configuration u s e d for pulsed T R 3 m e a s u r e m e n t s .
Monochromators
In TR 3 as well as in normal resonance Raman spectroscopy, the signal intensity is weak, compared to the intensity of the exciting laser beam (a typical ratio of the intensity of elastically scattered light to inelastically scattered light is on the order of 10 4 t o l 0 8 for most chromophores). Moreover, high spectral resolution is necessary to extract vibrationalfrequency information from scattered light in the visible region of the spectrum. Consequently, the requirements for a useful Raman monochromator are several, and considerable effort has gone into the design of the monochromator in commercially available Raman spectrometers. Primary considerations include the spectral dispersion and optical path length of the monochromator, which together determine wavelength resolution and stray-light rejection. In addition, owing to the low Raman signal intensities, the signal throughput of the monochromator is usually of major importance. In practice, single, double, and triple monochromators have all been used in both normal and time-resolved Raman measurements. In double and triple monochromators, two and three gratings, respectively, are used
416
SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S
[17]
sequentially to disperse the scattered light. High-efficiency rejection of the incident laser frequency can be achieved by these monochromators. For the double monochromator, the two gratings are arranged to provide additive dispersion of the radiation; by using a long optical path (typically 1 m), a suitable double monochromator can record useful Raman spectra to within 50 cm- 1of the excitation frequency. For a triple monochromator, the first two gratings are arranged in subtractive dispersion to provide an extremely effective filter for the incident laser beam. All of the dispersion results from the third grating, which is usually used with a 0.5-I m optical path. Triple monochromators generally allow measurements to be made closer to the Rayleigh wavelength than the double monochromators, and they are the most satisfactory choice when information on low-frequency modes is required. Unfortunately, double and triple monochromators suffer significant loss of signal as the scattered radiation passes through the successive optical elements involved in the dispersion process. Triple monochromators typically have a throughput of 3%, whereas the corresponding throughput is less than 10% for a double monochromator. For high dispersion, a single 0.75 m or 1 m single monochromator with throughput efficiency of more than 30% is ideal for TR 3 experiments. However, the single monochromator does not offer sufficient rejection of the Rayleigh scattering to allow effective measurement at frequencies less than 500 cm- 1, even when a long-pass filter is placed between the sample and the monochromator to increase the discrimination against Rayleigh scattering. A single monochromator, in conjunction with a notch filter and ruled holographic grating, can be used to increase the sensitivity of Raman spectral measurements. The efficiency of a ruled holographic grating is generally lower but flatter than a ruled grating. However, its efficiency is much higher than that of a simple holographic grating (JobinYvon, Metuchen, N J). Holographic notch filters provide spectral bandwidth and sharper edges without the unwanted secondary reflection bands associated with dielectric filters. These filters allow the detection of Stokes and anti-Stokes Raman spectra within 100 cm-1 of the laser line, without the need to readjust the filter angle. More recently, supernotch filters have been constructed that provide detection of Raman lines within 50 cmof the excitation frequency. Detectors
Traditionally, Raman spectra have been recorded by using a photomultiplier tube and a scanning monochromator for wavelength dispersion.
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
417
For time-resolved resonance Raman spectroscopy, however, optical multichannel analyzers (OMAs) are routinely used as part of the detection system. The major advantage of the OMA detection system is that the entire spectrum is monitored simultaneously, so snapshots of the vibrational Raman spectrum can be recorded during each pair of pump-probe pulses. The detection sensitivity necessary for time-resolved spectroscopy is obtained by the application of intensifiers in conjunction with the diode array. Typical quantum efficiency for a modern, intensified diode-array based OMA is 20% at 400 nm. Moreover, the intensifier on the OMA detector can be controlled by a high-voltage pulser, so as to be synchronized with the laser pulses. For example, an open-gate pulse can be applied when the probe laser fires, but a close-gate situation can be maintained when the pump laser fires. This gating arrangement is useful in minimizing interference from scattered light and fluorescence that is generated by the pump pulse. Charge-coupled device (CCD), two-dimensional, optical array detectors that are based on silicon metal oxide semiconductor technology, have been applied in time-resolved Raman spectroscopy. The major advantages of CCDs over other multichannel detectors are the high quantum efficiency and the low readout noise. The peak quantum efficiencies of the CCDs exceed 70% at 650 nm, and the spectral window can range from 120 to 1000 nm. Peak sensitivity is centered in the red or near-IR spectral region, making the CCD applicable to Raman spectroscopy. The readout noise of a modem CCD is less than 10 photoelectrons, when operated at a readout rate of 20 t~sec/pixel. This is roughly two orders of magnitude less than other multichannel detectors, such as a photodiode array. However, there are some limitations to these detectors. The CCDs are sensitive to cosmic rays and other high-energy photons. These events, which show up as very narrow spikes in the spectrum, can be removed with available software from the manufacturers.
Sample Cells The choice of sample cells in time-resolved Raman spectroscopy is of importance. Spinning cells, circulating sample cells, and jet streams have been successfully applied to avoid damage to photolabile samples by the exciting radiation, a general problem in biological applications of resonance Raman spectroscopy. The application of flowing sample cells, under conditions in which sample recycling is feasible, has the advantage that long acquisition times can be achieved with fairly minimal sample consumption.
418
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
The most extensively used apparatus for probing transient species and catalytic reactions is the continuous-flow technique. This technique has been applied to the study of fast reactions. Mathies e t al. 26 and Terner e t al. 27 used the jet-stream sampling system to obtain the resonance Raman spectra of rhodopsin, isorhodopsin, and bacteriorhodopsin. The essence of this experimental technique is that the sample is rapidly flowed through the laser beam so that the residence time of the sample in the scattering volume is short, with respect to the time it takes the prepared state to evolve into subsequent products and the time required for significant photodamage of the sample to occur. Woodruff and Spiro 28used an alternative method to avoid photodamage of the sample. This technique involved a circulating sample cell in an ordinary capillary and a constant temperature bath to control the temperature of the sample. Woodruff and Spiro extended the continuous-flow technique and, by introducing rapid-mixing methods, measured the Raman spectra of transient intermediates during the horseradish peroxidase/H202 reaction. 29 Common techniques for achieving rapid mixing are based on Gibson-type mixers 3°or on adapting commercially available stopped-flow accessories.31 Kincaid and co-workers 32 have extended this rapid-mix/flow approach by developing an elegant microdrop technique. In the experimental setup (Fig. 3), which has been used to study the peroxidase reaction, two regularly spaced, high-velocity streams form uniformly sized droplets that are oriented so that the individual droplets from one stream collide and coalesce with droplets from the second stream. The resulting stream is made of uniformly sized droplets approximately 100/zm in diameter that then pass through the focused laser beam. The mixing apparatus can be moved, relative to the focused laser beam, so as to probe the stream at definite distances; thus, precise times after mixing can be determined. The emerging stream of droplets has a linear velocity of approximately 20 m/sec, which corresponds to a 3/zsec residence time in the CW laser. The time lapse between initial mixing of the two solutions and Raman detection is typically 10-50/~sec. For systems that are amenable to photoinitiation, a two-beam approach is feasible, and both CW and pulsed experiments can be carried out (Fig. 26 R. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, 1 (1976). 27 j. Terner, C.-L. Hsieh, A. R. Burns, and M. A. E1-Sayed, Proc. Natl. Acad. Sci. U.S.A. 76, 3046 (1979). 28 W. H. Woodruff and T. G. Spiro, Appl. Spectrosc. 28, 74 (1974). 29 W. H. Woodruff and T. G. Spiro, Appl. Spectrosc. 28, 576 (1974). 3o Q. H. Gibson and L. Milnes, Biochem. J. 91, 161 (1964). 31 S. Han, Y.-C. Ching, and D. L. Rousseau, J. Am. Chem. Soc. 112, 9445 (1990). 32 S. F. Simpson, J. R. Kincaid, and J. F. Holler, J. Am. Chem. Soc. 108, 3136 (1986).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
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FIG. 3. (A) System schematic of the microdroplet experiment. (B) Apparatus for droplet generation and collision. The horizontal linear translator adjusts the relative phase of the droplet streams, whereas the vertical translator is used to collide the streams. (From Simpson
et al? 2)
420
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
1). Han e t al. 3~ used rapid-mix/flow/CW laser methodology to study the reaction between 02 and cytochrome oxidase, which can be initiated by photodissociating CO from the reduced enzyme in the presence of 02. Three time regimes are critical in the flow apparatus. The first one is the t i m e , t l , that it takes for the sample to pass from the mixer to the photolysis beam. The second time, t2, is the time required for the sample to flow through the pump or the probe beam. The third time, t3, involves how long it takes for the sample to flow from the pump beam to the probe beam. In the experimental arrangement, Han e t al. set t 1 at 0.15 sec and t 2 at 25/zsec. The probe-pump delay time, t3, c a n be varied from zero to several milliseconds. For the corresponding two-beam, pulsed experiment, we developed a rapid-mixing/jet apparatus that allows light scattering from a sample jet in air 33 (Fig. 4). The cell is designed to minimize sample consumption and is well suited to pulse-laser excitation and multichannel detection. The mixer/jet cell provides a continuous flow of sample in air in the scattering volume at flow rates as low as 0.3 ml/min, from which high-quality resonance Raman spectra at laser pulse energies as low as 0.1 mJ can be obtained. At this flow rate and with a repetition rate of 10 Hz, the total sample volume per laser shot is 0.5/zl. The photon flux on the sample molecules depends directly on pulse energy. Therefore, an increase in flow rate above 0.2 ml/min will not decrease photon flux, as is the case for continuous-wave excitation, and the high flow rates necessary in other jet systems do not offer any advantage to the pulse application. To illustrate some of the features of the cell design, and particularly the advantages of the jet in overcoming the difficulty of detecting lowfrequency vibrations of large molecules like cytochrome oxidase, we show in Fig. 5 the low-frequency resonance Raman spectra of cytochrome oxidase in its oxidized (inset) and 10 nsec photoproduct states. The mode observed at 220 cm -1 has been assigned to the v(FeE+-His) stretching vibration. The relaxation pathways of many heme proteins including cytochrome oxidase predict that changes in the heme core site depend on the iron motion out of the heme plane and, hence, on the position of the histidine in a direct way. The application of our mixer/flow cell has allowed us to monitor the position of the v(Fe-His) band as a function of time, which provides insight into the mechanisms involved in heine-protein interactions. Applications Nanosecond TR 3 spectroscopy is adaptable for use in monitoring dynamic interactions between protein active sites and their local en33 C. Varotsis, W. A. Oertling, and G. T. Babcock,
Appl. Spectrosc. 44, 742 (1990).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
421
Z
MIXER/
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Z
FIG. 4. Apparatus for room temperature resonance Raman spectra of rapidly mixed/flowing samples. (From Varotsis eta/. 33)
vironment and in the elucidation of the excited state properties of a variety of chromophores. Examples of data from both pioneering and current applications of TR 3 spectroscopy to study photophysical and chemical intermediates formed in the nanosecond to microsecond time range are presented here to demonstrate the versatility of the technique.
422
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
CO
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Fro. 5. Low-frequencyresonanceRaman spectra of cytochromeoxidasein its oxidized (inset) and 10 nsec photoproductstates.
Excited State Dynamics Transient absorption studies of Zn(TPP) (TPP, tetraphenylporphyrin) have shown that excitation into either singlet states S 1 or $2 is followed by intersystem crossing to the lowest triplet state, TI. However, neither the singlet nor the triplet state were characterized vibrationally. Figure 6 shows the two-pulse, pump-probe TR 3 spectrum from the triplet (T]) state of Zn(TPP) in tetrahydrofuran (THR) and methylcyclohexane obtained with a low-temperature ( - 80°) backscattering apparatus. 34 The first conclusion from this work is that the greater number of vibrational modes observed in the lowest ~r-~r * triplet state of Zn(TPP), relative to the ground state, indicates lowering of the point group symmetry of the porphyrin core from the D4h symmetry of the ground electronic state. The symmetrylowering phenomenon was attributed to the Jahn-Teller effect in the excited triplet state. The second conclusion is that the electronic excitation 34 V, A. Walters, J. C. de Paula, G. T. Babcock, and G. E. Leroi, J. Am. Chem. Soc. 111, 83OO (1989).
[17]
423
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY T
E
o to
to
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FIG. 6. Time-resolved resonance Raman spectrum of the lowest triplet state of Zn(TPP) in (a) tetrahydrofuran and (b) methylcyclohexane. Spectrum C is the ground-state species. For the T 1 spectrum in T H F (Alfa, ultrapure 99%), 100 m W of the pump pulse (532 nm) and 40 m W of the probe pulse (460 nm) were used. The delay between the p u m p and probe beams was 50 nsec. (From Walters eta/. 34)
424
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
appears to be localized on the porphyrin ring alone and does not extend to the phenyl rings. Reed et al. 35 used the same technique to characterize the anion radical and the photoexcited triplet state of Zn(TPP). Sato et al. 36 have reported the T R 3 of the free base octaethylporphyrin (OEPH2) in its lowest excited singlet and triplet states. Relaxation Pathways in Hemoglobin and Myoglobin Hemoglobin (Hb) and myoglobin (Mb), oxygen transport and storage proteins, respectively, have been studied extensively by nanosecond timeresolved resonance Raman spectroscopy in an effort to understand the molecular and electronic foundations for the function of these proteins. A useful approach is to bind an exogenous ligand, such as CO, photodissociate the heme-bound ligand with pulsed laser radiation, and subsequently monitor relaxation to equilibrium. This provides a means by which to generate transient species and monitor nonequilibrium protein dynamics and recombination processes. Friedman and Lyons 37 used TR 3 spectroscopy to study the properties of the photodissociated Hb and Mb and the recombination processes. The two proteins exhibit very different properties. Photodissociation of HbCO produced a near-unity quantum yield of deoxyHb, followed by recombination of CO within 100 nsec. MbCO, however, shows no significant recombination for times less than 100/xsec. In addition, when pumped in the visible region, MbCO undergoes 97% photolysis, whereas HbCO undergoes 45-47%. The difference in the quantum yield for photodissociation was suggested to originate from differences in geminate recombination rates. Scott and Friedman 38 extended the TR 3 experiments to monitor the time evolution of the Raman band associated with the v(Fe-His) of deoxyHb*. The behavior of the Fe-His bond reflects the relaxation processes within the proximal heine pocket. Their work demonstrated that, in the transition from deoxyHbA to deoxyHb*, the v(Fe-His) frequency increases from 215 to 230 cm -l. This observation was attributed to the heme-histidine tilt, opening the way to the elucidation of the mechanisms involved in the protein control of ligand binding. In addition to these nanosecond dynamics, picosecond studies of both Mb and Hb have been c a r r i e d o u t . 39,40 35 R. A. Reed, R. Purrello, K. Prendergast, and T. Spiro, J. Phys. Chem. 97, 9720 (1991). 36 S.-I. Sato, M. Asano-Someda, and T. Kitagawa, Chem. Phys. Lett. 189, 443 (1992). 37 j. M. Friedman and K. B. Lyons, Nature (London) 284, 570 (1980). 38 T. W. Scott and J. M. Friedman, J. Am. Chem. Soc. 106, 5677 (1984). 39 R. G. Alden, M. D. Chavez, M. R. Ondrias, S. H. Courtney, and J. M. Friedman, J. Am. Chem. Soc. 112, 3241 (1990). 40 R. G. Alden, M. R. Ondrias, S. Courtney, E. W. Findsen, and J. M. Friedman, J. Phys. Chem. 94, 85 (1990).
[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
425
T o
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180
230
280
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330
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(cm
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)
FI~. 7. Low-frequency, time-resolved spectra of cytochrome oxidase subsequent to CO photolysis, in ascending order: (a) equilibrium reduced oxidase, (b) 10 nsec, (c) 125 nsec, (d) 1 /~sec, (e) 150/~sec, (0 750/zsec, (g) 1 msec, and (h) 5 msec. Spectra were obtained with 532 and 440 nm pump-probe beams, respectively, as described in the text. Spectra are the unsmoothed sums of three to five scans at 10 cm-[/min. Spectral band-pass was less than or equal to 7 cm -1 in all cases. (From Findsen et al. 42)
Cytochrome Oxidase Cytochrome oxidase, the terminal enzyme complex of the mitochondrial respiratory chain, has been the subject of intense study because of its electron transfer, proton-pumping function and Oz-reducing catalytic functions. 41 The complex intramolecular electron transfers that occur between cytochrome a/CuA and the binuclear center cytochrome a3/CuB, where the four-electron reduction of molecular oxygen to water occurs, 41 M. Wikstr6m, K. Krab, and M. Saraste, in "Cytochrome Oxidase--A Synthesis." Academic Press, New York and London, 1981.
426
[17]
SPECTROSCOPIC M E T H O D S FOR M E T A L L O P R O T E I N S
427 nm EXCITATION T = 298 K td : 10 ~ s
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[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
427
have been studied by various spectroscopic techniques. Two types of TR 3 experiments have been used to study the enzyme. The first experiment involves monitoring the evolution of the CO photolysis product of the cytochrome a3 transient species. The second has been undertaken to characterize intermediates that occur in dioxygen reduction, which is essential for elucidating the chemical mechanisms of the redox processes catalyzed by the enzyme. Cytochrome a3 Hemopocket Relaxation. In a series of pump-probe pulsed TR 3 measurements, Findsen et al. 42 monitored the full time evolution of cytochrome a 3 hemopocket relaxation subsequent to carbon monoxide photolysis from fully reduced cytochrome oxidase. In this work, a scanning monochromator and photomultiplier tube (PMT) detection was used. Figure 7 shows the low-frequency TR 3 spectra of cytochrome oxidase obtained at various delay times subsequent to CO photolysis from cytochrome a3. The band at 214 cm-1 in reduced cytochrome oxidase has been assigned to the F e - H i s stretching mode. The increased frequency of this vibration in the transient species was explained as a result of a strengthened bond. However, other degrees of freedom, such as histidine rotation relative to the heme plane, were postulated. The TR 3 spectra obtained from 10 to 125 nsec are indicative of protein relaxation prior to heme relaxation. At later times (t > 200 nsec), the v(Fe-His) mode decreases, owing to the rearrangement of the proximal heme pocket. Finally, at t = 1 msec, the frequency of the proximal Fe-His appears at 222 cm- 1, which indicates recombination of CO with cytochrome a 3 and subsequent photodissociation of CO by the probe pulse, which produces again the 10 nsec unligated, transient species. The first conclusion from the work of Findsen et al. 42 is that the correlation between the F e - H i s bond and the frequency of/)4 observed in hemoglobins is absent in cytochrome a3 transients. Moreover, geminate CO recombination is also absent in cytochrome a 3. The second conclusion is 42 E. W. Findsen, J. Centeno, G. T. Babcock, and M. R. Ondrias, J. A m . Chem. Soc. 109, 5356 (1987).
FIG. 8. Time-resolved, resonance Raman spectra of cytochrome oxidase, following initiation of the reaction with oxygen at room temperature. The energy of the 532 nm photolysis pump pulse was 1.3 mJ, sufficient to photolyze the enzyme-CO complex and initiate the Oz-reduction reaction. The energy of the probe beam was 0.3 mJ for spectra A and B, 26 and 1.0 mJ for spectra C - E . The repetition rate for both the pump and probe pulses (10 nsec duration) was 10 Hz. The pump-probe delay was 10/~sec for spectra A - D and 10 nsec for transient spectrum E. The accumulation time was 110 min for spectrum A, 70 min for spectrum B, 5 min for spectra C and D, and 15 rain for spectrum E. (From Varotsis eta/. 48)
428
[171
SPECTROSCOPIC METHODS 'FOR METALLOPROTEINS
427 n m EXCITATION T = 298 K
" cO Ou3
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[17]
TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY
429
that heme pocket relaxation is biphasic, with no change in Fe-His frequency during the first 200 nsec subsequent to CO photolysis. In recent TR 3 and time-resolved IR measurements, Woodruff et al. 43 have found transient CO binding to Cu B following its photodissociation from cytochrome a 3 and rapid binding (< 10 psec) of a photolabile endogenous ligand to cytochrome a 3 . They suggested that the photolabile ligand is transferred from Cu Bto cytochrome a 3 , when CO binds to Cu B, forming a cytochrome a 3 species having axial ligation that differs from the reduced unligated enzyme. The implications of this work for 0 2 binding pathways have attracted considerable recent attention. 44 Dioxygen Intermediates. Although the reaction between cytochrome oxidase and 0 2 occurs too quickly (tl/2 1 msec) to be studied by conventional stopped-flow techniques, Gibson and Greenwood45 used photolysis of the cytochrome a32+-CO complex of the enzyme in the presence of 0 2 to circumvent this limitation. Babcock et al. 9'46'47 adapted the Gibson-Greenwood technique and used time-resolved resonance Raman spectroscopy to study the reaction of fully and partially reduced oxidase with 02. We have extended the flow/flash time-resolved approach so as to be able to observe the low-frequency region of the Raman spectrum. Figure 8 shows the time-resolved resonance Raman spectra of cytochrome oxidase at 10/xsec subsequent to carbon monoxide photolysis in the presence of 0 2 .48'49 Spectrum E (Fig. 8) is that of the photodissociation product of the 43 W. H. Woodruff, O. Einarsdottir, R. B. Dyer, K. A. Bagley, G. Palmer, S. J. Atherton, R. A. Goldbeck, T. D. Dawes, and D. S. Kliger, Proc. Natl. Acad. ScL U.S.A. 88, 2588 (1991). 44 G. T. Babcock and M. Wikstr6m, Nature (London) 356, 301 (1992). 45 Q. Gibson and C. Greenwood, Biochemistry 86, 541 (1963). 46 G. T. Babcock, J. M. Jean, L. N. Johnston, G. Palmer, and W. H. Woodruff, J. Am. Chem. Soc. 106, 8305 (1984). 47 G. T. Babcock, J. M. Jean, L. N. Johnston, W. H. Woodruff, and G. Palmer, J. lnorg. Biochem. 23, 243 (1985). 48 C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Am. Chem. Soe. 111, 6439 (1989); C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Am. Chem. Soc. 112, 1297 (1990). 49 C. Varotsis, W. H. Woodruff, and G. T. Babcock, J. Biol. Chem. 265, 11131 (1990).
FIG. 9. Time-resolved resonance Raman spectra of fully reduced cytochrome oxidase at the indicated times. The energy of the 532 nm photolysis pump/pulse was 1.3 m J, sufficient to photolyze the enzyme-CO complex and initiate the O2-reduction reaction. The energy of the 427 nm probe beam was 0.8 mJ for spectra A and F and 0.3 mJ for spectra B - E . The repetition rate for both the pump and probe pulses (10 nsec duration) was 10 Hz. The accumulation time was 15 rain for both spectra A and F and 50 rain for spectra B - E . The enzyme concentration was 50/~M after mixing, pH 7.4. (From Varotsis and Babcock. 55)
430
SPECTROSCOPIC METHODS FOR METALLOPROTEINS
[17]
reduced carbonmonoxy enzyme (pump-probe delay 10 nsec). Spectrum A (Fig. 8) obtained with a low-energy, defocused beam (0.3 mJ), is similar to the 10 nsec spectrum with the exception that a new mode appears at 571 cm -1. Figure 8B shows that the 571 cm -1 mode in the 160z spectrum is downshifted to 546 cm-1 when the experiment is repeated with 1802 . This allowed us to assign it as the iron-oxygen stretching motion in the cytochrome a32+-O2 complex, as the 24 cm -1 shift is in agreement with that expected from the two-body harmonic oscillator approximation for Fe2÷-O2 . The v(Fe2+-O2) in the oxycytochrome a32+ species is very close to the v(FeZ+-O2) frequency of an imidazole-heme a2+-dioxygen complex model compound. Spectra C and D were obtained with relatively high energies (1 mJ), and the absence of modes located at 571 and 546 cm-1 indicates photodissociation of the oxy ligand, as was observed in the high-frequency experiments. Han e t al. 31'5°'51 and Ogura et al., 52-54 using time-resolved CW techniques, also observed the oxy species. Fig. 9 shows the high-frequency resonance Raman spectra of fully reduced t,,2+., ~- -3 2+~j cytochrome oxidase at various delay times subsequent to carbon monoxide photolysis in the presence of O2.55 In the I0 nsec photoproduct spectrum, the oxidation state marker, v4, is at 1355 cm -1, establishing that both cytochrome a and cytochrome a 3 are in the ferrous state. The core expansion regions shows two vibrations at 1570 and 1586 cm -1. The 1622 cm -1 mode arises from the C~---C stretching vibration of cytochrome a 2+ and a32+. Spectrum B (Fig. 9) shows that no significant changes are detected at 2/,sec in the reaction. As the reaction proceeds, oxidation of cytochrome a 3 and a occurs. At 50/,sec, the oxidation state marker has shifted to 1371 cm- 1, and the shoulder at 1358 cm- 1is substantially decreased, indicating that the oxy adduct of cytochrome a 3 is formed in this reaction. The decrease in intensity of the 1613 cm -1 mode and the concomitant increase in scattering at 1650 cm -1 indicate that partial oxidation of cytochrome a has also occurred at 50 ~sec. Oxidation of cytochrome a remains partial, as indicated by the 1358 cm-1 shoulder in the v4 region in the 500/~sec spectrum. The second phase of cytochrome a oxidation occurs in the 500 >sec to 5 msec time range to produce the oxidized enzyme. Insights into 02 activation and energy conservation in 50 S. Han, Y.-C. Ching, and D. L. Rousseau, Proc. Natl. Acad. Sci. U.S.A. 87, 8408 (1990). 51 S. Han, Y.-C. Ching, and D. L. Rousseau, Nature (London) 348, 89 (1990). 52 T. Ogura, S. Yoshikawa, and T. Kitagawa, J. Am. Chem. Soc. 112, 5630 (1990). s3 T. Ogura, S. Takahashi, K. Shinzawa-Itoh, S. Yoshikawa, and T. Kitagawa, J. Biol. Chem. 265, 14721 (1990). 54 T. Ogura, S. Takahashi, K. Shinzawa-Itoh, S. Yoshikawa, and T. Kitagawa, Bull. Chem. Soc. Jpn. 64, 2901 (1991). s5 C. Varotsis and G. T. Babcock, Biochemistry 29, 7357 (1990).
[18]
R E S O N A N C E R A M A N SPECTRA OF M E T A L L O P R O T E I N S
431
cell respiration that can be obtained by combining time-resolved techniques with biochemical approaches have been discussed in detail by Babcock and Wikstr6m. 44 Conclusions In summary, T R 3 spectroscopy is a technique that can be used to probe structural and conformational as well as kinetic properties of transient species. It appears that the two-pulse, pump-probe, time-resolved Raman approach, in conjunction with a single monochromator and a CCD detector, provides the most reliable configuration to record the time evolution of transient species.
[18] T e c h n i q u e s for O b t a i n i n g R e s o n a n c e R a m a n S p e c t r a of Metalloproteins
By THOMAS M. LOEHR and JOANN SANDERS-LOEHR Introduction The focus of this chapter is pragmatic. It provides the reader with tested laboratory procedures that illustrate how resonance Raman spectra of metalloproteins are obtained and verified, how active-site structural and/or mechanistic information is probed by isotopic labeling, and how spectral data interpretation is initiated. The methodology described here, including the majority of illustrative examples, is based on the investigation of copper- and iron-containing proteins carried out in the authors' laboratory. This specific-methods approach discusses sample preparation and temperature control, choice of scattering geometries, data collection, data quality, and the initiation of data analysis. It describes common precautions and pitfalls, as well as criteria for judging sample integrity. It shows how one confirms the resonance Raman phenomenon and also discusses the utility of excitation profiles. This chapter is not intended to review the resonance Raman literature on copper and iron l~roteins. The general theory of Raman spectroscopy, and resonance Raman spectroscopy in particular, including a description of Raman instrumentation, laser sources, and general applications of the technique, is presented in [14] in this volume. The special area of UV resonance Raman spectroscopy, probing ultraviolet-absorbing amino acids and nucleic acids, is covered in [15]. The use of micro (resonance) METHODS IN ENZYMOLOGY, VOL. 226
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