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J. Phys. Chem. 1995, 99, 4842-4846
Vibrational Relaxation of the CO Stretch Vibration in Hemoglobin-CO, Myoglobin-CO, and Protoheme-CO J. C. Owrutsky3 M. Li, B. Locke, and R. M. Hochstrasser" Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania I91 04 Received: September 23, 1994; In Final Form: January 24, 1995@
Two-color infrared pump-infrared probe spectroscopy has been used to measure vibrational relaxation times ( T I ) for the C O stretching vibrations of carboxyhemoglobin (HbCO), carboxymyoglobin (MbCO), and carboxyprotoheme, all in D20 at 300 K. The times are fast (17-31 ps) compared to most previously measured metal carbonyl TI times. The C O relaxation is found to be somewhat faster in HbCO and MbCO than in PCO. The transient infrared spectra and anisotropy were also measured.
Introduction The desire to understand the nature of cooperative ligand binding and dissociation of heme proteins has inspired many experimental and theoretical studies on this system. It is clearly useful to examine various environmental factors that are involved, including the ligand binding strength and geometry, as well as more subtle influences, such as those from the solvent and protein host. Efforts to explore these issues have motivated recent ultrafast studies on heme proteins both with and without ligands such as CO. These experiments typically involve photoexcitation of the heme followed by transient optical, Raman, or IR detection to monitor the temporal evolution of species such as ligands andor the heme in order to explore various aspects of the dynamics, such as the ligand dissociation and rebinding, heme cooling, and energy transfer to the surrounding solvent and protein. 1-6 In addition to population relaxation studies, polarization-dependent transient IR detection provides anisotropy measurements which indicate the ligand geometry relative to the heme plane. These measurements have been made using picosecond7 and femtosecond8 transient IR. The vibrational population relaxation time ( T I )of the ligand should be sensitive to its environment, so that T1 measurements for various heme proteins might reveal evidence about the ligand bond and perhaps about the heme-ligand bond dynamics, where the latter is of crucial importance in ligand binding processes. In this work we report vibrational relaxation times and transient spectra for the CO stretch in carboxyhemoglobin (HbCO, AI), carboxymyoglobin (MbCO), and carboxyprotoheme IX (PCO) by two-color transient infrared spectroscopy. The measured relaxation times 53'( ps) are among the fastest for metal carbonyls and are not much slower than those measured for adsorbates on clean metal surfaces. The anisotropy was also measured to probe the rigidity of the CO orientation. The CO ligand vibrational frequencies (-1950 cm-l) in heme-CO molecules are similar to those for terminal CO moieties (-1950-2030 cm-') in metal carbonyls. The IR absorption coefficient for the CO stretch vibration is greatly increased by the metal-CO bonding, but the vibrational frequencies are not as strongly affected. The heme CO and terminal metal carbonyl (and top-site surface adsorbate CO) frequencies are only somewhat lower than that of the free carbon +Present address: Code 6111, Chemistry Division, Naval Research Laboratory, Washington, D.C. 20375. Abstract published in Advance ACS Abstracts, March 1, 1995. @
monoxide (2143 cm-I), while organic carbonyls (1600- 1700 cm-'), bridged metallocarbonyl, and bridge surface groups (-1700- 1800 cm-I) are considerably 10wer.~ The question of interest in this work is the mechanism of coupling vibrational energy in the CO bond into other degrees of freedom of the metal carbonyl compound (IVR) and the competing process in which energy is transferred directly to surrounding solvent motions (Le., by external vibrational redistribution, EVR) in CO bound to heme and heme proteins. There have already been several vibrational relaxation time measurements for metal carbonyls.1° In these experiments an infrared pulse was used to excite the v = 1 level of a CO stretch, and the time dependence of the recovery of the bleached v = 0 1 transition or of the decay of the v = 1 2 excited state absorption was studied by probing with a delayed pulse. The measured relaxation times vary considerably from molecule to molecule. Some examples are as follows: for WCO6, 800 ps in CC4 and 70 ps in C& (ref loa); 101 ps for Rh(C0)z(C5H702) in CHC13 (ref 10e); and oom, S . K.; Stoutland, P. 0.; Dyer, R. B.; Woodruff, W. H. J . Am. Chem. SOC.1992, 114, 3133. (12) Owrutsky, J. C.; Diller, R.; Iannone, M.; Cowen, B.; Maiti, S.; Li, M.; Sarisky, M.; Kim, Y.;Locke, B.; Hochstrasser, R. M. Proc. SHE-lnt. Soc. Opt. Eng. 1992, 1599, 52. (13) Owrutsky, J. C.; Li, M.; Culver, J. P.; Sarisky, M. J.; Yodh, A. G.; Hochstrasser, R. M. In Time-Resolved Vibrational Spectroscopy W,Lau, A., Siefert, F., Wernke, W., Eds.; Springer-Verlag: Berlin, 1993. (14) Hochstrasser, R. M. Proc. SPZE-Znt. SOC.Opt. 1992, 1921, 16. (15) Hill, J. R.; Tokmakoff, A.; Peterson, K. A.; Sauter, B.; Zimdars, D.; Dlott, D. D.; Fayer, M. D. J . Phys. Chem. 1994, 98, 11213. (16) Oxtoby, D. W. Adv. Chem. Phys. 1981, 47, 487. (17) Owrutskv. J. C.: Rafterv. D.:. Hochstrasser. R. M. Annu. Rev. Phvs. Chem.'1994, 45,'519. ' (18) (a) Zinth. W.: Kolmeder. C.: Bena. B.; Irgens-Defregger A.; Fischer, S. F.; Kaiser, W. J . Chem. Phys. 1983, 78,3916: (b) FendCA.; Fischer, S. F.; Kaiser, W. Chem. Phys. 1981,57,55. (c) Ambroseo, J. R.; Hochstrasser, R. M. J . Chem. Phys. 1988,89,5956. (d) Graener, H.; Ye, T. Q.; Laubereau, A. J. Chem. Phys. 1989, 90, 3413. (19) Li, M.; Owrutsky, J.; Sarisky, M.; Culver, J. P.; Yodh, A.; Hochstrasser, R. M. J . Chem. Phys. 1993, 98, 5499. (20) Graener, H.; Seifert, G.; Laubereau, A. Chem. Phys. 1993, 175, 193. (21) (a) Proceedings of the Conference on Vibrations at Surfaces. J . Electron Spectrosc. Relat. Phenom. 1990,54/55. (b) Cavanagh, R. R.; King,
D. S.; Stephenson, J. C.; Heinz, T. F. J . Phys. Chem. 1993, 97, 786. (c) Eisenthal, K. B. Annu. Rev. Phys. Chem. 1992.43, 627. (22) (a) Whitnell, R. M.; Wilson, K. R.; Hynes, J. T. J . Phys. Chem. 1990,94, 8625. (b) Whitnell, R. M.; Wilson, K. R.; Hynes, J. T. J . Chem. Phys. 199,96,5354. (c) Bruehl, M.; Hynes, J. T. Chem. Phys. 1993,175, 205. (d) Ferrario, M.; Klein, M. L.; McDonald, I. R. Chem. Phys. Len. 1993,213,537. (e)Benjamin, I.; Whitnell, R. M. Chem. Phys. Lett. 1993, 204, 45. (23) (a) Harris, C. B.; Shelby, R. M.; Comelius, P. A. Phys. Rev. Lett. 1977,38, 1415. (b) Shelby, R. M.; Harris, C. B.; Comelius, P. A. J . Chem. Phys. 1979, 70, 34. (c) Marks, S.; Comelius, P. A.; Harris, C. B. J . Chem. Phys. 1980, 73, 3069. (24) Li, X. Y.; Spiro, T. G. J . Am. Chem. SOC. 1988, 110, 6024. (25) As indicated in refs 12 and 13, these T I times were measured using two-color IR pump-probe for various CO stretches of FeCp(C0)z dimer in CC4. For the cis isomer, the terminal CO stretch (near 2004 cm-I), the T I time is 30 ps, and for a spectrally unresolved isomeric mixture of cis and trans, the bridge CO stretch (near 1785 cm-I), it was 18 ps. (26) It recently came to our attention that the anisotropy measurements in refs 7 and 8 are subject to a small but significant error that casts doubt on the magnitudes of the bending and tilting angles reported in these papers. See: Locke, B.; Lian, T.; Hochstrasser, R. M. Chem. Phys. 1995,190,155. (27) McCalley, R. C.; Shimshick, E. J.; McConnell, H. M. Chem. Phys. Lett. 1972, 13, 115. (28) Szabo, A. J . Chem. Phys. 1984, 81, 150.
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