The Journal of
Physical Chemistry
0 Copyright, 1982, by the American Chemical Society
VOLUME 86, NUMBER 6
MARCH 18, 1982
LETTERS Plcosecond Resonance Raman Spectrum of the Oxyhemoglobin Photoproduct. Evidence for an Electronically Excited State James Terner,t* David F. Vom,$ Carolyn Paddock,$ Richard B. Miles,$ and Thomas 0. Spiro't Depertment of chemistry, and Depemnent of Mechenlcel and Amspace Englneed~,Mnceton Untverstty, Mnmton, New Jersey 08544 (Rmelved: October 7, 1981; I n F h l F m : November 30, 1981)
The resonance Raman (RR) spectrum of the HbOzphotoproduct has been obtained with 30-ps, 532-nm pulses from a 0.1-mJ/pulse mode-locked NdYAG laser. This spectrum differs appreciably from those reported previously for HbCO, and for Hb02with longer, weaker pulses. These differences are attributed to a prompt photoproduct,which can be associated with a previously observed Hb02spectral intermediate that decays within 90 ps. The prompt intermediate is inferred, on the basis of the RR band frequencies, to be an electronically excited state of deoxyHb with substantial T-T* character. It might be a triplet T-A* state, formed via the dissociation of triplet O2 from Hb02
Introduction There is much current interest in the time course of the photoinduced deligation of hemoglobin (Hb) and myoglobin (Mb). The absorption of picosecond laser pulses by the O2 or CO complexes of these proteins is followed by rapid absorptivity changes leading to spectral intermediates that resemble, but are not identical with, deoxyHb.'* The absorption spectra are essential for identifying the intermediates but provide little information about their molecular structures. Resonance Raman (RR) spectroscopy, which can be time-resolved with pulsed laser sources, is capable of giving detailed structural information about chromophoric molecules, and in particular the heme group of heme
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proteins. Excitation of HbCO,with 10-ns laser pulses, using coherent' or spontaneouss~Raman techniques, gave photoproduct spectra resembling that of deoxyHb, but with small frequency down~hifts.~ Similar spectra were observed with -30-ps pulses,l*J1 and the downshifts were ~
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(1) Shank, C. B.; Ippen, E. P.; Bersohn, R Science 1976, 183, 50. (2) Noe, L. J.; Eisert, W. G.; Rentzepis, P. M. R o c . Natl. Acad. Sci. U.S.A. 1978, 75,573. (3) Noe, L. J.; Eisert, W. G.; Rentzepis, P. M. Biophys. J. 1978, 24, 379-81. (4) Greene, B. I.; Hochstrasser, R. M.; Weisman, R. B.; Eaton, W. A. R o c . Natl. Acad. Sci. U.S.A. 1978, 75, 5256-9. (51 Eisert. W. G.; Deaenkolb, E. 0.: Noe, L. J.; Retzepis, P. M. Biophye. J. 1979,25,455. (6) Chernoff, D. A.; Hochstrasser, R. M.; Steele, A. W. Proc. Natl. Acad. Sci. U.S.A. 1980. 77,5606-10. (7) Dallinger, R. F.; Nestbr, J. R.; Spiro, T. G. J . Am. Chem. SOC.1978 100,6251-2.
'Current address: Department of Chemistry, Virginia Commonwealth University, Richmond, Va 23284. *Department of Chemistry. fl Department of Mechanical Engineering. 0022-3654182f2088-Q859$Q1.2510
(8) Woodruff, W. H., Farquhmon, S. Science, 1978, 901,831-3.
(9) Lyons, K.B.;Friedman, J. N.; Fleury, P. A. Nature (London) 1978, 275, 565-6.
(IO) Terner, J.; Spiro, T. G.; Nagumo, M.; Nicol, M. F.; El-Sayed, M. A. J. Am. Chem. SOC.1980,102, 3238-9.
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The Journal of Physical Chemistty, Vol. 86, No. 6, 1982
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interpreted as reflecting a high-spin heme with the Fe atom closer to the heme plane than in deoxyHb, presumably due to unrelaxed protein forces. The relaxation time for the disappearance of these downshifts, reflecting the movement of the Fe atom to its out-of-plane position in deoxyHb,12has subsequently been determined to be in the 20-300-11s range,13 a time consistent with a large-scale tertiary structure change, but shorter than the known relaxation time for the R-T quaternary structure change in deoxyHb.I4 The RR results on HbCO are consistent with the early time absorption spectrum of the HbCO photoproduct, which resembles that of deoxyHb, but is broader; it is developed within 10 ps and persists for a t least 680 ps.* The 10-ps spectrum of Hb02 is appreciably different but relaxes within 90 ps to a spectrum like that of the HbCO photoproduct.6 A 30-ps RR spectrum of Hb02, obtained by Coppey et al.l5 with 10-Hz, 40-mJ, 532-nm YAG pulses, showed some photolysis, with a deoxy-like contribution, but the resolution and signal-to-noise were insufficient to determine the photoproduct spectrum. Recently, Nagumo et al.)6 using the approach previously applied to HbCO'OJ' (0.8-MHz, 5-nJ, 575-nm pulses from a synchronously pumped mode-locked dye laser), produced a -50-ps RR spectrum of the Hb02 photoproduct which was within experimental error the same as that of the HbCO photoproduct.16 We have now reexamined the -30-ps YAGexcited Hb02photoproduct, and find that its RR spectrum is distinctly different from that reported by Nagumo et al.16 We tentatively identify it as arising, in part, from electronically excited de0xyHb.l'
Experimental Section Hemoglobin was isolated from human whole blood, obtained from a local hospital, by the following procedure: The red cells were washed repeatedly in 0.9% NaCl, and then lysed in distilled water. The resultant suspension was centrifuged repeatedly to remove particulates. When the supernatant hemoglobin was clear, it was deoxygenated by gentle stirring under water-vapor-saturated nitrogen gas at 4 "C, and stored. Experimental samples were diluted to approximately 1mM in 0.5 M phosphate buffer at pH 7.0. The detection system was an optical multichannel analyzer (Princeton Applied Research 1205A) with extended delay accessory (1207) and a silicon-intensified vidicon (1205D) in a dry ice cooled housing (1212). The vidicon was attached to a Spex Model 1401 0.75-m double spectrometer equipped with a 0.85-m extended focal length exit mirror and a 10-mm central slit. Data were output onto paper tape and processed by a MINC (Digital) computer. Frequency doubled (5320 A) pulses from a passively mode-locked YAG laser were used to excite the RR spectra and simultaneously to produce partial photolysis of HbOP Single pulses of 30-ps duration were switched out, at the 10-Hz laser repetition rate, and amplified with a double pass YAG amplifier,18before frequency doubling. The
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(11) Terner, J.; Stong, J.; Spiro, T. G.; Nagumo, M.; Nicol, M. F.; El-Sayed, M. A. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,1313-7. (12) Perutz, M. F. Br. Med. Bull. 1976,32,196-207. (13) Stein,P.; Terner, J.; Spiro, T. G. J.Phys. Chem. 1982, SS,166-70. (14) (a) Sawicki, C. A.; Gibson, Q. H. J. B i d . Chem. 1975,251,1533. (b) Cho, K.C.; Hopfield, J. J. Biochemistry 1979, 18, 6826. (16) Coppey, M.; Tourbez, H.; Valat, P.; Alpert,B. Nature (London) 1980,284,668-70.
(16) Nagumo, M.; Nicol, M.; El-Sayed, M. A. J.Phys. Chem. 1981,&, 2436-8. (17) Cornelius, P. A.; Steele, W. A,; Chernoff, D. A.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. In press. (18) Miles, R. B.; Laufer, G.; Paddock, C.; Faris, G. App. Opt. 1980, 19, 3595.
Figure 1. The -30-ps frequency doubled (5306 A) YAG laser excitedRaman spectra of (a) partlaHy phobolyzedHb02,obtained with tlght focussing of the laser (- 100-pm spot); (b) Unphotolyzed HbO,, obtalned with loose focussing; (c) Hb02 photoproduct, obtained by substracting spectrum b from spectNm a, with a scale factor adjusted to blank out the 1640-cm-' band; (d) deoxyHb. See text for experimental details.
TABLE I: Resonance Raman Band Frequencies (cm-' ) v10
deoxyHb HbCO photoproduct (and delayed HbO, photoproduct) HbO, photoproduct (prompt)
*19
VI1
1605 1556 1603 1552
1549 1543
1590
1538
1550
pulse energy was 0.2 mJ, giving an average power of 2 mW. The oxyhemoglobin sample was pumped through a 30gauge syringe needle at a velocity of 5 m/s and recirculated. Since the laser pulse frequency was 10 Hz, the sample volume was excited by only one pulse and had several seconds to relax and recombine with oxygen before being pumped again through the syringe needle. The method of obtaining the picosecond resonance Raman spectrum of photolyzed oxhemoglobin was similar to that described previously for CO-hemoglobin.lo With high oxyhemoglobin sample concentrations and diffusely focussed laser excitation, a negligible fraction of the sample was photolyzed. To increase this fraction, the sample concentration was lowered by dilution with buffer and the laser excitation was focussed to 100 um. In this manner
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The Journal of Physical Chemistty, Voi. 86, No. 6, 1982 881
we were able to photolyze approximately 20-309'0 of the irradiated volume. If the laser excitation was focussed more tightly, stimulated processes were observed. This resulted in a broad emmission and/or burning of the sample. The resonance Raman spectrum of deoxyhemoglobin was obtained by recirculating deoxyhemoglobin (0.5 mM) through a 1-mm glass capillary, with the reservoir stirred gently under water-vapor-saturated nitrogen gas.
Results and Discussion Figure 1,a and b, show the YAG-excited spectra of Hb02 with strong and weak focussing of the laser beam. Spectrum l b is that of essentially unphotolyzed Hb02 The strong bands at 1640 (depolarized, dp) and 1586 (anomalously polarized, ap) cm-l are identifiable as the vl0 (Bk) and v19 (A ) porphyrin modes.lg The shoulder (dp) at -1560 cm3 is mainly vll (BIJ with a small contribution from the photoproduct. This contribution increases substantially upon tightly focussing the laser beam, as shown in spectrum la. The YAG-excited RR spectrum of deoxyHb, Figure Id, shows a strong band (dp) at 1605 cm-', and a pair of overlapping bands, which can be deconvoluted via polarization spectral' into ap and d p components at 1556 and 1549 cm-'. These three bands are also v10, vlg., y d v11. They all respond to the core size of the porphyrin ring,lgb which is larger in high-spin (deoxyHb) than in low-spin (Hb02) hemes, due to the population of antibonding d, orbitals. The RR spectrum of the HbCO product at early and of the Hb02 product excited with weak 50-ps pulses,16is quite similar to that of deoxyHb, except for small, but reproducible, downshifts: 1605 1603,1556 1552, and 1549 1543 cm-'. The downshifts were as reflecting an expanded porphyrin core, consistent with the Fe atom being closer to the heme plane than in deoxyHb. Indeed, a model complex (THF),FenPP (THF, tetrahydrofuran; PP, protoporphyrin), in which the high-spin Fen is expected to be in the porphyrin plane, as in the structurally characterized (THF),FeTPP (TPP, tetraphenylporphine),20 gave vl0 and vlg (vll was unresolvable) frequencies within 1 cm-l of those seen for the photoproduct." The spectrum of the Hb02photoproduct obtained in the present experiment, Figure IC,was obtained by substracting spectrum l b from spectrum l a with a weighting factor adjusted to blank out the well-isolated Hb02 band at 1640 cm-'. This spectrum is quite different from that of the previously observed HbCO and HbOa photoproduct spectra There is no band at 1603cm-', but there is a band at 1590 cm-'. The other peak, at 1540 cm-', is part of a broad band which clearly has additional components both at higher and lower frequencies. Bands at 1538 (dp) and 1550 (ap) cm-' were resolved with the aid of polarization spectra. This polarization pattern indicates that the 1590and 1538-cm-' peaks may arise from vl0 and vll downshifted from the earlier photoproduct spectra. The frequencies are summarized in Table I. The additional downshifts cannot readily be ascribed to further porphyrin core expansion. In (THF)2FeTPP,20 whose PP analogue gives the same vll and v19 frequencies as the HbCO photoproduct,l' the Fe atom is exactly in the porphyrin plane, and the porphyrin is expanded (2.065 A
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(19)(a) A b , M.;Kitagawa, T.; Kyogoku, Y. J. Chem. Phys. 1978,69, 4526. (b) Choi, S.;Spiro, T. G.; Langry,K. C.; Smith, K. M.;Budd, D. L.; LaMar, G.N. J. Am. Chem. SOC. In preee. (20) Reed, C. A.; Mashiko,T.; Scheidt, W. R.; Spartalian, K.; Lang, G. J . Am. Chem. SOC.1980,102, 2302-6.
center to pyrrole N distance) as far as in any known Fe porphyrin complex. There is no obvious mechanism for producing a larger expansion. However, vl0 and vll are also sensitive to the effects of electron donation into the lowest Thus both unoccupied ?r orbital of the porphyrin, v10 and vll are strongly downshifted in (ImH)2Fe11MP (ImH, imidazole; MP, mesoporphyrin), relative to [(ImH)2Fen1MP],+reflecting the d, eg* back-bonding in the former, and the downshifts are progressively diminished as ImH is replaced by a-acid ligands, which compete with the porphyrin for the d, electrons.23 Moreover bands of ZnEP (EP, etioporphyrin) which are assignable to ul0 and vll, at 1613 and 1562 cm-', have been observed to shift down, by 32 and 25 cm-l, upon formation of the porphyrin dianion, ZnEP2- (partial downshifts are seen for the monoanion as well).24 These considerations lead us to attribute the 1590- and 1538-cm-' Hb02photoproduct bands to vl0 and vll arising from an electronidy excited high-spin heme. If this state has appreciable T-A* character, the 12- and 5-cm-' downshifts, relative to the HbCO photoproduct, would be consistent with the sensitivity of vl0 and vll to e * orbital occupancy. This assignment is supported by t8e recent observation by Cornelius et al.17 of a common prompt spectral intermediate upon excitation of Mb02, MbCO, and deoxyMb, the extent of formation decreasing in the order Mb02 > deoxyMb > MbCO. The intermediate was suggested to be an excited state of deoxyMb, which could be formed directly from Mb02 but not from MbCO (the small amount of intermediate formed from MbCO could have been generated from photoproduced deoxyMb). It is plausible that in the present experiment we are observing the rapidly relaxing spectral intermediate observed for Hb02 by Chernoff et a1.: and that this intermediate is electronically excited deoxyHb, with substantial m*character. Thii might be a triplet m*state, formed directly by the dissociation of triplet O2from photoexcited HbOz (and therefore not formed via dissociation of CO from HbCO). Hochstrasser and co-workerP have recently found evidence for a triplet state of ClFeTPP, which can be pumped with intense picosecond laser pulses, despite the rapid relaxation of Fe porphyrins via the low-lying ligand field states. The envelope of the Hb02 photoproduct RR bands is broad, and it seems likely that we are observing a mixture of prompt and delayed photoproducts. This is consistent with the disappearance of the prompt spectral intermediate within 90 ps,6 and with the observation by Nagumo et a1.I6 of a HbCO-like photoproduct RR spectrum with weak pulses of -50-ps duration. It is expected that electronic relaxation of excited deoxyHb would rapidly generate ground-state high-spin heme; the Fe atom would be constrained near the heme plane by protein forces, as in the HbCO photoproduct. Acknowledgment. We thank Eric Lobenstine and Siddharth Dasgupta for technical assistance and helpful discussions, and Professors M. A. El-Sayed and R. M. Hochstrasser for communicatingresults of their work prior to publication. This work was supported by NIH Grant HL 12526 (to T.G.S.) and NSF Grant ECS-8017655 (to R.B.M).
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(21) Spiro, T. G.; Strekas, T. C. J. Am. Chem. SOC. 1974,96,338-45. (22) Kitagawa, T.;Iizuka, T.;Saito, M.;Kyogoku, Y. Chem. Lett. 1975, 849.
(23) Spiro, T. G.; Burke, J. M.J . Am. Chem. SOC.1976, 98,5482. (24) Ksenoforitova, N. M.;Madov, V. G.; Sidorov, A. N.; Bobovich, Ya S.Opt. Spectrosc. 1976, 40, 462-5. (25) Cornelius, P. A.; Steele, A. W.; Chernoff, D. A.; Hochstrasser, R. M. Chem. Phys. Lett. 1981,82, 9-14,
