J. Phys. Chem. 1982, 86, 168-170
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Hemoglobin R-State Iron-Imidazole Frequency Observed by Time-Resolved Resonance Raman Spectroscopy Paul Steln, James lerner, and Thomas 0. Splro' Depattment of Chemistty, Wnceton Universitj', Wnceton, New Jersey 08544 (Received: September 24, 1981; In Final Form: December 1, 198 I )
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Resonance Raman (RR) spectra have been obtained of the photolysis product of CO-hemoglobin (COHb) formed within 0.3 ps, before the R T quaternary structure switch of deoxyhemoglobin(deoxyHb). The band due to iron-imidazole (Im) stretching was observed to be more symmetric and was shifted to higher frequency, relative to that of deoxyhemoglobin. The oxidation-state marker band was observed to be shifted to lower energy, 1355 cm-l, aicompared to stable deoxyhemoglobin at 1357 cm-'. The porphyrin core-size marker bands, however, were at essentially the same frequencies as for deoxyhemoglobin, showing that the shifts observed previously on a shorter time scale are associated with tertiary, rather than quaternary, structure and reflect a different molecular process than the R T transition. --+
Introduction The dynamics of CO-hemoglobin photodissociation have recently been studied with time-resolved resonance Raman The heme resonance Raman spectra of the CO-hemoglobin photoproduct on the nano~econdl-~ and picosecondH time scales have been shown to be similar to that of deoxyhemoglobin, but with small downshifta in frequencies knowngto be sensitive to the prophyrin core size. These results have been interpreted5 as implying a fast (less than 30 ps) spin-state change, via intersystem crossing to a dissociative ligand field state of COhemoglobin'O and a slower (greater than 20 ns) relaxation to the out-of-plane heme structure characteristic of deoxyhemoglobin. The slowness of this latter relaxation was suggested to result from constraints of the globin tertiary s t r u ~ t u r e . In ~ the present study we have observed the resonance Raman spectrum of photolyzed CO-hemoglobin in the R state, produced during a 0.3-ps laser photolysis interval. Experimental Section The experiment made use of a continuous wave (CW) laser beam focused through a fine microscope objective (Zeiss Neofluar 40X) onto a vertically flowing sample jet, as diagrammed and described in ref 11. Hemoglobin was prepared from fresh human red blood cells, deoxygenated by stirring under argon at 4 "C, followed by stirring under carbon monoxide (0.8 mM, pH 7.4). The CO-hemoglobin was pumped through a 30-gauge syringe needle (inside diameter, 150 pm) at a bulk flow rate of 0.1 m L / s resulting in a stream velocity of 6 m/s. The CW beam from an argon or krypton ion laser was focused to a 1-pm spot onto (1) Dallinger, R. F.; Nestor, J. R.; Spiro, T. G. J. Am. Chem. SOC. 1978, 100,6251-2. (2) Woodruff, W. H.; Farquharson, S. Science, 1978,201,831-3. (3) Lyons, K. B.; Friedman,J. M.; Fleuy, P. A. Nature (London) 1978, 275, 565-6. (4) Terner, J.; Spiro, T. G.; Negumo, M.; Nicol, M. F.; El-Sayed, M. A. J. Am. Chem. Soc. 1980,102,3238-9. (5) Terner, J.; Stong, J. D.; Spiro, T. G.; Nagumo, M.; Nicol, M. F.; El-Sayed, M. A. R o c . Natl. Acad. Sci. U.S.A. 1981, 78, 1313-7. (6)CoPpeY. M.: Tourbez, H.; Valet, P.: Alpert, B. Nature (London) 1980,284, -568-9. (7) Friedman, J. M.; Lyons, K. B. Nature (London) 1980,284, 570. (8) Lvone. K. B.: Friedman.J. M. 'Svmweium on Interaction Between Iron and Proteina h Oxygen &d Elect;on'Transport"; Ho, C., Eaton, W. A., Eds.; Elsevier: in press. (9) Spiro, T. G.; Stong, J. D.; Stein, P. J.Am. Chem. SOC. 1979,101, 2648-55. (10) Greene, B. I.; Hochstraaser, R. M.; Weisman, R. W.; Eaton, W. A. R o c . Natl. Acad. Sci. U.S.A. 1978, 75, 5255-9. (11) Temer, J.; Hsieh, GI.; Bums,A. R.; El-Sayed, M. A. Proc. Natl. Acad. Sei. U.S.A. 1979, 76, 3046-50. 0022-3654/82/2086-0168$01.25/0
the vertical jet, providing both a photolysis and Raman scattering source. Allowing a factor of 2 for the spot size uncertainity, we estimate that the laser interaction time with the flowing sample was less than 0.3 M. The Raman spectra represent an integration over this time interval. Two data acquisition systems were used. A fast detection system consisted of a Princeton Applied Research Corp. Model 1205A optical multichannel analyzer ( O M ) with an extended delay accessory, and a SIT vidicon (1205D) contained in a dry-ice-cooled housing. The operation of this system is described in ref 5. The vidicon housing was mounted on a Spex Model 14010.85-m double spectrometer equipped with two 600 grooves/" ruled gratings and a 1-m extended focal length output mirror. For low-energy resonance Raman spectra, and to obtain accurate frequency calibrations, a conventional photoncounting, scanning double monochromator was used; it consisted of a Spex 1401 with ruled 1200 grooves/mm gratings, an RCA C31034 photomultiplier tube, and Ortec photon counting electronics. Results and Discussion Hemoglobin cooperativity is associated with the transition between two quaternary structures, T (tense) and R (relaxed), upon binding O2to deoxyHb.12 The T state, which has a low O2affinity, is more stable for deoxyHb while the R state, which has a high O2affinity, is stabilized by successive O2binding to the hemes. It is possible to generate R-state deoxyHb by photolyzing 02Hb,or COHb, which has a higher quantum yield for photolysis. This form of deoxyHb reacts rapidly with ligands, and the subsequent relaxation to a slowly reacting form, presumably the T state, has been studied extensively by Gibson and co-workers.'& The relaxation time is in the 2-100-ps range, depending on ~0nditions.l~ The laser interaction time of our experiment, 10.3 ps, assures that we are examining R-state deoxyHb from COHb photolysis. The molecular mechanism of the R-T transition has been the focus of much research and discussion. Perutz12 has drawn attention to the bond between the heme iron atom and the proximal imidazole ligand and has suggested that tension on this bond in the T state may be responsible for the lowered O2affiity. With the recent assignmentlP17 (12) Perutz, M. F. Br. Med. Bull. 1975,32, 195-207. (13) (a) Sawicki, C. A; Gibson, Q. H. J. Biol. Chem. 1966,164,703. (b) Cho, K. C.; Hopfield, J. J. Biochemistry 1979, 18, 5826. (14) Kincaid, J.; Stein, P.; Spiro, T. G. Roc. Natl. Acad. Sci. U.S.A. 1979, 76, 544-52, 4146. (15) Kitagawa, T.; Nagai, K.; Tsubaki, M. FEBS Lett. 1979, 104, 376-8.
0 1982 American Chemical Society
The Journal of Physical Chemlstty, Vol. 86, No. 2, 1982 169
Letters (a)
RN I
photolyzed COHb
a
zoo
n
8::
Photolynd HbCO
I. Low-frequency Raman spectra, Showing vFbIm 220 cm-l, wlth B band excitation, X, = 4545 A, of (a) HbCo (1 mM, p~ 7) owing in a free jet through (0.3-ps interaction time) the laser beam, which Is both the photolysis and the Raman wee, and (b) deoxytlb (0.3 mM, pH 7) in a recirculatingcapillary. Spectra obtained in scanning mode: 5-cm-' increments. 1 slincrement. of the stretching vibration of the iron-imidazole bond, N 220 cm-' in the RR spectra of deoxyHb and model compounds, much interest attaches to the low-frequency RR spectrum. Figure 1shows this region of the spectrum for the COHb photoproduct and for deoxyHb, obtained with 454.5-nm excitation, where vFeh enhancement is strong. A preliminary report of this result has appeared elsewhere.ls This peak is seen to be symmetric, and centered at 223 cm-l, in the photoproduct spectrum, whereas for deoxyHb it is asymmetric, with a dominant component at 200 cm-l and a shoulder at 207 cm-'. The same change has been observed by Nagai et a1.,16 for the chemically modified hemoglobins, NES des-Argl4laHb and des-Hi~l~fl-Arg'~~", which are believed to adopt the R quaternary state.lg Nagai and Kitagawa20 have shown, using valency hybrid Hb's, that the 220- and 207-cm-l components of the deoxyHb band can be assigned to the 6 and a chains, respectively. The substantial R T shifts, particularly for the a chains, were interpreted20 in terms of molecular tension in the T state,12 although possible changes in proximal imidazole H bonding provide an alternative explanation.21 The present kinetic experiment confirms the validity of the modified hemoglobins as models for R-state Hb A, at least as far as the ironimidazole interaction is concerned. There is definitely a downshift in v F e h in T- vs. R-state deoxyHb. A major goal of this study was to examine the relaxation of the heme structure itself, following COHb photolysis. It has been established via pulsed laser RR studiess5 that at shorter times, 30 ps-20 ns, the porphyrin core-size marker bands V, IV,and IIIs were similar in frequency to those of deoxyHb but were slightly downshifted. The frequencies were within experimental error ( f 2 cm-') of those observed5for a high-spin 6-coordinate Fen heme, in which the iron atom is constrained to remain in the porphyrin planeF2 It was therefore suggested that the low
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(16) Nagai,K.; Kitagawa, T.; Morimoto,H. J. Mol. Biol. 1980, 136, 271-89. (17) Hori, H.; Kitagawa, T. J. Am. Chem. SOC.1980, 102, 3608-13. (18) Stein,P.; Terner, J.; Spiro, T. G. Biophys. J. 1981, 33, 8OA. (19) (a) Perutz, M. F.; Heidner, E. J. Ladner, J. E.; Beetlestone, J. G.; Ho, C.; Slade, E. F. Biochemistry 1974,13,2187-200. (b) Fung,L.W.-M.; Ho,C. Zbid. 1976,14,2526-35. (20) Nagai,K.; Kitagawa, T. R o c . Natl. Acad. Sci. U.S.A. 1980, 77, 2033-7. (21) Stein,P.;Mitchell, M.; Spiro, T. G. J. Am. Chem. SOC.1980,102, 7795-7. (22) Reed, C. A.; Maahiko, T.; Scheidt, W. R.; Spartalian, K.; Lang, G. J . Am. Chem. SOC.1980,102, 2302-6.
,,
Figure 2. High-frequency Raman spectra with Q band excitation, A,, = 5682 A, of the same samples as in Figure 1. Bands 111, IV, and V, at 1545, 1556, and 1605 cm-' for deoxyHb, are within 1 cm-' of the same frequencies for the COHb photoproduct; the 1584- and 1633-cm-' bands are remnant bands (IV and V) of unphotoiyred COHb. Spectra obtained with a silicon-intensified vidicon ( M A I) at 0.2 Ahhannei.
photolyzed
0
deoxyHb
Figure 3. High-frequency Raman spectra with B band excltation, A, = 4545 A, of the same samples as In Flgue 1. Band I , at 1359 cm-? for deoxyHb, is 2 cm-' lower in the COHb photoproduct. Scanning mode, conditions as in Figure 1.
high spin conversion occurred within 30 ps of COHb photolysis, presumably via intersystem crossing of excited COHb, but that the subsequent movement of the iron atom out of the heme plane to its deoxyHb position took longer than 20 ns. The slowness of this relaxation was attributed to coupling of the iron motion with the tertiary structure change of the protein between COHb and deoxyHb. Figure 2 shows the RR spectra of deoxyHb and the
