1224 zyxwvutsrqpo Biochemistry zyxwvuts 1995,34, 1224-1237 Evidence for Sub-picosecond Heme Doming in Hemoglobin and Myoglobin: A Time-Resolved Resonance Raman Comparison of Carbonmonoxy and Deoxy Species+ S. Franzen,t B. Bohn,s C. Poyart,@ and J. L. Martin*,+ Laboratoire d'Optique AppliquLe, INSERM U27.5, Ecole Polytechnique-ENSTA, 91 120 Palaiseau, France, and INSERM U299, zy Le Kremlin-Bicetre, France Received February zyxwvutsr 1, 1994; Revised Manuscript Received November 8, 1994@ ABSTRACT: Separation of the photophysical aspects of the sub-picosecond (sub-ps) time-resolved resonance Raman signal from contributions due to conformation has been achieved by comparing deoxyhemoglobin (Hb) in the T state with (carbonmonoxy)hemoglobin (HbCO), deoxy-P4 @CO) (all R state), and monomers deoxymyoglobin and (carbonmonoxy)myoglobin (MbCO) 104 consists of a tetramer of four P-subunits and shows no cooperativity]. In all photolyzed species, Hb*(CO), Mb*(CO), and P4*(CO), the iron- histidine out-of-plane mode (YFe-His), indicative of heme doming, achieves 90% of its full intensity in 1 ps. The frequency of this mode (223-228 cm-') is shifted significantly relative to equilibrium deoxy- Hb (210-216 cm-') in the T state, but not with respect to either equilibrium deoxy-Mb or deoxy-/34. A correlation between the +12 cm-' bandshift of VFe-His and the -2 cm-' shift of the electron density marker band ( ~4 at 1370 cm-l) relative to T-state deoxy-Hb is shown to hold on all time scales, including the sub-picosecond time scale. Photolyzed Hb*(CO) consists of R-state or weakly interacting tetramers on the picosecond time scale and is shown to have properties similar to those of photolyzed Mb*(CO) and /34*(CO> on the picosecond time scale. These results establish that heme doming occurs as an ultrafast reaction to ligand dissociation and that heme doming is the primary event in the sequence of conformational changes leading to the cooperative R zyxwvuts - T transition. In the oxygen transport protein hemoglobin, the formation or breakage of a single chemical bond between the heme iron and a diatomic ligand is transmitted to the protein and expressed in terms of intersubunit structural changes, which ultimately modify the binding affinity of all four heme irons in the protein. The X-ray crystal structure of hemoglobin shows that the heme iron is nearly coplanar with the heme in ligated hemoglobin and more than 0.4 A from the heme plane in deligated hemoglobin (Perutz, 1979). The out-of- plane iron displacement or heme doming, which results from the breaking of the iron-ligand bond, is thought to initiate the series of conformational changes that ultimately leads to a quaternary structure change, inducing cooperative behavior among the heme iron binding sites in hemoglobin (Perutz, 1970; Baldwin & Chothia, 1979). Hemoglobin is known to undergo a cooperative transition from a relaxed state, R, with high affinity for a diatomic ligand, such as 02, CO, or NO, to a tense state, T, with low ligand binding affinity. The R - T transition is a quaternary transition that occurs on the microsecond time scale following ligand photodissociation(Sawicki & Gibson, 1976; Hofrich- ter et al., 1983). Prior to the quaternary transition, there are changes in tertiary structure over a range of time scales from picosecond to microsecond. Time-resolved resonance Ra- man (TRRR) spectroscopy has played an important role in measuring the time scale of these structural changes and S.F. was the recipient of a European Molecular Biology Organiza- tion fellowship. * INSERM U275. * INSERM U299. @ Abstract published in Advance ACS Abstracts, January 1, 1995. elucidating specific models of the reaction coordinate (Scott & Friedman, 1984). In the high-frequency region of the resonance Raman (RR) spectrum of Hb, the electron density marker, or v4 Raman band, observed at 1355 cm-' in deoxy Hb exhibits a time- dependent shift in photodissociated HbCO (denoted Hb*- (CO) or simply Hb*). The time scale of relaxation of the ca. -2 cm-' v4 bandshift to the equilibrium deoxy-Hb frequency has been used to estimate the time scale of tertiary and quaternary structural changes in Hb* that result in the formation of deoxy-Hb in the T state (Friedman et al., 1982a). In the low-frequency region of the RR spectrum, it has been shown that the Fe-His out-of-plane mode, VF~-H~~, is absent from HbCO, but appears at a shifted position of 223-228 cm-' in photolyzed (R state) HbCO as measured at 30 ps and 10 ns, later relaxing to the equilibrium (T state) deoxy position of 210-216 cm-' on the 100 ns to microsecond time scale (Scott & Friedman, 1984). It has been shown that the frequencies of VF~-H~~ in the a- and P-subunits of T-state hemoglobin are 203 and 220 cm-', respectively; the R-state values are nearly the same at 223 and 224 cm-' (Nagai & Kitagawa, 1980). This suggests that the single value of 212-216 cm-' reported in this paper and elsewhere is the average of the value obtained in the different subunits. Studies have established that the zyxw YF~-H~~ mode is indicative of a domed heme (Nagai & Kitagawa, 1980; Argade et al., 1984). Heme doming has been hypothesized to be the primary event in the series of conformational changes responsible for the cooperative R - T transition in hemoglobin (Perutz, 1970). At first this hypothesis was based on the correlations between quaternary structure and heme iron position (and 0006-2960/95/0434-1224$09.00/0 0 1995 American Chemical Society