Metastable photoproducts from carbon monoxide myoglobin.

Abstract

The photoproduct of carbon monoxide myoglobin generated at 4 K and lower has a resonance Raman spectrum characteristic of a high-spin heme but in which the high-frequency core size-sensitive lines are at lower frequency than those in the deoxy preparation. Such differences are not detected in the photoproduct generated at higher temperatures (50 K) or in that generated at room temperature with 10-nsec pulses. The data indicate that at the low temperature (4 K), the heme in the photoproduct is not fully relaxed, and from the data we conclude that the photoproduct has an expanded porphyrin core. We infer that the core size exceeds that in deoxymyoglobin because the rigid protein prevents the highspin iron atom from moving to its full out-of-plane displacement at the very low temperatures.To gain an understanding of the basic mechanisms of ligand association in heme proteins, the elementary steps in the ligand binding and release process must be uncovered. An approach to the elucidation of these steps is to photodissociate a bound ligand and follow the relaxation of the deoxy heme that has been generated. At room temperature the evolution of this photoproduct occurs over a wide temporal scale ranging from the subpicosecond absorption of the photon (1) to structural relaxation of the protein residues, which may occur on a time scale of 10s of microseconds (2,3). The specific relaxation pathway of the photoproduct is very complex and depends on the protein as well as the ligand.An alternate approach in the identification of the intermediate structures in the photoproduct relaxation is through the use of cryogenic temperatures, where metastable states may be isolated (4). By this method changes in the properties of photodissociated heme proteins were detected many years ago with optical absorption techniques (4-6), and studies of the metastable species have been since extended with many other techniques. The relationship between the structure, as inferred from the resonance Raman spectra, of the lowtemperature photoproduct and the transient photoproduct generated at 300 K was discussed recently, and in carbon monoxide hemoglobin, HbCO, it was observed that the differences in the Raman spectra between the stabilized photoproduct and the deoxy preparation at 80 K were the same as those obtained transiently at room temperature with 10-nsec pulses (7). These findings led us to investigate the low-temperature (1.6-100 K) photodissociation of carbon monoxide myoglobin, MbCO, with resonance Raman scattering to determine the properties of the unrelaxed photoproduct and its relationship to the room-temperature transient photoproduct. The experiments were carried out by freezing CO-bound Mb to low-temperature and then directing a laser beam on the sample. The laser photodissociates the CO, resulting in a deoxy heme trapped in the frozen protein matrix. At the lowest temperatures we find significant differences in the resonance Raman spectra ofthe photoproduct and the equilibrium deoxy species. We interpret these results...

Spectroscopic evidence for conformational relaxation in myoglobin.

“…Raman spectroscopy offers support for the hindered mobility of the heme iron in the photoproduct Mb* at low temperatures. From the shifts of Raman lines in the high-frequency region ("1500 cm-'), Rousseau and Argade (8) concluded that the heme core of Mb* is expanded compared to deoxyMb (2.027 versus 2.020 A at 4.2 K) (8). This expansion can be explained by the increased repulsion between the occupied (d.2-y2) orbital of the high-spin iron and the pyrrole nitrogen orbitals because of incomplete relaxation.…”

“…The rate coefficient K*(T) does not follow an Arrhenius relation but can be described by K*(T) = A*e-[E*/RTf. [8] The relaxation parameters for MbCO in glycerol/buffer, pH 6.8, were determined from fitting the rebinding kinetics between 160 and 210 K by Eqs. 7 and 8 (13).…”

“…A proteinquake then releases the stored energy and leads to the Mb structure. For the present discussion, we assume that the large-scale motions are described by the scheme (1,8,9) hpb FIM 2…”

Probing the electronic and geometric structure of ferric and ferrous myoglobins in physiological solutions by Fe K-edge absorption spectroscopy

“…[12][13][14] Important in the context of these studies is that hemoproteins are photosensitive, leading to a wide range of time-resolved optical and X-ray spectroscopic studies ranging from milliseconds to femtoseconds. [15][16][17][18][19][20][21][22][23][24][25][26][27] In these experiments, the biological function of ligand detachment from the heme is triggered by an optical (so-called pump) pulse, while the evolution of the system is probed by a second (typically optical or X-ray) pulse, with a tunable time delay with respect to the pump pulse. While optical tools can reach femtosecond resolution they do not provide direct structural information.…”

Transient and Time‐resolved Optical Studies of Photolyzed Carbonmonoxy Hemoglobin and Myoglobin

“…The behavior of v2 at cryogenic temperatures corroborates these results. Rousseau and Argade (1986) found that the position of v2 was -4 em-• lower in the metastable photoproduct of MbCO. They interpreted this as an indication of an expanded heme core-size and further evidence of an unrelaxed heme.…”