1.F Photo-dissociation reaction of carboxymyoglobin

　　The relationship between thestructural properties of proteins and their reactions is essentially importantfor understanding the protein biological functions. Myoglobin has been used as a model system for experimental and theoretical studies of such a kinetics-structural relation. A heme is embedded within the protein and a small ligand (e.g., O2, CO, NO) is reversibly bind tot he sixth coordination site, on the distal side, of the heme. By the photoexcitation of the heme, the ligand-metal bond is photodissociated. Since there is no route prepared for the ligand to pass through the protein to the outer solvent, the protein structure has to change if the ligand is removed from the protein. This photoreaction can be used, therefore, to trigger a perturbation to the protein by pulsed laser light. Since most of kinetic studies have been conducted by optical spectroscopies, which use the optical transition of the heme, they probed only the local structural change around the heme but give little information about the global structural change of the molecule such as protein motion. Therefore, there are many unresolved questions in the dissociation-association process of MbCO in spite of a large number of studies. For example, although the 180 ns-dynamics at 20 °C was sometimes regarded as the CO escaping process from the protein to solvent, it could be just CO moving inside the protein. How fast does the CO escape from the protein matrix to the solvent? Relating with this question, we might have another one; how many intermediates are there in the time course from the photodissociation to the CO escape? If there are several intermediates, how much energy do the intermediates have? The energies of the intermediates are very important information, but our knowledge of this problem is very limited even after the long history of the Mb studies. These difficulties may be solved by using a time-resolved spectroscopy that does not use the optical transition of the heme. We performed a quantitative investigation on the dynamics of the protein of horse heart carboxymyoglobin (MbCO) after photodissociation of CO from 10 ns to milliseconds continuously by a new technique; the laser induced transient grating (TG) and photoacoustic (PA) hybrid method. We found that initially, ~10 ns after thephotodissociation, a small volume contraction takes place (5 ml/mol) and an energy of 61 kJ/mol is stored in the system. This energy is smaller than the Fe-CO bond energy. This fact indicates a rather large protein structural relaxation in this fast time scale. Subsequently a structural change, volume expansion of 14.7 ml/mol, occurs with a lifetime of 700 ns at 20 C.This indicates the presence of the second intermediate state, from where the ligand cannot return back to the heme, and the 700 ns process is attributed to the escaping of CO from the protein to the outer solvent phase. Interestingly we found that this volume change is actually depend on the temperature. The energies and the structural relaxation of the protein are discussed.
　　We also investigated the photodissociation kinetics of sperm whale (SW) MbCO by the TG and PA hybrid method. SW-Mb is the most frequently studied Mb, and the dynamics as well as the enthalpy change and volume change of this protein are important information. Besides this, there are mainly three purposes. We developed a new method of the TG analysis of the CO diffusion process under various grating wavenumbers to determine the rate of the ligand escape from the protein interior. Using this analysis, we can definitively show that the 700 ns-dynamics represents the CO escaping process. From these results, we conclude that there are at least two intermediate states during the course of the CO dissociation. Hence, a four-state model is appropriate to describe the ligand dissociation reaction even at room temperature. The dynamics of SW-MbCO is compared with that of HH-MbCO. There are 21 amino acid residues that are different between HH- and SW-Mb. Comparing these two proteins, we can examine how the observed dynamics is sensitive to these amino acid residues. In particular, the comparison will give us a clue on the role of salt bridge between Arg45 and heme 6-propionate side chain. We examined the participation of the salt bridge dynamics in the CO dissociation reaction.
　　Protein dynamics observed by the transient grating (TG) method were studied for some site-directed mutants at the distal histidine of myoglobin (H64L, H64Q, H64V). The time profiles of the TG signals were very sensitive to the amino acid residue of the 64 position. It was found that the sensitivity is mostly caused by the different rates of the ligand escape from the protein to solvent and the magnitude of the molecular volume change. Several molecular origins of the volume difference between MbCO and Mb were proposed. Interestingly, the volume difference between the CO-trapped Mb inside the protein interior and Mb is similar to that of the partial molar volume of CO in solvent. Nature of the CO trapped cite was discussed.

　　　　　　　　　　　　　　　　　　　　　　 (Back)

---