This thesis is written to summarize investigations of the mechanisms that underlie the kinetics of diatomic ligand rebinding to the iron atom of the heme group, which is chelated inside heme proteins.
The family of heme proteins is a major object of studies for several branches of scientific research activity. Understanding the ligand binding mechanisms and pathways is one of the major goals for biophysics. My interests mainly focus on the physics of this ligand binding process. Therefore, to investigate the problem, isolated from the influence of the protein matrix, Fe-protophorphyrin IX is chosen as the prototype system in my studies. Myoglobin, the most extensively and intensively studied protein, is another ideal system that allows coupling the protein polypeptide matrix into the investigation.
A technique to synchro-lock two laser pulse trains electronically is applied to our pump-probe spectroscopic studies. Based on this technique, a two color, fs/ps pump-probe system is developed which extends the temporal window for our investigation to 13ns and fills a gap existing in previous pump-probe investigations. In order to apply this newly-developed pump-probe laser system to implement systematic studies on the kinetics of diatomic ligand (NO, CO, O2) rebinding to heme and heme proteins, several experimental setups are utilized.
In Chapter 1, the essential background knowledge, which helps to understand the iron-ligand interaction, is briefly described.
In Chapter 2, in addition to a description of the preparation protocols of protein samples and details of the method for data analysis, three home-made setups are described, which include: a picosecond laser regenerative amplifier, a pump-probe application along the bore (2-inch in diameter) of a superconducting magnet and a temperature-controllable cryostat for spinning sample cell.
Chapter 3 presents high magnetic field studies of several heme-ligand or protein-ligand systems. Pump-probe spectroscopy is used to study the ligand recombination after photolysis. No magnetic field induced rate changes are observed in any of these ligand recombination processes within the experimental detection limit. A magnetic field dependent CO rebinding behavior is observed for the FePPIX-CO sample in 80%glycerol/20%water environment. Careful data analysis indicates that this magnetic field induced change is due to the amplitude difference of a "fast" (<10ps) response with and without the magnetic field application (the amplitude changes from ∼55% at 0 Tesla to ∼45% at 10 Tesla). Kinetics of CO rebinding to FePPIX in 80%glycerol at the extremes of the magnetic field intensities (0Tesla vs. 10 Tesla) can be decomposed into a ligand rebinding process plus two 5ps decays heme cooling with different amplitudes. It leads to suggest a magnetic field induced change of a short-lived heme cooling response after photolysis. Also, CO rebinding kinetics to different heme compounds demonstrates a wide range for the Arrhenius pre-factors. This work reveals that the "spin-selection rule" does not play a key role in the recombination process of CO to heme iron.
In Appendix 1, the recombination of oxymyoglobin and its mutants is investigated in the temperature range from 275K to 318K, using a home-made cryostat. Quite surprisingly, the O2 molecule rebinds to heme iron inside myoglobin with dramatically different behavior as the temperature is varied, depending on the protein environment. It shows little dependence (Mb), no dependence (V68W Mb mutant) and large dependence (L29W Mb mutant) in this 40K temperature window. To expand this temperature window, since the motor inside the cryostat is capable to work as low as 230K, glycerol is introduced into the protein preparation. It is observed that protein samples in a glycerol/water mixture, even with only 20% glycerol (in weight), the temperature dependences of the O2 rebinding to heme iron are dramatically altered. The O 2 rebinding behavior also shows a high dependence on the glycerol concentration in the solution.
In Appendix 2, the absorption spectra of Fe-protoporphyrin IX in different monomeric complexes are investigated and compared. This work may suggest that, inside the CTAB micelles, ferrous Fe-PPIX exists in an equilibrium state of different species, CO probably can bind to FeII-PPIX with different trans ligand (H2O or OH-), and the trans effect, exhibiting while NO binds to H93G Mb mutants might also happen when NO binds to FeII-PPIX inside CTAB micelles.
In Appendix 3, several ultrafast kinetics studies of CO rebinding to FePPIX are listed. They might be helpful for future studies on such systems.
In Appendix 4, a series of systematic studies of the NO recombination to FeIIPPIX is performed with temperature and environment variation. A four-state model is proposed to explain the kinetics of NO-FeIIPPIX after photolysis. (Abstract shortened by UMI.)