Resonance Raman Investigation of Protein Dynamics Studies on Myoglobin: Information Transmission and Energy Funneling Mechanisms

The iron protoporphyrin IX（b type heme）exists as a reaction center in most of theheme proteins as a prosthetic group which bound with the protein matrix. It carries outvarious functions including the diatomic gaseous ligand storage and transport，electrontransfer，oxidization，peroxidization，catalysis and signaling process and so on．For investigating the active center, heme-iron-ligand complex，IR and visible RR spectroscopyhave revealed the heme environment structure and the ligand discrimination mechanismfor many heme proteins．Furthermore，electron paramagnetic resonance spectroscopyand quantum mechanism study have been used to revealing the reaction mechanism ofheme enzyme．However, in any cases the accompanying conformational changes inprotein moiety always occur for regulating protein function as revealed by x-raycrystallography. It appears quite important to establish a correlation between the activecenter, heme，and the protein matrix for understanding the essential mechanism forheme proteins. Also，this issue has attracted a lot of concerns from many fields. Thenature utilizes b type heme as the most common structure among the four kinds of hemesthat contain same framework but altered substitutions. Therefore, it is easy to proposethat the side chain of heme could play important role in regulation of protein structureand function, and this propose is consistent with the discoveries from more and moreexperimental data that side chains are involved in many reactions related with proteinfunctions. In this study, the interactions between heme and protein matrix as well as solvent leadingto intramolecular transduction of structural information and intermolecular energy transfer weresystematically investigated by using Mb. Those interactions include the covalent bond and thehydrogen bonds between heme and globin and spatial collision between heme side chains and watermoleculs． In order to investigate the transmission of a binding slgnal of a gaseous ligand from theligand binding site?heme，to protein moiety in gas sensory heme proteins，we applied UVRRspectroscopy to myoglobin as a model. UVRR spectroscopy is known as an excellent tool formonitoring protein conformational changes. First of all，we determined the changes of conformationin globin that occur upon binding of CO，NO，or O2to heme. Specifically，NO induces spectral changes in Trp residues of A-helix that are significantly different from those induced by O2or CObinding. On the other hand，binding of O2 to heme produces spectral changes in the Tyr residues of H-helix that are difftrent from those induced by CO or NO binding. The UVRR results demonstratethat the heme discriminates among different ligands by driving corresponding conformational changes in the globin matrix. In order to explore the signaling pathway through His93 covalent bond，and 6-or7-propionate hydrogen bonding network，we extended measurements to mutant-and heme-modifiedMbs in a similar way to native Mb, and investigated how they are responsible for transmittingstructural changes from ligand binding site--heme to globin for difftrent ligands. The experimentalresults demonstrate that the cleavage of Fe-His93 covalent bond eliminates communication to theC-terminal of the H-helix and that 7-propionate hydrogen-bonding network is essential fortransmitting the CO or NO binding signal to the N-and C-termini. Finally，6-propionate is importantonly for NO binding. Thus，the hydrogen-bonding network in the protein appears to be critical forintramolecular slgnal transduction in gas sensory heme proteins. Furthermore, pathway of vibrational energy dissipation from the heme tosurrounding protein matrix and solvent following CO photolysis in the fast timecomponent (≤10 ps）was investigated by using picosecond time-resolved anti-StokesRaman spectroscopy. The modified-and mutant Mbs，in which the 6- or 7-propionate isselectively replaced by a methyl group or related hydrogen bonds is eliminated bymutagenesis was used as model systems. The time constants of population decay ofvibrationally excited states for two modified Mbs became significantly larger comparedwith those of native Mb．However the corresponding values of mutants are not differentfrom those of the native Mb．This work indicates that the two heme-propionate sidechains are highly involved in the energy transfer from the heme to solvent through thecollision with surrounding water molecules and contribute equally. But the hydrogenbonding interactions with protein matrix seem to contribute scarcely to this fast energytransfer process．This is the first experimental data estimating the contribution ofindividual heme-propionate side chains to the vibrational energy transfer from the hemeto the surroundings