Detection of Fast Conformation Changes of Proteins by Time-Resolved Resonance Raman Spectroscopy

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Fast conformation changes of proteins in addition to the catalytic reaction at the activie site play an important role in enzymic reactions, since the incorporation of substrates and release of products are indispensable for enzymes to function repeatedly. They are achieved by rapid rearrangements of side chains or main chain of proteins. It is the purpose of this study to reveal such fast conformation changes of protein accompanied by functioning of proteins, and in practice, we investigated resonance Raman spectra of reaction intermediates of cytochrome c oxidase and myoglobin.Cytochrome c oxidase is the terminal enzyme of mitochondrial respiration chain and catalyzes the reduction of molecular oxygen coupled with proton translocation. This reaction is inhibited upon binding of CO to the heme iron. The enzymic reaction was initiated by photolyzing CO from CO-bound enzymes in O_2-saturated buffer solution, and transient Raman spectra were observed at time DELTAt following the initiati … More on of reaction. On the basis of the order of appearance of oxygen-isotopesensitive bands, it has been established that the reaction proceeds in the following way ; Fe^<III>-O_2*Fe^<III>-O-OCu^<II><tautomer>Fe^V=O*Fe^<IV>=O*Fe^<III>-OH.Thus, this study has made a breakthrough in 60 years of research history of cytochrome c oxidase.The molecular structures of myoglobin has been revealed with x-ray crystallography at the level of 1.5 A resolution for both deoxy- and CO-bound forms. According to them, there is no pathway for migration of CO into the heme iron from outside of the protein. Therefore, rapid rearrangements of protein structures should be accompanied by the ligand binding. In order to reveal transient structures of proteins, we carried out the pump/probe time-resolved resonance Raman experiments for recombination of photodissociated CO-myoglobin and its distal histidine mutants. It was found that the species with the Fe-CO stretching mode (nu_<Fe-CO>) around 490 cm^<-1> recover much faster than those with nu_<Fe-CO> around 510 cm^<-1>. However, the 490 cm^<-1> species was not generated as a precursor of the 510 cm^<-1>species. To understand the protein dynamics and their relation with the nu_<Fe-CO> frequency, we carried out ab initio MO calculations and molecular dynamics. Less