Structural Study on Electron Tunneling and Proton Relay in Proteins by Using Ultraviolet Resonance Raman Spectroscopy

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Molecular mechanism of the vectorial proton transportation through biological membrane was investigated by using resonance Raman spectroscopy, and a system for measuring UV resonance Raman spectra of protein moieties was constructed. Two typical systems were picked up; one is a light driven proton pumping system having bacteriorhodopsin and the other is an electron-driven proton pumping system containing cytochrome c oxidase. For bacteriorhodopsin intermediates involved in a time region when the Schiff base proton of the chromophore was transferred to the protein moiety were examined and discussion was focused on what would happen when the protein moiety did not accept the proton. For cytochrome c oxidase the occurrence of photoreduction was confirmed and the electron transfer rate from the a heme to the <a_3> heme was determined. It was found that the aerobic photoreduction generates a photosteady state composed mainly of <a^(2+)> <a(_3^(3+))> , and that its <a^(2+)> and <a(_3^(3+))> hemes can be selectively probed with the 441.6 and 406.7 nm excitations, respectively. Sensitivity of the Fe-His stretching Raman intensity to the enzymic activity was pointed out, and transient Raman spectra of oxygenated intermediates were discussed. A system to observe Raman spectra excited by UV light around 200-240 nm was set up with a SPEX 1260 single monochromator equipped with a 1200 gr/mm and 200 nm blaze grating. Excitation light was derived from Nd:YAG laser and Raman shifter. The system brought about spectra of good quality for aromatic amino acid residues.