Site-selective spectroscopy of myoglobin

Abstract

Site-selective spectroscopy of myoglobinTakashi Kushida, Atusi Kurita and Yasuo KaneinatsuDepartment of Physics, Osaka University,Toyonaka, Osaka, 560 JapanABSTRACTSite-selective fluorescence spectroscopy and hole-burning spectroscopy have beenperformed for Zn-substituted inyoglobin (ZnMb) in the red spectral region at lowtemperatures. The site-energy distribution of the chromophore and the fluorescencespectrum of ZnMb in a single site have been obtained from the analysis of laser-induced fluorescence spectra. It has been found that this single-site fluorescencespectrum can be regarded as the homogeneous spectrum. The comparison of the holespectra between freeze-dried ZnMb powder and ZnMb in glycerol-water mixture showsthat the phonon-sideband profiles of these spectra as well as that of the single-site fluorescence spectrum reflect the density of states of vibrational modes of theprotein itself. The density of states of low-frequency vibrational modes of ZnMbweighted by the coupling strength between the electrons of the chroinophore and thevibrations of the polypeptide chain has been determined from the above-obtainedsingle-site fluorescence spectrum by solving an integral equation numerically. Thisweighted density of vibrational states has been compared with that calculated on thebasis of the linear electron—phonon coupling theory using the data of the normal-mode analysis of myoglobin by a computer simulation. The result has been found to bein fair agreement except for the low-energy region below 10 cm1. The distributionof the barrier height between the conformational substates obtained from the depend-ence of the hole recovery process on the sample temperature will also be discussed.1. INTRODUCTIONWhen many localized centers of the same kind are doped in condensed matter, thetransition energy may be different for individual centers, because the microscopicenvironment is not completely the same for each center. If the transition energy isthe same for two centers, they are said to be in the same site. Because of thisdistribution of the transition energy, the optical spectrum is usually broadened.This is called inhomogeneous broadening. When the absorption spectrum of a localizedcenter is dominated by this inhoutogeneous broadening, it may be possible to excitethe centers in a given site selectively by shining the sample with monochromaticlight. The study of optical spectra of fluorescence, absorption and so on under sucha condition is called site-selective spectroscopy. The representatives are site-selective fluorescence spectroscopy and hole-burning spectroscopy. These techniquesenable us to obtain the optical spectra of the center without the effect of theinhomogeneous broadening, the distribution of the number of the localized centers asa function of the site-energy, the effective density of states of vibrational modesof the host material, the distribution of the barrier-height between the localminima on the potential surface, and so on.In order to investigate low-frequency vibrational modes and structural dynamicsof the protein, we have performed site-selective fluorescence spectroscopy andhole-burning spectroscopy for mnyoglobin in the lowest optical absorption band at low