Selective spectroscopy of biomolecules

Various pigmented molecules, among them chlorophylls in different chemical forms are involved in the photosynthetic process of plants and bacteria. The photoactive part of most photosynthetic complexes constitutes from closely packed, non-covalently bound pigments whose electronic properties are modified by the binding sites of the surrounding protein. To advance our comprehension of the photosynthetic light harvesting and charge separation processes the aggregated pigments are involved in, the properties of the individual pigments should be understood in greater detail than available so far.

We have applied a variety of conventional and selective spectroscopy techniques for studying vibronic spectra of chlorophyll a and bacteriochlorophyll a molecules in the solvents where the pigments are known to be either in the penta- or hexa-coordinated state. Breakdown of a mirror symmetry rule for the conjugate Qy absorption and fluorescence emission spectra, expected from the basic model of vibronic spectra, has been observed both at room and at cryogenic temperatures. In some solvents the coordination state changes can be induced by lowering temperature or with increasing pressure. Transformations of the chlorophyll a absorption spectra with lowering temperature that reveal the disputed Qx band position were demonstrated. A site-selective technique advanced in this laboratory and referred to as difference fluorescence line-narrowing (delta-FLN) spectroscopy was used for a detailed description of the electron-phonon and vibronic coupling strengths. The electron-phonon coupling strength was found to depend strongly on the excitation wavelength within the inhomogeneously broadened Qy origin absorption band. In the chlorophyll a-doped propanol-1, for example, the Huang-Rhys factors Sph increase almost linearly from about 0.4 at 669 nm to about 0.6 at 693 nm, showing an unexpected direct correlation between the electron-phonon coupling strength and the electronic transition energy. In contrast, the vibronic coupling strengths, although different in the conjugate absorption and fluorescence emission spectra, were found to be independent on the excitation wavelength.

The inhomogeneously broadened low-temperature absorption (blue line) and fluorescence (red line) spectra of chlorine-doped 1-propanol glass at 4.5 K.  The insets show a series of narrow deep holes in the hole burning spectrum, and a delta-FLN spectrum (excited at 632 nm), consisting of a narrow zero-phonon line (ZPL), which is accompanied by a broad phonon sideband (PSB) and several vibronic replicas of ZPL+PSB.