Identification of metal-sensitive structural changes in the Ca-binding photocomplex from Thermochromatium tepidum by isotope-edited vibrational spectroscopy.

Calcium ions play a dual role in expanding the spectral diversity and structural stability of photocomplexes from several Ca-requiring purple sulfur phototrophic bacteria. Here, metal-sensitive structural changes in the isotopically labeled light-harvesting 1 reaction center (LH1-RC) complexes from the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were investigated by perfusion-induced attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The ATR-FTIR difference spectra induced by exchanges between native Ca and exogenous Ba exhibited interconvertible structural and/or conformational changes in the metal binding sites at the LH1 C-terminal region. Most of the characteristic Ba/Ca difference bands were detected even when only Ca ions were removed from the LH1-RC complexes, strongly indicating the pivotal roles of Ca in maintaining the LH1-RC structure of Tch. tepidum. Upon N-, C- or H-labeling, the LH1-RC complexes exhibited characteristic N/N-, C/C-, or H/H-isotopic shifts for the Ba/Ca difference bands. Some of the N/N or C/C bands were also sensitive to further H-labelings. Given the band frequencies and their isotopic shifts along with the structural information of the Tch. tepidum LH1-RC complexes, metal-sensitive FTIR bands were tentatively identified to the vibrational modes of the polypeptide main chains and side chains comprising the metal binding sites. Furthermore, important new IR marker bands highly sensitive to the LH1 BChl a conformation in the Ca-bound states were revealed based on both ATR-FTIR and near-infrared Raman analyses. The present approach provides valuable insights concerning the dynamic equilibrium between the Ca- and Ba-bound states statically resolved by x-ray crystallography.