Spectroscopic and Thermodynamic Characterization of the Metal-Binding Sites in the LH1–RC Complex from Thermophilic Photosynthetic Bacterium Thermochromatium tepidum

The light-harvesting 1 reaction center (LH1–RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermostability and an unusual LH1 <i>Q_y</i> transition, both induced by Ca²⁺ binding. In this study, metal-binding sites and metal–protein interactions in the LH1–RC complexes from wild-type (B915) and biosynthetically Sr²⁺-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca²⁺, Sr²⁺, or Ba²⁺ to the LH1 αβ-subunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca²⁺ and Sr²⁺ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca²⁺ and Sr²⁺ (or Ba²⁺) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca²⁺ depletion and Sr²⁺ substitution demonstrated that dissociation and binding of Ca²⁺ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr²⁺ (or Ba²⁺) exists in the vicinity of the Ca²⁺-binding site, a part of which is shared in both metal-binding sites