Temperature and Light-Induced Structural Changes in Photosynthetic Reaction Center Proteins Probed by X-ray Absorption Fine Structure

Structures of the Fe ²⁺ binding site in photosynthetic reaction center (RC) proteins from Rhodobacter sphaeroides R26 were obtained by Fe K-edge X-ray absorption fine structure (XAFS) spectroscopy. Measurements were taken at cryogenic and room temperatures, in the dark as well as in the light-induced charge-separated state P ⁺Q _AQ _B ^-. Evidence for protein matrix expansion near the Fe ²⁺ site because of lowering the temperature from 290 to 15 K was observed as elongation of Fe ²⁺ to neighboring atom distances. Upon formation of the charge-separated state P ⁺Q _AQ _B ^-, the distances between the Fe ²⁺ and neighboring atoms were reduced slightly. Replacing the native Fe ²⁺ with either Mn ²⁺ or Zn ²⁺ resulted in distinct differences in local structural responses to both temperature and charge separation, suggesting that the conformation changes caused by these factors may follow different paths in the potential energy landscape of the protein. The XAFS measurements of Fe ²⁺ site structure revealed a more symmetrically oriented imidazole ligands arrangement around Fe than those in current crystal structures. These measurements for high-precision Fe site local structure indicate that XAFS is a useful technique for probing structural changes in the metal site environment because of temperature change, metal replacement, and light-induced charge separation in the reaction center proteins.