Structures of the Fell 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(A)Q(B)(-). Evidence for protein matrix expansion near the Fe2+ site because of lowering the temperature from 290 to 15 K was observed as elongation of Fe2+ to neighboring atom distances. Upon formation of the charge-separated state P(+)Q(A)Q(B)(-), the distances between the Fe2+ and neighboring atoms were reduced slightly. Replacing the native Fe2+ with either Mn2+ or Zn2+ 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 Fe2+ 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.