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The Fe²⁺ Site of Photosynthetic Reaction Centers Probed by Multiple Scattering X-Ray Absorption Fine Structure Spectroscopy: Improving Structure Resolution in Dry Matrices

Giulia Veronesi ^* , Lisa Giachini ^*, Francesco Francia , Antonia Mallardi , Gerardo Palazzo ^¶, Federico Boscherini ^*  and Giovanni Venturoli  

^* Dipartimento di Fisica, Università di Bologna, Bologna, Italy; Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, Bologna, Italy; Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, Bologna, Italy; Istituto per i Processi Chimico-Fisici, CNR, Bari, Italy; and ^¶ Dipartimento di Chimica, Università di Bari, Bari, Italy

Correspondence: Address reprint requests to Giovanni Venturoli, Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, 40126 Bologna, Italy. E-mail: ventur{at}alma.unibo.it.

We report on the x-ray absorption fine structure of the Fe²⁺ site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe²⁺ is ligated with four N atoms from four histidine (His) residues and two O atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N distances, and two distinct Fe-O bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N distances, and in one Fe-O bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.