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Multiple Scattering X-Ray Absorption Studies of Zn²⁺ Binding Sites in Bacterial Photosynthetic Reaction Centers

Lisa Giachini ^* , Francesco Francia , Antonia Mallardi , Gerardo Palazzo ^¶, Emilio Carpenè ^||, Federico Boscherini ^*  and Giovanni Venturoli  

^* Dipartimento di Fisica, Università di Bologna, Bologna, Italy; Istituto Nazionale per la Fisica della Materia (INFM), UdR di Bologna, Bologna, Italy; Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, Bologna, Italy; Istituto per i Processi Chimico-Fisici, CNR, Bari, Italy; ^¶ Dipartimento di Chimica, Università di Bari, Bari; and ^|| Dipartimento di Biochimica, Sezione Biochimica Veterinaria, Università di Bologna, Ozzano Emilia, Bologna, Italy

Correspondence: Address reprint requests to Giovanni Venturoli, E-mail: ventur{at}alma.unibo.it.

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q_B. On the basis of x-ray diffraction at 2.5 Å resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd²⁺ or Zn²⁺. Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn²⁺ ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn²⁺ binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.

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