X-ray absorption studies of the local environment of zinc ions bound to the bacterial photosynthetic reaction center

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides slows the light-induced electron and proton transfer to the secondary quinone, QB (Utschig et al., 1998, Biochemistry 37, 8278; Paddock et al., 1999, Proc. Natl. Acad. USA 96, 6183). On the basis of X-ray diffraction (XRD) at 2.5 A ° resolution a site has been identified at the protein surface which binds Cd(II) or Zn(II). Both metal ions binds to the same cluster formed by AspH124, HisH126 and HisH128. A water molecule was also proposed to interact with the Zn (Axelrod et al., 2000, Proc. Natl. Acad. Sci. USA 97, 1542). Recent data suggest that inhibition of proton transfer by Cd(II) is predominantly a consequence of competing with protons for binding to HisH126 and HisH128, thus hampering the function of these residues as proton donor/acceptors along the proton pathway to the QB site (Paddock et al., Biochemistry 42,9626, 2003). Determination of the local structure of the bound metal ions is expected to contribute significantly to elucidate the details of the inhibition mechanism. For this reason we performed Zn K-edge X-ray absorption measurements on Zn-doped RCs embedded into polyvinyl alcohol films at both room and liquid nitrogen temperature. Data analysis has been performed using ab-initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) of significant amplitude have been included. Results for complexes characterized by a Zn to RC stoichiometry close to 1 indicate that Zn binds two O and two N atoms in the first coordination shell. The two N atoms come from His, and only one of the two O atoms comes from an amino acid (Asp or Glu); the second O atom belongs to a water molecule. Complexes characterized by approximately two Zn ions per RC show a second structurally distinct binding site, involving three N and one O atom, all coming from nonaromatic residues. The results obtained for the higher affinity site nicely fit the coordination proposed on the basis of XRD data. The second binding site, revealed by our investigation on noncrystalline samples, is most probably located in a more disordered domain of the RC protein and might have a hitherto not appreciated role in charge transfer inhibition.