In Rhodobacter sphaeroides reaction centers, mutation of proline L209 to aromatic residues in the vicinity of a water...

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobacter sphaeroides obtained at high resolution has revealed a number of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, H. (1994) Structure 2, 925-936; Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816). Some of them are organized into distinct hydrogen-bonded water chains that connect Q(B) (the terminal quinone electron acceptor of the reaction center) to the aqueous phase. To investigate the role of the water chains in the proton conduction process, proline L209, located immediately adjacent to a water chain, was mutated to the following residues: F, Y, W, E, and T. We have first analyzed the effects of the mutations on the kinetic and thermodynamic properties of the rate constants of the second electron transfer (k(AB)(2)) and of the coupled proton uptake (k(H)+) at the second flash. In all aromatic mutants, k(AB)(2) and k(H)+ are notably and concomitantly decreased compared to the wild-type, while no effect is observed in the other mutants. The temperature dependence of these rates shows activation energy values (DeltaH) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, except for the L209PW and L209PF mutants. The analysis of the enthalpy factors related to the electron and proton-transfer processes in the L209PF and the L209PW mutants allows to distinguish the respective effects of the mutations for both transfer reactions. It is noteworthy that in the aromatic mutants a substantial increase of the free energies of activation is observed (DeltaG(L209PY) < DeltaG(L209PF) < DeltaG(L209PW)) for both proton and electron-transfer reactions, while in the other mutants, DeltaG is not affected. The salt concentration dependence of k(AB)(2) shows, in the L209PF and L209PW mutants, a higher screening of the protein surface potential experienced by Q(B). Our data suggest that residues F and W in position L209 increase the polarizability of the internal water molecules and polar residues by altering the organization of the hydrogen-bond network. We have also analyzed the rates of the first electron-transfer reaction (k(AB)(1)), in the 100 micros time domain. These kinetics have previously been shown to reflect protein relaxation events possibly including proton uptake events (Tiede, D. M., Vazquez, J., Cordova, J., and Marone, P. M. (1996) Biochemistry 35, 10763-10775). Interestingly, in the L209PF and L209PW mutants, k(AB)(1) is notably decreased in comparison to the wild type and the other mutants, in a similar way as k(AB)(2) and k(H)+. Our data imply that the dynamic organization of this web is tightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the protein.