In Rhodobacter sphaeroides reaction centers, mutation of proline L209 to aromatic residues in the vicinity of a water channel alters the dynamic coupling between electron and proton transfer processes

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobacter sphaeroides obtained at high resolution has revealed a num ber of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, I-I. (1994) Structure 2, 925-936; Stowell, M. H. B., McPhillip s, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Sci ence 276, 812-816). Some of them are organized into distinct hydrogen-bonde d 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 immedi ately 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 el ectron 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 c oncomitantly decreased compared to the wild-type, while no effect is observ ed in the other mutants. The temperature dependence of these rates, shows a ctivation energy values (Delta H double dagger) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, ex cept for the L209PW and L209PF mutants. The analysis of the enthalpy factor s related to the electron and proton-transfer processes in the L209PF and t he L209PW mutants allows to distinguish the respective effects of the mutat ions for both transfer reactions. It is noteworthy that:in the aromatic mut ants a substantial increase of the free energies of activation is observed (Delta G double dagger(L209PY) < Delta G double dagger(L209PF) < Delta G do uble dagger(L209PW)) for both proton and electron-transfer reactions, while in the other mutants, Delta G double dagger is not affected. The salt conc entration dependence of kAB(2) shows, in the L209PF and L209PW mutants, a h igher screening of the protein surface potential experienced by Q(B) Our da ta suggest that residues F and W in position L209 increase the polarizabili ty of the internal water molecules and polar residues by altering the organ ization of the hydrogen-bond network. We have also analyzed the rates of th e first electron-transfer reaction (k(AB)(1)), in the 100 mu s time domain. These kinetics have previously been shown to reflect protein relaxation ev ents possibly including proton uptake events (Tiede, D. M., Vazquez, J., Co rdova, J., and Marone, P. M. (1996) Biochemistry 35, 10763-10775). Interest ingly, in the L209PF sind L209PW mutants, kAB(I) is notably decreased in co mparison 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 t ightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the prot ein.