INTERRUPTION OF THE WATER CHAIN IN THE REACTION-CENTER FROM RHODOBACTER-SPHAEROIDES REDUCES THE RATES OF THE PROTON UPTAKE AND OF THE 2ND ELECTRON-TRANSFER TO Q(B)

A chain of bound water molecules was recently identified in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides by X-ray crystallography [Ermler et al. (1994) Structure 2, 925-936]. The possible role of the chain in proton transfer from the solution to the secondary quinone (Q(B)) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy. Pro L209, situated along the water chain about 9 Angstrom from Q(B), was changed into the aromatic residues Phe and Tyr in order to interrupt the chain. In the PL209Y (Pro L209 --> Tyr) mutant, the very small changes in the Q(A)(-)Q(B) <-> Q(A)Q(B)(-) equilibrium constant (K-2) and the first electron-transfer rates (K-AB((1))) indicate that the mutation does not lead to large structural changes. In the PL209F (Pro L209 --> Phe) mutant, a 7-fold decrease of k(AB)((1)) is observed. It follows a pH dependence parallel to that of the wild type. It is consistent with no modification of the pK of the Glu L212 determined from the pH dependence of K-2. The decreased k(AB)((1)) may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position. The major effect of the mutations observed is a concomitant decrease of the rates of the second electron transfer, k(AB)((2)), and of the proton uptake upon the second flash. The relative decrease of the k(AB)((2)) rate values in the mutants is more pronounced above pH 8. Our results indicate that the mutations have specifically altered the pathway of proton transfer to Q(B). We propose that in the wild type the chain of bound water molecules provides the most efficient pathway for the proton transfer from cytoplasm to the close vicinity of Q(B). This pattern might be a common behavior in proton-translocating membrane proteins.