Effect of binding of Cd2+ on bacterial reaction center mutants: proton-transfer uses interdependent pathways.
In bacterial reaction center of Rhodobacter sphaeroides, Cd2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (kAB(1)) and second flash (kAB(2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogen-bond networks involving water molecules. We show that (1) the combined effects of Cd2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd2+] titration curves of kAB(2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S.A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd2+ binds; (2) in the L209PY mutant, the pH titration curves of kAB(1), kAB(2), and k(H)(+), the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 microM Cd2+, similarly to the wild type RCs (Gerencser, L., and Maroti, P. (2001) Biochemistry 40, 1850-1860). We propose that Cd2+ binding influences the electrostatics of interdependent ways of proton penetration within the protein, involving at least, directly or indirectly, L209P, L210D, and M17D, probably in conjunction with hydrogen-bonded connected water molecules.
University of Szeged, Szeged, H-6722, Hungary, and Centre de Génétique Moléculaire, Bat. 24, 91198, CNRS, Gif/Yvette, France.
Laszlo
L
Gerencser
Antoine
A
Taly
Laura
L
Baciou
Peter
P
Maroti
Pierre
P
Sebban
D002104:Cadmium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons
D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D002104:Cadmium