Bacterial reaction centers (RCs) convert light energy into chemical free energy Via the double reduction and protonation of the secondary quinone electron acceptor, Q B , to the dihydroquinone Q B H 2 . Two RC mutants (M266His f Leu and M266His f Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, Q A , and Q B . However, in the M266HL mutant a destabilization of ∼40 meV of the free energy level of Q A -was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the Q A region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of Q A /Q A -in M266HL caused accelerated recombination of the charges in the P + Q A -state of the RCs where the native Q A was replaced by a low potential anthraquinone (AQ A ). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P + AQ A -f PAQ A charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon Q A -and Q B -formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either Q A -or Q B -. We surmise that the presence of an extended protonated water H-bond network providing protons to Q B is responsible for these effects.Bacterial reaction centers (RCs 1 ) convert light excitation energy into chemical free energy in a similar way as the photosystem II of oxygen evolving complexes (1). RCs are membrane proteins composed of three subunits, L, M, and H, with a total molecular weight of about 100 kDa. The initial electronic excitation of a dimer of bacteriochlorophylls, P, situated on the periplasmic side of the RCs produces its electronic excited singlet state of lowest energy P*. Within about 200 ps an electron is sent from P* Via intermediate carriers (a monomer of bacteriochlorophyll and a bacteriopheophytin, H) to a ubiquinone acceptor named Q A , located on the cytoplasmic side of the complex. The electron is then transferred, in the hundred microseconds time scale, to the seconda...