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Abstract

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_BH₂. Two RC mutants (M266His → Leu and M266His → 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^- → 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.