Bacterial reaction centers (RCs) convert light energy into chemical free energy
via the doublereduction and protonation of the secondary quinone electron acceptor, Q
B, to the dihydroquinone Q
BH
2.Two RC mutants (M266His
Leu and M266His
Ala) with a modified ligand of the non-he
me ironhave been studied by flash-induced absorbance change spectroscopy. No important changes were observedfor 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 ofQ
A- was observed, at variance with the M266HA mutant. The superposition of the three-di
mensionalX-ray structures of the three proteins in the Q
A region provides no obvious explanation for the energymodification in the M266HL mutant. The shift of the midpoint redox potential of Q
A/Q
A- in M266HLcaused accelerated recombination of the charges in the P
+Q
A- state of the RCs where the native Q
A wasreplaced by a low potential anthraquinone (AQ
A). As previously reported for the native RCs, in the M266HLwe observed a biphasicity of the P
+AQ
A- PAQ
A charge recombination. Interestingly, both phasespresent a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependenciesof the proton uptake upon Q
A- and Q
B- formations are superimposable in both mutants but very differentfrom those of native RCs. The data
measured in mutants are similar to those that we previously obtainedon strains modified at various sites of the cytoplasmic region. The similarity of the response to thesedifferent mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidicresidues resulting in strongly anticooperative proton binding. The unspecific disappearance of the highpH band of proton uptake observed in all these mutants appears as the natural consequence of removingany
member of an interactive proton cluster. This long range interaction also accounts for the similarresponses to mutations of the proton uptake pattern induced by either Q
A- or Q
B-. We surmise that thepresence of an extended protonated water H-bond network providing protons to Q
B is responsible forthese effects.
