Influence of Polar Mutations on the Electronic and Structural Properties of Q A -· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the Q A -· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the Q A site. All three mutants decrease the redox midpoint potential ( E m ) of Q A by ∼0.1 V, yet the mechanism for this drop in E m is unclear. In this work, we examine (i) the hydrogen bonding interactions between Q A - · and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13 C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in E m for the Q A mutants. Instead, 14 N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 N δ may be involved in the observed lowering of the Q A midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered Q A - · spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 N δ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in Q A E m .