Recent studies have shown that only quinones with a 2-methoxy group can act simultaneously as the primary (Q_A) and secondary (Q_B) electron acceptors in photosynthetic reaction centers from purple bacteria such as Rb. sphaeroides. ¹³C HYSCORE measurements of the 2-methoxy group in the semiquinone states, SQ_A and SQ_B, were compared with DFT calculations of the ¹³C hyperfine couplings as a function of the 2-methoxy dihedral angle. X-ray structure comparisons support 2-methoxy dihedral angle assignments corresponding to a redox potential gap (ΔE_m) between Q_A and Q_B of 175–193 mV. A model having a methyl group substituted for the 2-methoxy group exhibits no electron affinity difference. This is consistent with the failure of a 2-methyl ubiquinone analogue to function as Q_B in mutant reaction centers with a ΔE_m of ∼160–195 mV. The conclusion reached is that the 2-methoxy group is the principal determinant of electron transfer from Q_A to Q_B in type II photosynthetic reaction centers with ubiquinone serving as both acceptor quinones.