Influence of QA site redox cofactor structure on equilibrium binding, in situ electrochemistry, and electron-transfer performance in the photosynthetic reaction center protein.

The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.