Contributions of the protein environment to the midpoint potentials of the A₁ phylloquinones and the F_X iron-sulfur cluster in photosystem I

Electrostatic calculations have predicted that the partial negative charge associated with D575_PsaB plays a significant role in modulating the midpoint potentials of the A_1A and A_1B phylloquinones in photosystem I. To test this prediction, the side chain of residue 575 _PsaB was changed from negatively charged (D) to neutral (A) and to positively charged (K). D566_PsaB, which is located at a considerable distance from either A_1A or A_1B, and should affect primarily the midpoint potential of F_X, was similarly changed. In the 575_PsaB variants, the rate of electron transfer from A_1A to F_X is observed to decrease slightly according to the sequence D/A/K. In the 566_PsaB variants, the opposite effect of a slight increase in the rate is observed according to the same sequence D/A/K. These results are consistent with the expectation that changing these residues will shift the midpoint potentials of nearby cofactors to more positive values and that the magnitude of this shift will depend on the distance between the cofactors and the residues being changed. Thus, the midpoint potentials of A_1A and A_1B should experience a larger shift than will F_X in the 575_PsaB variants, while F_X should experience a larger shift than will either A_1A or A_1B in the 566_PsaB variants. As a result, the driving energy for electron transfer from A_1A and A_1B to F_X will be decreased in the former and increased in the latter. This rationalization of the changes in kinetics is compared with the results of electrostatic calculations. While the altered amino acids shift the midpoint potentials of A_1A, A_1B, and F_X by different amounts, the difference in the shifts between A_1A and F_X or between A_1B and F_X is small so that the overall effect on the electron transfer rate between A_1A and F_X or between A_1B and F _X is predicted to be small. These conclusions are borne out by experiment.