The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetryrelated protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll-protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to Ϸ60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron-nuclear-nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from Ϸ2:1 to Ϸ5:1 in the mutants compared with Ϸ2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation-reduction midpoint potential.The reaction center is the site of the primary process in photosynthesis, which is the conversion of light energy into a charge-separated state (for reviews, see ref. 1). The electron transfer process in the reaction center has the remarkable aspect that the quantum yield is near unity, that is, for every photon absorbed, one electron is transferred. The reaction center isolated from the purple bacterium Rhodobacter sphaeroides is particularly useful for the study of electron transfer because this complex has been well characterized biochemically and spectroscopically, and the three-dimensional structure has been determined by x-ray diffraction (2-5). The reaction center has two core subunits, L and M, that form the binding sites for the cofactors and are related by an Ϸ2-fold symmetry axis. The primary electron donor (P) consists of two symmetry-related bacteriochlorophyll (BChl) a molecules labeled P L and P M that overlap at the ring I position and are separated by Ϸ3.5 Å (Fig. 1). Light absorption causes the excitation of P, and within 3.5 ps, an electron is transferred to a bacteriopheophytin acceptor. Electron transfer proceeds to the primary quinone acceptor, Q A , in Ϸ200 ps and to the secondary quinone, Q B , in Ϸ200 s. After reduction of the donor by cytochrome c 2 , absorption of a second photon leads to the transfer of a second electron to Q B , which then leaves the protein carry...