Energetics for Oxidation of a Bound Manganese Cofactor in Modified Bacterial Reaction Centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn²⁺ with a dissociation constant of 1 M at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P⁺ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn²⁺ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P⁺. The extent of P⁺ reduction was found to systematically range from a full reduction in the mutants with high P/P⁺ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn²⁺ oxidation on the P/P⁺ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn²⁺/Mn³⁺ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn²⁺/Mn³⁺ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn²⁺ oxidation ranging from 30 to 120 s^-1 as the P/P⁺ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s^-1, reflecting an increase in the free energy difference due to the lower Mn²⁺/Mn³⁺ midpoint potential. This dependence of the rate constant on the P/P⁺ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s^-1 and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn₄Ca cofactor of photosystem II.