| Reaction center (RC) function and electron transfer kinetics were measured following changes in the protein environment at the QB site by mutation and changes in the QA-- energy level by quinone exchange.; A mutant with M220-M261 from the QA site substituted for L193-1,227 in the QB site in Rb. capsulatus RCs were studied. Despite the huge change in the QB pocket, secondary quinone function still can be reconstituted. The UQ4 dissociation constant for the QB site in the mutant is only 2 times larger than in the wild type RCs. The electron transfer rate of the mutant from QA-- to QB is 14/s, 3 orders of magnitude slower than the wild type RCs. The equilibrium constant between QA--Q B and QAQB-- is bigger than 200 in the mutant compared to 10 in the wild type, showing a large stabilization of QB--.; Electron transfer from P+QA--QB to P+QAQB-- was measured in R-26 Rb. sphaeroides RCs where QA was substituted with different naphthoquinones (NQA), QB was ubiquinone (UQB) in all cases. These substitutions change the redox midpoint potential of Q A and therefore change the driving force (DeltaGAB) for electron transfer. The absorption change provides a direct monitor of the electron transfer from NQA-- to UQB. The electron transfer rate is heterogeneous. In RCs with 2-methyl-3-phytyl-1,4-naphthoquinone (MQ) substituted at QA, three rates are observed: tau1 ≈ 3mus +/- 0.9mus, tau2 = 80 +/- 20mus, and tau 3 = 0.4 +/- 0.2ms at pH=8 and 22°C. Analysis of the time correlated spectra shows that the changes at taul are mostly due to electron transfer, electron transfer and charge compensation are mixed in tau 2, while little or no electron transfer occurs at tau3 in MQAUQB RCs. The activation energy for electron transfer is AG=3.5 kcal/Mol for both taul and tau2. With different NQs at QA, taul is found to be free energy dependent with a reaction reorganization energy of 850 +/- 100 meV. The slower phase tau2 is free energy independent. |
