Transient Electron Paramagnetic Resonance Spectroscopy on Photosystem I Reaction Centers
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The reason for the different rates of electron transfer from phylloquinone (PhQ) to the iron-sulfur cluster FX in the two branches of Photosystem I (PSI) is not fully understood. Techniques such as Transient Electron Paramagnetic Resonance (TREPR) have allowed further understanding of the electron transfer rates in these two branches and the role of D575PsaB and Q588PsaA in determining the different rates. Room temperature 9.5 GHz TREPR measurements were performed on PSI from the wildtype and three mutant strains of the cyanobacterium Synechocystis sp. PCC6803, Q588DPsaA (A-mutant), D575QPsaB (B-mutant), Q588DPsaA/ D575QPsaB (AB-mutant) to determine the effects of the mutations on the rates of the electron transfer. The goal of these experiments is to test the model proposed by Ishikita and Knapp, (J. Biol. Chem. 278, 52002–52011 (2003)) that explains the differences in the electron transfer rates as resulting from differences in the amino acid sequences of the two main protein subunits PsaA and PsaB which lead to different midpoint potentials of the two PhQs. The model also proposes that aspartate D575PsaB changes its protonation state during electron transfer. The model suggests that D575PsaB and Q588PsaA play significant roles in determining the potentials, but a previous study found that mutations to D575PsaB caused only small changes in the kinetics (Karyagina, Pushkar, Stehlik, van der Est, Ishikita, Knapp, Jagannathan, Agalarov, Golbeck (Biochemistry 46, 10804-10816 (2007)). It was proposed that this is because the mutations caused similar changes in the potentials of both PhQ and Fx. In this thesis, this proposal is tested using the double mutant Q588DPsaA/ D575QPsaB. Because the two point mutations are symmetrical with respect to FX but not with respect to the quinones, it is postulated that the potentials of the quinones should be changed while that of FX should be unaffected and therefore, the kinetics of the A- and B- branches should be significantly altered. The transient EPR data show evidence that the rates of electron transfer in the two branches have been swapped in the double mutant Q588DPsaA/ D575QPsaB and this indicates the important roles of D575PsaB and Q588PsaA in determining the electron transfer rate.