Molecular insights into evolution and mechanisms of binding, release and electron transfer between plastocyanin or cytochrome c6 and photosystem I
Final Report Abstract
The binding and electron transfer between plastocyanin (pc) and photosystem I (PSI) can be described by hydrophobic as well as electrostatic interactions. The two α helices, l and l’ in PsaB and PsaA, respectively, are involved in forming the hydrophobic interaction site at the oxidizing site of PSI. To obtain mechanistic insights into the function of the two negatively charged residues D612 and E613, present in the α helix l of PsaB, we took advantage of site-directed mutagenesis to alter both residues into His and transformed a PsaB-deficient mutant of Chlamydomonas reinhardtii. Flash-induced spectroscopy revealed that PSI harboring the changes D612H and E613H developed a high affinity towards binding of the electron donors and possessed an altered pH-dependence of electron transfer with the electron donors. Despite optimized binding and electron transfer between the altered PSI and its electron donors, the mutant strain PsaB-D612H/E613H showed a strong light sensitive growth phenotype. Furthermore we identified phosphorylation of pc as a potential modulator of electron transfer in vivo. Functional studies of genetically engineered pc suggest that phosphorylation of S50 of pc might accelerate electron transfer in vivo. It is also possible that phosphorylation of pc facilities the long-range electron transfer from grana localized cyt b6f complexes to stromal PSI. Two residues, R647 and D648 in the α helix l' of PsaA were changed to R647D, D648R or D647D/R648D, by using site-directed mutagenesis and transformation of a PsaA-deficient mutant of C. reinhardtii. All three mutant strains are showing a strong light sensitive growth phenotype. By using Flash-induced laser spectroscopy all altered photosystems reveal a reduced second order electron transfer-rate in vitro as compared to wild type. More importantly no proper affinity coefficient (KD) due to a non-increasing amplitude of the first component of the P700+ reduction transient was found, suggesting, that only a small fraction of PSI is capable of pre-binding reduced pc. Therefore, the luminal helix residues R647 and D648 of the PSI subunit PsaA are required for efficient binding of pc.
Publications
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Allmer J, Kuhlgert S, Hippler M
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Winkler M, Kuhlgert S, Hippler M, Happe T
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Busch A, Hippler M