Wright, Derek William; (1999) Analysis of site-directed mutants of the FX-binding region of photosystem I and second site revertants in Chlamydomonas reinhardtii. Masters thesis (M.Phil), UCL (University College London).

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Abstract

This thesis reports structural, biochemical and biophysical analyses of photosystem I (PSI), using site-directed mutants and second-site revertants of Chlamydomonas reinhardtii. The principal techniques used were non-denaturing gel electrophoresis, biochemical assays of electron transport, continuous wave and pulsed electron paramagnetic resonance spectroscopy (EPR) and electron-nuclear double resonance spectroscopy (ENDOR). Site-directed mutants of the conserved region of PsaA which is thought to form the FX binding site have been previously generated (Hallahan et al., 1995): C575D, C575H, C575S and D576L, all of which are non-photosynthetic. Photosynthetic second-site revertants have been generated from D576L (Evans ct al., 1999). The secondary mutations are in nuclear genes. Non-denaturing polyacrylamide gel electrophoresis of thylakoid membranes indicated that PSI did not assemble in C575D. C575H, C575S and D576L assembled PSI at reduced levels. Continuous wave EPR showed no photoreduction of iron-sulphur centres in C575H. Spectra of FA/FB in D576L and the revertants showed an altered electron distribution. NADP+ photoreduction was abolished in the site-directed mutants and restored in the revertants. Photoreduction of methyl viologen took place with thylakoid membranes of C575S and D576L. Photoreduction of neutral red but not of methyl viologen took place with C575H. EPR and ENDOR spectra of A1- indicated no significant differences in electronic structure or binding site structure between wild type and D576L, and only very small differences between C. reinhardtii and spinach. Rates of electron transfer were determined using time-resolved pulsed EPR. Forward electron transfer from A1 did not take place in C575H and C575S. The rate of forward electron transfer from A1 was considerably slower in D576L than in wild type. The revertants showed rates similar to wild type. C575H and C575S do not bind FX. In D576L, PsaC is incorrectly bound. The structure of the FX binding site is incorrect in D576L, and is corrected in the revertants.

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