Targeted Mutations in the psaC Gene of Chlamydomonas reinhardtii: Preferential Reduction of FB at Low Temperature Is Not Accompanied by Altered Electron Flow from Photosystem I to Ferredoxin
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Abstract:
The terminal part of the electron pathway within the photosystem I (PSI) complex includes two [4Fe-4S] centers, FA and FB, which are coordinated by the PsaC subunit. To gain new insights into the electron transfer mechanisms through PsaC, we have generated three mutant strains of the alga Chlamydomonas reinhardtii in which two positively charged residues, K52 and R53, near the FA center have been altered in different ways. The mutations K52S/R53D and K52P/R53D lead to a strong destabilization of PSI. The third mutant K52S/R53A accumulates PSI to 30% of wild-type levels and shares the same residues between two of the cysteine ligands of FA as the PsaC homologue in the green sulfur bacterium Chlorobium limicola, in which FB has a higher redox potential than FA [Nitschke, W., Feiler, U., & Rutherford, A. W. (1990) Biochemistry 29, 3834-3842]. Low-temperature electron paramagnetic resonance (EPR) studies reveal that, in contrast to wild type, FB is preferentially photoreduced in this mutant, as was also observed for C. limicola. The preferential photoreduction of FB could be due to changes in the redox potential of FA and/or to slight structural modifications of the PsaC subunit. However, room temperature optical measurements show that stable charge separation still occurs and, surprisingly, that electron transfer from PSI to ferredoxin proceeds at normal rates in the mutant. As C. limicola, the K52S/R53A and K52S/R53D C. reinhardtii mutants are photosensitive when grown aerobically, but can grow photoautotrophically under anaerobic conditions.Keywords:
Chlamydomonas reinhardtii
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Cytochrome b6f complex
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Activities related to photosystem I and photosystem II are cytochemically localized at the electron microscope level by using suitable artificial electron donors and acceptors with, or without, factors uncoupling the light-induced electron flow between the two systems. Photo-oxidation of DAB, insensitive to DCMU, is linked to photosystem I activity. It occurs within the grana and stroma membranes of the chloroplasts. Photo-reduction of TC-NBT sensitive to DCMU, is a photosystem II mediated reaction. It is trapped within the grana membranes.
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Chlamydomonas reinhardtii is a unicellular green alga, which is used as a model organism in many fields of molecular biology. In order to understand more about the optimum conditions necessary for the growth of C. reinhardtii in a lab setting, we focused on how temperature can affect the abundance of both wild-type and mutant forms. We used the CC-1690 wild-type strain and the CC-3913 - pf9-3 mutant strain. In our experiment, we used three temperature treatments: 11°C, 17°C, 25°C. We had three replicates at each treatment temperature for each and took cell density measurements over ten days. Based on observations and data that we collected, we observed that the wild type generally reaches the greatest cell density at 17°C, where as the mutant type has the highest cell density at 25°C. The results of these data allow us to reject both of our null hypotheses. We suggest that this is because the wild type C. reinhardtii are better capable of accumulating the energy necessary for growth due to the functions of flagella and the presence of certain enzymes, whereas the mutant type lacks these traits beneficial for their living (Falk et al. 2006).
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PROTEIN COMPOSITION OF SPINACH CHLOROPLASTS AND THEIR PHOTOSYSTEM I AND PHOTOSYSTEM II SUBFRAGMENTS*
Abstract— The proteins of spinach chloroplasts and their subfragments containing photosystem I and photosystem II, obtained by Triton X‐100 treatment or French‐pressure rupture, were separated by sodium dodecyl sulfate (SDS)‐acrylamide electrophoresis at pH 7·0 in phosphate buffer. The individual protein bands were identified where possible by comparing them with known, isolated and characterized proteins from chloroplasts, and their molecular weights were determined. The protein composition of the chloroplast fragments were correlated to the functional properties of these fragments. Distinct patterns were obtained for photosystem I and photosystem II particles. The major protein of photosystem II is expressed in the 23 kilodalton range and photosystem I proteins seem to be clustered mainly in the 50–70 kilodalton range.
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Abstract With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4°C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.
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