CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.

CAH3 is the only carbonic anhydrase (CA) present in the thylakoid lumen of the green algae Chlamydomonas reinhardtii. The monomer of the enzyme has a molecular weight of ~29.5 kDa with high CA activity. Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex. Both proposed roles are considered in this review, together with a description of the enzymatic parameters of native and recombinant CAH3, the crystal structure of the protein, and the possible use of lumenal CA as a tool for increasing biomass production in higher plants. The identified involvement of lumenal CAH3 in the function of PSII is still unique among green algae and higher plants and can be used to understand the mechanism(s) of the functional interconnection between PSII and the proposed CA(s) of the thylakoid lumen in other organisms.

Action of 2,6-Dichloro-1,4-benzoquinone on the O2-Evolving Activity of Photosystem II in Chlamydomonas reinhardtii Cells with and without Cell Wall: Inhibitory Effect of Its Oxidized Form.

Chlamydomonas reinhardtii is a widely used object in studies on green algae concerning both photosynthesis aspects and possible biotechnological approaches. The measurement of the maximum O2 evolution by photosystem II (PSII) in living algal cells in the presence of artificial acceptors is one of the commonly used methods for determining the photosynthetic apparatus state or its change as compared to a control, parent strain, etc., because PSII is the most sensitive component of the thylakoid membrane. The present study shows the need to use low concentrations of 2,6-dichloro-1,4-benzoquinone (DCBQ) paired with potassium ferricyanide (FeCy) for achieving the maximum O2 evolution rate, while a DCBQ concentration above certain threshold results in strong suppression of O2 evolution. The required DCBQ concentration depends on the presence of the cell wall and should be exactly ~0.1 mM or in the range of 0.2-0.4 mM for cells with and without a cell wall, respectively. The inhibition effect is caused, probably, by a higher content of DCBQ in the oxidized form inside cells; this depends on the presence of the cell wall, which influences the efficiency of DCBQ diffusion into and out of the cell, where it is maintained by FeCy in the oxidized state. The possible mechanism of DCBQ inhibition action is discussed.

Involvement of Carbonic Anhydrase CAH3 in the Structural and Functional Stabilization of the Water-Oxidizing Complex of Photosystem II from Chlamydomonas reinhardtii.

The involvement of carbonic anhydrases (CA) and CA activity in the functioning of photosystem II (PSII) has been studied for a long time and has been shown in many works. However, so far only for CAH3 from Chlamydomonas reinhardtii there is evidence for its association with the donor side of PSII, where the CA activity of CAH3 can influence the functioning of the water-oxidizing complex (WOC). Our results suggest that CAH3 is also involved in the organization of the native structure of WOC independently of its CA activity. It was shown that in PSII preparations from wild type (WT) the high O2-evolving activity of WOC was observed up to 100 mM NaCl in the medium and practically did not decrease with increasing incubation time with NaCl. At the same time, the WOC function in PSII preparations from CAH3-deficient mutant cia3 is significantly inhibited already at NaCl concentrations above 35 mM, reaching 50% at 100 mM NaCl and increased incubation time. It is suggested that the absence of CAH3 in PSII from cia3 causes disruption of the native structure of WOC, allowing more pronounced conformational changes of its proteins and, consequently, suppression of the WOC active center function, when the ionic strength of the medium is increased. The results of Western blot analysis indicate a more difficult removal of PsbP protein from PSII of cia3 at higher NaCl concentrations, apparently due to the changes in the intermolecular interactions between proteins of WOC in the absence of CAH3. At the same time, the values of the maximum quantum yield of PSII did not practically differ between preparations from WT and cia3, indicating no effect of CAH3 on the photoinduced electron transfer in the reaction center of PSII. The obtained results indicate the involvement of the CAH3 protein in the native organization of the WOC and, as a consequence, in the stabilization of its functional state in PSII from C. reinhardtii.

The Main Structural and Functional Characteristics of Photosystem-II-Enriched Membranes Isolated from Wild Type and cia3 Mutant Chlamydomonas reinhardtii.

Photosystem II (PSII)-enriched membranes retain the original PSII architecture in contrast to PSII cores or PSII supercomplexes, which are usually isolated from Chlamydomonas reinhardtii. Here, we present data that fully characterize the structural and functional properties of PSII complexes in isolated PSII-enriched membranes from C. reinhardtii. The preparations were isolated from wild-type (WT) and CAH3-deficient mutant cia3 as the influence of CAH3 on the PSII function was previously proposed. Based on the equal chlorophyll content, the PSII-enriched membranes from WT and cia3 have the same amount of reaction centers (RCs), cytochrome b559, subunits of the water-oxidizing complex, Mn ions, and carotenes. They differ in the ratio of other carotenoids, the parts of low/intermediate redox forms of cytochrome b559, and the composition of outer light-harvesting complexes. The preparations had 40% more chlorophyll molecules per RC compared to higher plants. Functionally, PSII-enriched membranes from WT and cia3 show the same photosynthetic activity at optimal pH 6.5. However, the preparations from cia3 contained more closed RCs even at pH 6.5 and showed more pronounced suppression of PSII photosynthetic activity at shift pH up to 7.0, established in the lumen of dark-adapted cells. Nevertheless, the PSII photosynthetic capacities remained the same.

Carbonic anhydrase CAH3 supports the activity of photosystem II under increased pH.

The lumenal carbonic anhydrase (CA) CAH3 from green alga Chlamydomonas reinhardtii is the only one CA identified so far in close association with the photosystem II (PSII) multi-subunit protein complex. It was proposed earlier, that CAH3 could facilitate the H+ removal from the active center of the PSII water-oxidizing complex (WOC) under the light, thereby increasing its activity. In the present work, using PSII enriched membranes from the wild type of C. reinhardtii and from the CAH3-deficient mutant cia3, we demonstrate, that the suppression of the photosynthetic activity of PSII by increased pH is more pronounced in preparations from cia3 as compared to the wild type. Experiments with CA inhibitors show that the activity of CAH3 supports the function of PSII and prevents its irreversible inactivation under light upon increased pH. The photosynthetic activity of PSII from cia3 can be restored to the wild type level upon increased pH if an excess of HCO3- is added. These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO3- dehydration reaction. Measurements of the photoinduced electron transfer rate in PSII from water or from an artificial electron donor indicate, that CAH3 has a direct influence on the WOC function. Based on the data obtained in this work we conclude, that in vivo CA-activity of CAH3 may support the photosynthetic activity of PSII at increased pH in the thylakoid lumen and can be observed under the dark to light transition.