We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.6 times more than wild type). We present compelling evidence that the mutant has an average of two manganese atoms per PSII reaction centre. When bicarbonate is added to mutant thylakoids or PSII particles, the O2 evolution rates exceed those of the wild type by up to 50%. The donor side of PSII in the mutant also exhibits a much higher sensitivity to overexcitation than that of the wild type. We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water-oxidizing complex in a functionally active state. The possibility that two manganese atoms are enough for water oxidation if bicarbonate ions are available is discussed.
Previous studies of the mitochondrial carbonic anhydrase (mtCA) of Chlamydomonas reinhardtii showed that expression of the two genes encoding this enzyme activity required photosynthetically active radiation and a low CO 2 concentration. These studies suggested that the mtCA was involved in the inorganic carbon-concentrating mechanism. We have now shown that the expression of the mtCA at low CO 2 concentrations decreases when the external NH 4 ϩ concentration decreases, to the point of being undetectable when NH 4 ϩ supply restricts the rate of photoautotrophic growth. The expression of mtCA can also be induced at supra-atmospheric partial pressure of CO 2 by increasing the NH 4 ϩ concentration in the growth medium. Conditions that favor mtCA expression usually also stimulate anaplerosis. We therefore propose that the mtCA is involved in supplying HCO 3 Ϫ for anaplerotic assimilation catalyzed by phosphoenolpyruvate carboxylase, which provides C skeletons for N assimilation under some circumstances.In algae and plants, the tricarboxylic acid (TCA) cycle plays a fundamental biosynthetic role (Beardall and Raven, 1990). The removal of intermediates from this cycle to feed other biosynthetic pathways (of which amino acid synthesis is often quantitatively the most important) requires that the cycle is replenished of its intermediates via anaplerotic reactions (Beardall and Raven, 1990; Norici and Giordano, 2002). The anaplerotic reactions make use of inorganic carbon to build the C4 compounds in demand in the TCA cycle, via -carboxylation (Beardall and Raven, 1990; Norici and Giordano, 2002). The provision of inorganic carbon for these reactions can be crucial to sustain amino acid and protein synthesis (among others; Norici and Giordano, 2002). Anaplerotic -carboxylation therefore represents a pivotal intersection among the metabolisms of C and N. Consequently, mechanisms must exist to ensure that there is sufficient inorganic carbon to maintain anaplerosis at an appropriate rate, especially in conditions in which the dissolved inorganic carbon (DIC) in the cytosol may be limited, competition with other DIC-requiring pathways (mostly photosynthesis) is significant, and N assimilation is fast. Respiration and photorespiration are conveniently located (spatially and functionally) sources of CO 2 to supply reactions that replenish the TCA cycle. A mechanism that recovers respiratory CO 2 would therefore be a very effective way to ensure an appropriate flux of C to the TCA cycle via anaplerosis. However, respiration and photorespiration produce DIC in the form of CO 2 , whereas many -carboxylases such as phosphoenolpyruvate carboxylase (PEPc) and pyruvate carboxylase (PC) require HCO 3 Ϫ (Chollet et al., 1996; Norici and Giordano, 2002). Thus, if the uncatalyzed rate of CO 2 conversion to HCO 3 Ϫ is not sufficiently high, enzymatic hydration of CO 2 to HCO 3 Ϫ may be necessary (Raven and Newman, 1994; Huang and Chapman, 2002). Reactions of this sort are catalyzed by carbonic anhydrases (CAs), whose activity is wi...
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