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Structural properties of the isolated extrinsic regulatory 33 kDa protein of the water-oxidizing complex were analyzed at different pH values. It was found that (a) titrations of the buffer capacity reveal a characteristic hysteresis effect that is unique for the 33 kDa subunit and is not observed for the other extrinsic proteins, (b) changes of the emission from the fluorescence probe 1,8-ANS are indicative of an increased accessibility of the hydrophobic core of the 33 kDa protein to the dye at lower pH, (c) the near-UV circular dichroism spectrum of the polypeptide is altered owing to a pH decrease from 6.8 to 3.8 and becomes drastically changed at pH 2.8, and (d) the content of secondary structure elements remains virtually constant in the range 3.8 < pH < 6.8, with the following values gathered from far-UV CD spectra: approximately 8% alpha-helix, approximately 33% beta-strand, approximately 15% turns, and approximately 44% random coil. Further acidification down to pH 2.8 gives rise to a decreased alpha-helix and increased beta-strand and random coil content. A theoretical model [Ptitsyn, O., & Finkelstein, A. (1983) Biopolymers 2, 15-22] was used to predict the probability and location of secondary structure elements within the protein sequence. On the basis of these calculations, an extended hydrophobic beta-sheet domain could exist in the center of the protein and an alpha-helix in the C-terminal region. From these data, the 33 kDa protein is inferred to change its tertiary structure in vitro upon acidification of the aqueous environment. Possible implications of these features are discussed.

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.

Cyanobacteria, algae, and plants oxidize water to the O 2 we breathe, and consume CO 2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO 2 /HCO − 3 in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO − 3 acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast's lumen, resulting in a light-driven production of O 2 and CO 2 . Depletion of HCO − 3 from the media leads, in the absence of added buffers, to a reversible down-regulation of O 2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO 2 / HCO − 3 in photosynthetic water oxidation.O xygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO 2 to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O 2 ) and protons. This reaction is catalyzed by a penta-μ-oxo bridged tetra-manganese calcium cluster (Mn 4 CaO 5 ) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1-4). The possible roles of inorganic carbon, C i ðC i = CO 2 ; H 2 CO 3 ; HCO − 3 ; CO 2− 3 Þ, in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO 2 and therefore has to be based on the photolysis of H 2 CO 3 ("Kohlensäure") and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6-8). Several research groups followed up on these initial results and identified two possible sites of C i interaction within PSII (reviewed in refs. 9-12). Functional and spectroscopic studies showed that HCO − 3 facilitates the reduction of the secondary plastoquinone electron acceptor (Q B ) of PSII by participating in the protonation of Q 2− B . Binding of HCO − 3 (or CO 2− 3 ) to the nonheme Fe between the quinones Q A and Q B was recently confirmed by X-ray crystallography (3,13,14). Despite this functional role at the acceptor side, the very tight binding of HCO − 3 to this site makes it impossible for the activity of PSII to be affected by changing the C i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the watersplitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C i near the Mn 4 CaO 5 cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound HCO − 3 affects the activity of PSII at the donor side remains a viable option (reviewed i...