Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

Abstract

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...

Bicarbonate-induced redox tuning in Photosystem II for regulation and protection

“…A role of HCO 3 − in photoassembly of the cluster has been suggested previously, and remains possible (5,16). A role of HCO 3 − , enhancing proton removal from the site of water splitting, was recently reported (10), and it was suggested that this could represent a regulatory link between CO 2 and water splitting (10). The present work also shows a mechanism in which the CO 2 level can control PSII activity (see below).…”

“…The "donor-side" studies of PSII are often based on the idea that the acceptor-side HCO 3 − has a single fixed binding constant for HCO 3 − bound to the Fe 2+ , keeping it permanently bound to PSII under physiological conditions (10). These results change that premise, and thus it may be worthwhile reassessing some phenomena attributed to the donor side.…”

“…Measurements of the dissociation constant of HCO 3 − (K d = 40-80 μM), compared with estimates of the HCO 3 − concentration in the stroma (reviewed in ref. 9), led to the assumption that HCO 3 − remains bound under physiological conditions (10). Recently, however, it was reported that the HCO 3 − bound to PSII in Chlamydomonas can be displaced by acetate when present in the culture medium (11).…”

“…The dissociation constant for HCO 3 − has been estimated to be 40-80 μM, whereas the concentration of HCO 3 − in the stroma at ∼pH 8 is estimated to be 220 μM (9,42). Thus, it has been generally assumed that the HCO 3 − should be constantly bound to the site (10). A weakening of the HCO 3 − binding, with the estimated K d increasing by ∼18 times (Fig.…”

Crystal Structure and Functional Characterization of Photosystem II-Associated Carbonic Anhydrase CAH3 in Chlamydomonas reinhardtii

“…If proton removal is slow, this leads to less efficient O 2 production and consequently may lead to donor side photoinhibition (Minagawa et al, 1996). That HCO 3 -acts as a mobile proton carrier has been recently demonstrated for spinach (Spinacia oleracea) PSII membrane fragments using membrane inlet mass spectrometry (MIMS; Koroidov et al, 2014). These results show that PSII possesses a light-and HCO 3 --dependent CO 2 production for up to 50% of the O 2 produced.…”