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

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO₂ and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO₂ fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO₃^– increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO₃^– from CO₂. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO₂/HCO₃^– on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO₂ concentration.Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyze the interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻), a reaction that otherwise proceeds slowly at physiological pH. CAs belong to three evolutionary distinct classes, α, β, and γ, which share no significant amino acid sequence identity and are thought to be the result of convergent evolution (Hewett-Emmett and Tashian, 1996; Supuran, 2008; Ferry, 2010; Rowlett, 2010). Animals have only the α-CA type, but as multiple isoforms. By contrast, higher plants, algae, and cyanobacteria may contain members of all three CA families. In algae, CAs has been found in mitochondria and chloroplasts and in the cytoplasm and apoplasm.Many fresh-water and soil-living microalgae face limiting concentrations of inorganic carbon (Ci) in their environments. To overcome this, the green microalga Chlamydomonas reinhardtii, as well as most other unicellular algae and cyanobacteria, actively accumulate Ci inside the cells. This mechanism is known as the carbon-concentrating mechanism (CCM; Raven, 1997; Wang et al., 2011; Meyer and Griffiths, 2013). CCM allows the algae to maintain a high concentration of CO₂ around the carboxylating enzyme, Rubisco, even under limiting external Ci. The increased concentration of CO₂ in the chloroplast increases the CO₂/O₂ specificity for Rubisco that leads to a decreased oxygenation reaction, and hence carboxylation becomes more efficient.CCM can be induced in C. reinhardtii cultures by bubbling air containing CO₂ at ambient or concentrations (≤0.04%; Vance and Spalding, 2005). Full metabolic adaptation is usually reached within 10 to 12 h after transfer to air CO₂ conditions (Renberg et al., 2010). Already within the first few hours after induction, several genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Fang et al., 2012). Surprisingly, the global changes in protein expression do not correspond to those in the gene expression; only few proteins are either up- or down-regulated during CCM induction (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). CAs are important components of the CCM. In C. reinhardtii, 12 genes are expressed that encode for CA isoforms (Moroney et al., 2011). Among the many genes that are significantly up-regulated during CCM induction, there is one encoding for an apoplastic CA (CrCAH1) and two encoding for mitochondrial CAs (CrCAH4 and CrCAH5; Fujiwara et al., 1990; Eriksson et al., 1996).An α-type CA (CrCAH3) located in the thylakoid lumen in C. reinhardtii has also been identified as important at low CO₂ levels (Karlsson et al., 1998). The sequence indicates that it is transported through the thylakoid membrane via the Twin Arg Translocation pathway (Albiniak et al., 2012). A mutant not expressing CrCAH3 (knockout of the cah3 gene) shows no or poor growth under air CO₂ levels (Spalding et al., 1983; Moroney et al., 1986) and has a severely impaired photosynthetic capacity under low Ci conditions. This mutant, called CrCIA3, has been a valuable tool for resolving the CrCAH3 function.It is also established that CrCAH3 is associated with PSII (Stemler, 1997; Villarejo et al., 2002; Blanco-Rivero et al., 2012). Using isolated PSII membranes from C. reinhardtii, Shutova et al. (2008) presented data suggesting that CrCAH3 is important for efficient water oxidation by facilitating the removal of protons that are produced when water is oxidized by PSII. This is in line with recent studies (Zaharieva et al., 2011; Klauss et al., 2012) showing that it is crucial to have alternating electron and proton removals from the oxygen-evolving complex (OEC) during the five-state catalytic cycle, i.e. the Kok cycle (Kok et al., 1970), of photosynthetic water oxidation. If proton removal is slow, this leads to less efficient O₂ production and consequently may lead to donor side photoinhibition (Minagawa et al., 1996). That HCO₃^– 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 HCO₃^–-dependent CO₂ production for up to 50% of the O₂ produced.Taken together, these data suggest that CrCAH3 plays an important role in regulating PSII reactions. In this work, we present further evidence for its function in PSII primary reactions, in particular at low Ci concentrations. We determined crystal structures of CrCAH3 at 2.6 to 2.7 Å resolution in complex with acetazolamide (AZM) or phosphate ions. Our results support a zinc-hydroxide catalytic mechanism of CrCAH3 similar to that of other α-CAs. CrCAH3 has, however, an activity optimum at lower pH values than CAs of the same type, which normally operate at pH 7.0 and higher (Demir et al., 2000). The activity optimum of CrCAH3 makes it more suitable for CO₂/HCO₃^– interconversion at the pH levels present in the thylakoid lumen under light exposure.