Role of a novel photosystem II-associated carbonic anhydrase in photosynthetic carbon assimilation in Chlamydomonas reinhardtii

Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2-concentrating mechanism (CCM), which helps to overcome CO2 limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II-mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular alpha-CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2 for carboxylation.     

A quantitative assessment of the carbonic anhydrase activity in photosystem II

Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O(2) evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.     

A quantitative assessment of the carbonic anhydrase activity in photosystem II

Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O₂ evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.     

Extrinsic photosystem II carbonic anhydrase in maize mesophyll chloroplasts

One form of carbonic anhydrase (CA) has been observed in maize (Zea mays) thylakoids and photosystem II (PSII)-enriched membranes. Here, we show that an antibody produced against a thylakoid lumen-targeted CA found in Chlamydomonas reinhardtii reacts with a single 33-kD polypeptide in maize thylakoids. With immunoblot analysis, we found that this single polypeptide could be identified only in mesophyll thylakoids and derived PSII membranes, but not in bundle sheath thylakoids. Likewise, a CA activity assay confirmed a large amount of activity in mesophyll, but not in bundle sheath membranes. Immunoblot analysis and CA activity assay showed that the maximum CA can be obtained in the supernatant of the PSII-enriched membranes washed with 1 M CaCl(2), the same procedure used to remove all extrinsic lumenal proteins from PSII. Because this CA reacts with an antibody to lumen-directed CA in C. reinhardtii, and because it can be removed with 1 M CaCl(2) wash, we refer to it tentatively as extrinsic CA. This is to distinguish it from another form of CA activity tightly bound to PSII membranes that remains after CaCl(2) wash, which has been described previously. The function of extrinsic CA is not clear. It is unlikely to have the same function as the cytoplasmic CA, which has been proposed to increase the HCO(-)(3) concentration for phosphoenolpyruvate carboxylase and the C(4) pathway. We suggest that because the extrinsic CA is associated only with thylakoids doing linear electron flow, it could function to produce the CO(2) or HCO(-)(3) needed for PSII activity.     

A novel carbonic anhydrase II binding site regulates NHE1 activity

Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na(+)/H(+) exchanger. CAII did not bind to acidic sequences in NHE1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na(+)/H(+) exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail.     

Molecular evolution of the carbonic anhydrase genes: Calculation of divergence time for mouse carbonic anhydrase I and II

Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Directed evolution of the promiscuous esterase activity of carbonic anhydrase II

A promiscuous activity of an existing enzyme can confer an evolutionary advantage by providing an immediate response to a new selection pressure and a starting point for the divergence of a new enzyme. This work seeks to examine how this process might take place. Human carbonic anhydrase II (hCAII) is an enzyme that evolved to catalyze the reversible hydration of CO(2) and performs this task at a remarkable rate (k(cat) approximately 10(6) s(-)(1)). hCAII also exhibits promiscuous activity toward highly activated esters such as 4-nitrophenyl acetate. We describe a much weaker esterase activity of hCAII toward the bulkier and much less activated ester substrate 2-naphthyl acetate (2NA). Directed evolution of hCAII produced a variant with 40-fold higher rates toward 2NA, owing to two mutations: one within the active site (Ala65Val) and one at its mouth (Thr200Ala). Structure-activity studies suggest that these mutations led to adaptation of the active site for bulkier substrates and for the catalysis of nonactivated esters. The mutations did not, however, significantly alter the native activity of hCAII. Our results support the notion that the evolution of a new function can be driven by mutations that increase a promiscuous function (which serves as the starting point for the evolutionary process) but do not harm the native function.     

A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.     

Quantitative assessment of intrinsic carbonic anhydrase activity and the capacity for bicarbonate oxidation in photosystem II

On the basis of equilibrium isotopic distribution experiments using (18)O-labeled water, it is generally accepted that water is the sole substrate for O(2) production by photosystem II (PSII). Nevertheless, recent studies indicating a direct interaction between bicarbonate and the donor side of PSII have been used to hypothesize that bicarbonate may have been a physiologically important substrate for O(2) production during the evolution of PSII [Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., and Tyryshikin, A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2170-2175]. To test out this hypothesis and to determine whether contemporary oxygenic organisms have the capacity to oxidize bicarbonate, we employed special rapid-mixing isotopic experiments using (18)O/(13)C-labeled bicarbonate to quantify the inherent carbonic anhydrase activity in PSII samples and the potential flux of oxygen from bicarbonate into the photosynthetically produced O(2). The measurements were made on PSII samples prepared from spinach, Thermosynechococcus elongatus, and Arthrospira maxima. For the latter organism, a strain was used that grows naturally in an alkaline, high (bi)carbonate soda lake in Africa. The results reveal that bicarbonate is not the substrate for O(2) production in these contemporary oxygenic photoautotrophs when assayed under single turnover conditions.     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.