Density Functional Theory Calculations of Molecular Hyperpolarizabilities

Recent density functional theory calculations of molecular hyperpolarizabilities are reviewed in order to try to assess the accuracy and reliability of DFT in the specified field by comparison with experiment and with ab initio HF and post-HF methods. In a table [1] (p. 157) containing results from the paper defining the compound electronic structure method Gaussian 2 [2], Foresman presents the relative accuracies of various 'model chemistries' for calculating thermochemical quantities. The Mean Absolute Deviation (MAD) from experiment, its Standard Deviation, and the largest positive and negative errors in the computed values, are the statistical means that allow the various models to be arranged in order of increasing MAD, and thus decreasing overall accuracy. These same statistical quantities, on a percent basis for size consistency and dimensionlessness, are used in this communication for quantifying the accuracies of various combinations of DFT functionals/basis sets/quantum chemical techniques/applied field strengths, in calculations of molecular mean dipole polarizabilities <>, first-order hyperpolarizabilities and second-order hyperpolarizabilities . The relative accuracies of DFT model chemistries are thus surveyed in three tables (including authors/references arranged in chronological order, compounds studied, and program codes employed), and can serve as guidelines for selecting optimal computational methodologies.     

Isolation and Characterization of a Carbonic Anhydrase Homologue from the Zebrafish (Danio rerio)

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost. Received: 31 March 1996 / Accepted: 8 September 1996     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Expression and Functional Study of Single Mutations of Carbonic Anhydrase 8 in Neuronal Cells

Cellular and Molecular Neurobiology - Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO2 to bicarbonate and proton....     

Up-Regulation of Carbonic Anhydrase Isozyme IV in CNS Myelin of Mice Genetically Deficient in Carbonic Anhydrase II

Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.     

Carbonic Anhydrase Polymorphism in Cattle and Swine

Two electrophoretically different carbonic anhydrase (Ca) isoenzymes have been demonstrated in cattle where their presence is presumed to be controlled by codominant allelic genes (Sartore & Bernoco 1966). Previous reports of Ca polymorphism in swine are not available.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

Carbonic Anhydrase and the Regulation of Photosynthesis

THE role of CO₂ in the regulation of photosynthetic and respiratory metabolism in plants is little understood in the unicellular alga Chlorella pyrenoidosa; for example, after autotrophic growth in high CO₂ (5·5% by volume), transfer to a CO₂ concentration about ten times less than the concentration in air results initially in low rates of photosynthesis characterized by the virtual absence of the Calvin cycle¹ of CO₂ fixation². An induction period of about 2 h is necessary before normal photosynthetic rates are established. Cells grown in air (0.03% CO₂) do not show this effect and photosynthesize at comparatively high rates even in very low concentrations of CO₂.     

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.     

Periplasmic Carbonic Anhydrase Structural Gene (Cah1) Mutant in Chlamydomonas reinhardtii

To survive in various conditions of CO₂ availability, Chlamydomonas reinhardtii shows adaptive changes, such as induction of a CO₂-concentrating mechanism, changes in cell organization, and induction of several genes, including a periplasmic carbonic anhydrase (pCA1) encoded by Cah1. Among a collection of insertionally generated mutants, a mutant has been isolated that showed no pCA1 protein and no Cah1 mRNA. This mutant strain, designated cah1-1, has been confirmed to have a disruption in the Cah1 gene caused by a single Arg7 insert. The most interesting feature of cah1-1 is its lack of any significant growth phenotype. There is no major difference in growth or photosynthesis between the wild type and cah1-1 over a pH range from 5.0 to 9.0 even though this mutant apparently lacks Cah1 expression in air. Although the presence of pCA1 apparently gives some minor benefit at very low CO₂ concentrations, the characteristics of this Cah1 null mutant demonstrate that pCA1 is not essential for function of the CO₂-concentrating mechanism or for growth of C. reinhardtii at limiting CO₂ concentrations.     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

杜氏盐藻两种碳酸酐酶基因启动子的克隆和功能研究

将克隆得到的杜氏盐藻DCA7和CA基因的启动子区与bar基因和NOS polyA终止子片段融合,分别构建成pMDDC-B和pMDC-B转基因杜氏盐藻表达载体。用基因枪法将两种表达载体转化人杜氏盐藻细胞,通过除草剂草丁膦筛选培养获得转化藻株,对转化藻株进行分析。对转化杜氏盐藻藻株的筛选培养结果表明：pMDDC-B和pMDC-B载体中的外源bar基因能在杜氏盐藻细胞中稳定或瞬时表达。同时在氦气压力为690kPa条件下,微弹轰击2次比微弹轰击1次或3次的效果更好。对pMDDC-B转化杜氏盐藻得到的稳定表达的转化藻株进行的PCR和Southern印迹分析的结果表明：外源的bar基因确已整合到杜氏盐藻基因组中。Northern印迹分析表明：DCA7基因启动子驱动bar基因在杜氏盐藻细胞中的表达效率受氯化钠浓度梯度调控。推测首次克隆得到的DCA7基因启动子可能是一种活性高、安全性好的高渗诱导性启动子;杜氏盐藻DCA7和CA基因启动子区的GT高度重复序列,可能与杜氏盐藻高度耐盐的分子机制有关。     

碳酸酐酶与临床医学

碳酸酐酶是一类含锌的蛋白酶,共发现了10种同工酶及三种碳酸酐酶相关蛋白,广布于人体各组织,能可逆性地催化C02的水合反应,参与调节pH、离子运输等多种生理过程,碳酸酐酶的缺乏和异常将可能会导致一系列的疾病。     

Guanidine Hydrochloride Unfolding of a Carbonic Anhydrase Molten Globule

Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution nuclear magnetic resonance spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters.     

Transition from exo- to endo- Cu absorption in CuSi(n) clusters: A Genetic Algorithms Density Functional Theory (DFT) Study

The characterization and prediction of the structures of metal silicon clusters is important for nanotechnology research because these clusters can be used as building blocks for nano devices, integrated circuits and solar cells. Several authors have postulated that there is a transition between exo to endo absorption of Cu in Si(n) clusters and showed that for n larger than 9 it is possible to find endohedral clusters. Unfortunately, no global searchers have confirmed this observation, which is based on local optimizations of plausible structures. Here we use parallel Genetic Algorithms (GA), as implemented in our MGAC software, directly coupled with DFT energy calculations to show that the global search of CuSi(n) cluster structures does not find endohedral clusters for n < 8 but finds them for n ≥ 10.     

微藻碳酸酐酶的特性及其环境调控

本文概述微藻碳酸酐酶的性质、种类、分布、分离纯化和环境调控的研究进展,并对未来有关微藻碳酸酐酶研究中需要探讨的问题作了展望。     

Carbonic Anhydrase Inhibitors. Arylsulfonylureido-and Arylureido-Substituted Aromatic and Heterocyclic Sulfonamides: Towards Selective Inhibitors of Carbonic Anhydrase Isozyme I

Abstract

Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydra-zino or hydroxyl group, with tosyl isocyanate or 3,4-dichlorophenyl isocyanate afforded two series of derivatives containing arylsulfonylureido or diarylureido moieties in their molecule respectively. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Potent inhibition was observed against all three isozymes but especially against CA I, which is generally 10-75 times less susceptible to inhibition by the classical sulfonamides in clinical use as compared to the other major red cell isozyme, CA II, or the membrane-bound one, CA IV. The derivatives obtained from tosyl isocyanate were generally more potent than the corresponding ones obtained from 3,4-dichlorophenyl isocyanate. This is the first reported example of selective inhibition of CA I and might lead to more selective drugs/diagnostic agents from this class of pharmacologically relevant compounds.     

pK(a) Calculations suggest storage of an excess proton in a hydrogen-bonded water network in bacteriorhodopsin.

Calculations of protonation states and pK(a) values for the ionizable groups in the resting state of bacteriorhodopsin have been carried out using the recently available 1.55 A resolution X-ray crystallographic structure. The calculations are in reasonable agreement with the available experimental data for groups on or near the ion transport chain (the retinal Schiff base; Asp85, 96, 115, 212, and Arg82). In contrast to earlier studies using lower-resolution structural data, this agreement is achieved without manipulations of the crystallographically determined heavy-atom positions or ad hoc adjustments of the intrinsic pK(a) of the Schiff base. Thus, the theoretical methods used provide increased reliability as the input structural data are improved. Only minor effects on the agreement with experiment are found with respect to methodological variations, such as single versus multi-conformational treatment of hydrogen atom placements, or retaining the crystallographically determined internal water molecules versus treating them as high-dielectric cavities. The long-standing question of the identity of the group that releases a proton to the extracellular side of the membrane during the L-to-M transition of the photocycle is addressed by including as pH-titratable sites not only Glu204 and Glu194, residues near the extracellular side that have been proposed as the release group, but also an H(5)O(2)(+) molecule in a nearby cavity. The latter represents the recently proposed storage of the release proton in an hydrogen-bonded water network. In all calculations where this possibility is included, the proton is stored in the H(5)O(2)(+) rather than on either of the glutamic acids, thus establishing the plausibility on theoretical grounds of the storage of the release proton in bacteriorhodopsin in a hydrogen-bonded water network. The methods used here may also be applicable to other proteins that may store a proton in this way, such as the photosynthetic reaction center and cytochrome c oxidase.     

Carbonic Anhydrase in Subchloroplast Particles of Pea Plants

The partitioning of carbonic anhydrase (CA) activity in chloroplasts isolated from 10–14-day-old pea (Pisum sativum L.) seedlings was investigated. The effect of CA inhibitors on the kinetics of chlorophyll fluorescence in photosystem II (PSII) preparations was also studied. The activity of CA was detected in fractions of soluble proteins and in the polypeptide complexes of the PSI and PSII. Isolated particles of photosystems retained a high photochemical activity similar to that of intact chloroplasts and the high level of polyunsaturated fatty acids. The association of CA with the particles of PSII (PSII-CA) was also tested by Western-blot analysis using antibodies against PSII-CA (Cah3) from Chlamydomonas reinhardtii. The PSII particles isolated with Triton X-100 (T-20) showed a higher activity of the enzyme as calculated on a protein basis than the DT-20 particles isolated with digitonin and Triton X-100. This difference seems to be related to the higher degree of nativity of the chloroplast T-20 fragments as compared to DT-20 particles. The higher level of chlorophyll per reaction center as well as the higher content of chlorophyll b and lipid fatty acids as calculated on protein basis, in particular of E-16:113 acid, which stabilizes the oligomeric structure of the light-harvesting complex of the PSII, also confirms this suggestion. The activity of CA was not detected in the DT-20 preparations treated with Tris–HCl to eliminate manganese ions. This is likely to indicate that one of the extrinsic polypeptides of PSII exhibits CA activity. Specific inhibitors of CA (acetazolamide and imidazole) inhibited the photoinduced yield of chlorophyll fluorescence (F). This might be determined by damaging the water-oxidizing system or its interaction with the PSII reaction centers. The functional role of PSII-CA for ₂-concentrating in carboxylation sites as well as its role in the coupling of light and dark reactions in chloroplasts is discussed.     

Immunological Study of Carbonic Anhydrase in C3 and C4 Plants Using Antibodies to Maize Cytosolic and Spinach Chloroplastic Carbonic Anhydrase

Carbonic anhydrase (CA) was purified to homogeneity from maizeleaves, and antibodies to the purified protein raised usingrabbits. Western blot analyses using antibodies to the purifiedmaize leaf CA indicated cross-reactivity with leaf extractsfrom a variety of C₃ and C₄ monocotyledonous and dicotyledonousplants. However maize leaf CA antibodies quantitatively titratedCA activity only in leaf extracts of maize and sorghum. Pretitrationof crude leaf extracts with maize CA antibodies and activitymeasurements, together with Western blot analyses of the resultantsamples, indicated that there are isozymic forms of CA in plantleaf tissues in both C₃ and C₄ plants which are similar in sizebut which differ antigenically from the maize leaf CA. (Received September 7, 1989; Accepted February 23, 1990)