International Journal of Molecular Sciences Review The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives Maria Giulia Lionetto *, Roberto Caricato, Maria Elena Giordano and Trifone Schettino Received: 2 December 2015; Accepted: 1 January 2016; Published: 19 January 2016 Academic Editor: Reinhard Dallinger Department of Biological and Environmental Science and Technologies (DiSTeBA), University of Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy; [email protected] (R.C.); [email protected] (M.E.G.); [email protected] (T.S.) * Correspondence: [email protected]; Tel.: +39-0832-298694; Fax: +39-0832-298626 Abstract: Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible ´ + hydration of CO2 to HCO3 and H . Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences. Keywords: carbonic anhydrase; metals; inhibition; expression; biomarker; bioassay 1. Introduction Carbonic anhydrase (CA) is a widely-distributed metalloenzyme, which catalyzes the reversible ´ + hydration of CO2 to HCO3 and H . This biochemical reaction plays a key physiological role in diverse biological systems. Six distinct and unrelated CA families (α-, β-, γ-CA, δ, ζ and η-CAs) have been identified in animals, plants, algae and bacteria [1,2]. They all catalyze the same reaction of CO2 hydration, but each family shows proper specific characteristics in primary amino acid sequence and 3D tertiary structure. In animals, CA isoforms play a fundamental role in a number of physiological processes involving ´ carbon dioxide and bicarbonate, such as transport of CO2 and HCO3 between body tissues and respiratory surfaces, pH homeostasis, electrolyte transport in various epithelia, biosynthetic reactions (gluconeogenesis, lipogenesis and ureagenesis), bone resorption and calcification. In algae, plants and some bacteria, CA isoforms are fundamental for photosynthesis [3,4]. The α-carbonic anhydrases are monomeric or dimeric and are found in animals, some fungi, bacteria, algae and green plants [5]. In mammals, at least 16 different α CA isoforms were isolated. Mammalian CA isoforms CAI, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and CA XV (not expressed in humans) have catalytic activity, while the remaining three CAs (CARP VIII, CARP X and CARP XI) have lost the catalytic activity and are known as CA-related proteins [6]. The β-carbonic anhydrases are dimers, tetramers or octamers and are expressed mainly in fungi, bacteria, archaea, algae and chloroplasts of monocotyledons and dicotyledons [7] and some prokaryotes [8]. The γ-anhydrase class is a homotrimer that has been described in bacteria, Archaea and plants [9]. It also includes a number of non-catalytically-active homologs present in diverse species. δ- and ζ-CAs are present in several classes of marine phytoplankton. The δ class has been described in diatoms, and Int. J. Mol. Sci. 2016, 17, 127; doi:10.3390/ijms17010127 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 127 2 of 14 Int. J. Mol. Sci. 2016, 17, 127 2 of 14 itsThe prototype ζ-CAs are is theprobably CA TWCA1 monomers from the and marine have diatom three Thalassiosiraslightly different weissflogii active[10 ,sites11]. Theon ζthe-CAs same are probablyprotein molecule monomers [12]. and have three slightly different active sites on the same protein molecule [12]. The ηη-CA-CA waswas recently recently found found in in a numbera number of speciesof species of the ofPlasmodium the Plasmodiumgenus. genus. These These are a groupare a ofgroup enzymes of enzymes previously previously ascribed ascribed to the α tofamily, the α but family, recently but demonstratedrecently demonstrated to have a numberto have a of number unique features,of unique including features, theirincluding metal their ion coordinationmetal ion coordination pattern [2]. pattern [2]. This review focuses on an interesting aspect of the research onon CA,CA, thethe relationshipsrelationships betweenbetween carbonic anhydraseanhydrase and and metals, metals, which which play aplay fundamental a fundamental role in the role bioactivity in the of bioactivity this metalloenzyme. of this Themetalloenzyme. review points The out thereview complexity points andout multi-aspectthe complexity nature and of thesemulti-aspect relationships, nature since of metalsthese canrelationships, be cofactors since of CA,metals but can also beinhibitors cofactors of CA, CA activitybut also andinhibitors modulators of CA ofactivity CA expression. and modulators New insightsof CA expression. and perspectives New areinsights discussed and encompassingperspectives are several discussed fields ofencompassing research from several biotechnological fields of applicationsresearch from to biotechnological environmental sciences. applications to environmental sciences. 2. Metals Metals and and CA CA Catalytic Catalytic Site Site + All CACA isoenzymesisoenzymes catalyzecatalyze thethe reversiblereversible hydrationhydration ofof COCO22 toto HCOHCO33 andand HH+ through a 3 2+ ´ metal-hydroxide [Lig[LigM3M2+(OH) −]] mechanism mechanism [[13–15]13–15] (Figure(Figure1 ).1). The The central central catalytic catalytic step step involves involves the ´ ´ reactionthe reaction between between CO2 COand2 theand OH the OHbound− bound to the to zinc the ion, zinc yielding ion, yielding a coordinated a coordinated HCO3 HCOion, which3− ion, 3 iswhich subsequently is subsequently displaced displaced from the metalfrom bythe Hmetal2O. In by the Hα2-,O.γ -In and theδ-CA α-, classes,γ- and Ligδ-CAis classes, represented Lig3 byis threerepresented key amino by three acid residues,key amino which acid areresidues, three histidineswhich are in threeα-CA, histidinesγ-CA and in δα-CA,-CA, one γ-CA histidine and δ-CA, and twoone histidine cysteines and in β -CAtwo cysteines and ζ-CA in and β-CA two and His ζ and-CA one and Gly two residues His and in oneη-CA Gly [16 residues]. A fourth in η histidine,-CA [16]. thatA fourth is His histidine, 64 in human that is CAII His (the64 in most human investigated CAII (the most CA isoform), investigated not directlyCA isoform), part ofnot the directly active part site, + contributesof the active tosite, the contributes catalytic process to the catalytic representing process the representing so-called “proton the so-called shuttle”. “proton This allowsshuttle”. the This H transferallows the from H+ thetransfer metal-bound from the water metal-bound molecule water to buffer molecule molecules to buffer located molecules outside located the active outside site and the ´ ´ ensuresactive site the and reaction ensures of thethe metal-boundreaction of the OH metal-boundwith CO2 OHto produce− with CO HCO2 to produce3 . HCO3−. 3 2+ ´ 2+ The metal (M) in the carbonic anhydrase anhydrase metal-hydroxide metal-hydroxide [Lig [Lig3MM2+(OH)(OH)−] mechanism] mechanism is Zn is2+ Zn for forall classes, all classes, but but other other transition transition metals metals have have been been demonstrated demonstrated to to bind bind to to the the catalytic catalytic site as physiologically-relevant metal metal cofactors cofactors or or displacers displacers of of the the native native cofactor, cofactor, producing producing in this in casethis newcase CAnew metallovariants CA metallovariants (Table (Table.1).1). Figure 1. The reversible hydration hydration of of carbon carbon dioxide dioxide to to bicarbonate bicarbonate catalyzed catalyzed by by CAs CAs by by means means of of a ametal metal (M)-hydroxide (M)-hydroxide mechanism. mechanism. Modified Modified from from Berg Berg [17]. [17]. (1 ()1 )The The release release of of a a proton proton from thethe zinc-bound water generates the zinc-bound OH−´; (2) A CO2 molecule binds to the active site and is zinc-bound water generates the zinc-bound OH ;(2) A CO2 molecule binds to the active site and ispositioned positioned for for optimal optimal interaction interaction with with the the zinc-bound zinc-bound OH OH−´; ;((3)3 )The The hydroxide hydroxide ion ion attacks thethe carbonyl of CO2, producing HCO3−´; (4) The release of HCO3− regenerates´ the enzyme. carbonyl of CO2, producing HCO3 ;(4) The release of HCO3 regenerates the enzyme. Int. J. Mol. Sci. 2016, 17, 127 3 of 14 Table 1. Metals as physiologically-relevant cofactors of CA. CA Families Metals as Physiologically-Relevant CA Cofactors Ref. α-CA Zn2+ [21] β-CA Zn2+ [7] γ-CA Fe2+; Zn2+ [9,22] δ-CA Zn2+ [10] ζ-CA Cd2+; Zn2+ [12] η-CA Zn2+ [2] Apart from zinc, other metals have been found to be physiologically-relevant cofactors of some CAs. 2.1. Metals as Physiologically-Relevant Cofactors of CA Zn2+ is one of the most widely-used metallic elements as enzyme cofactor in nature, and its presence in all of the CA families is a successful confirmation of its peculiar properties. The reason for its success lies in the filled d orbital (d10). Unlike other first-row transition elements (e.g., Sc2+, Ti2+,V2+, Cr2+, Mn2+, Fe2+, Co2+, Ni2+ and Cu2+), Zn2+ is not involved in redox reactions, but rather, it acts as a Lewis acid accepting a pair of electrons [18]. This makes zinc a good metal cofactor for biochemical reactions requiring a redox-stable ion to function as a Lewis acid-type catalyst [19], such as proteolysis and carbon dioxide hydration. Zinc complexes have low thermodynamic stabilities, as well as variable geometries, which in turn account for low activation barriers. This makes zinc a versatile and suitable as an active site metal [20]. Zinc is in the +2 state, and it is positioned in a cleft in the center of the CA molecule (Figure2). It is coordinated by the three key amino acid residues (see above).