A plant-type (beta-class) carbonic anhydrase in the thermophilic methanoarchaeon Methanobacterium thermoautotrophicum.

Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (beta-class) carbonic anhydrase in the archaea. The Methanobacterium thermoautotrophicum DeltaH cab gene was hyperexpressed in Escherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75 degrees C. No esterase activity was detected with p-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO(2) hydration activity with a k(cat) of 1.7 x 10(4) s(-1) and K(m) for CO(2) of 2.9 mM at pH 8.5 and 25 degrees C. Western blot analysis indicates that Cab (beta class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the gamma carbonic anhydrase from Methanosarcina thermophila was detected. These results show that beta-class carbonic anhydrases extend not only into the Archaea domain but also into the thermophilic prokaryotes.     

Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase

The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.     

Crystal structure of carbonic anhydrase CaNce103p from the pathogenic yeast <Emphasis Type="Italic">Candida albicans</Emphasis>

Background

The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the β-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants.

Results

Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker’s yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30–48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I β-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of β-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases.

Conclusions

CaNce103p is a β-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other β-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.

     

The active site structure of Thalassiosira weissflogii carbonic anhydrase 1

X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.     

Structure and function of carbonic anhydrases from Mycobacterium tuberculosis

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the alpha-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three beta-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 A, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.     

X-ray structure of beta-carbonic anhydrase from the red alga, Porphyridium purpureum, reveals a novel catalytic site for CO(2) hydration

The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated alpha-, beta-, and gamma-CAs based on their primary structure. beta-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of beta-CA from the red alga, Porphyridium purpureum, at 2.2-A resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an alpha/beta domain and three projecting alpha-helices. The motif is obviously distinct from that of either alpha- or gamma-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the beta-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in alpha-CAs, and possibly in gamma-CAs, is not directly applicable to the case in beta-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO(2) hydration reaction.     

The structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two

CsoSCA (formerly CsoS3) is a bacterial carbonic anhydrase localized in the shell of a cellular microcompartment called the carboxysome, where it converts HCO(3)(-) to CO(2) for use in carbon fixation by ribulose-bisphosphate carboxylase/oxygenase (RuBisCO). CsoSCA lacks significant sequence similarity to any of the four known classes of carbonic anhydrase (alpha, beta, gamma, or delta), and so it was initially classified as belonging to a new class, epsilon. The crystal structure of CsoSCA from Halothiobacillus neapolitanus reveals that it is actually a representative member of a new subclass of beta-carbonic anhydrases, distinguished by a lack of active site pairing. Whereas a typical beta-carbonic anhydrase maintains a pair of active sites organized within a two-fold symmetric homodimer or pair of fused, homologous domains, the two domains in CsoSCA have diverged to the point that only one domain in the pair retains a viable active site. We suggest that this defunct and somewhat diminished domain has evolved a new function, specific to its carboxysomal environment. Despite the level of sequence divergence that separates CsoSCA from the other two subclasses of beta-carbonic anhydrases, there is a remarkable level of structural similarity among active site regions, which suggests a common catalytic mechanism for the interconversion of HCO(3)(-) and CO(2). Crystal packing analysis suggests that CsoSCA exists within the carboxysome shell either as a homodimer or as extended filaments.     

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.     

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

The γ class of carbonic anhydrases

Homologs of the γ class of carbonic anhydrases, one of five independently evolved classes, are found in the genomic sequences of diverse species from all three domains of life. The archetype (Cam) from the Archaea domain is a homotrimer of which the crystal structure reveals monomers with a distinctive left-handed parallel β-helix fold. Histidines from adjacent monomers ligate the three active site metals surrounded by residues in a hydrogen bond network essential for activity. Cam is most active with iron, the physiologically relevant metal. Although the active site residues bear little resemblance to the other classes, kinetic analyses indicate a two-step mechanism analogous to all carbonic anhydrases investigated. Phylogenetic analyses of Cam homologs derived from the databases show that Cam is representative of a minor subclass with the great majority belonging to a subclass (CamH) with significant differences in active site residues and apparent mechanism from Cam. A physiological function for any of the Cam and CamH homologs is unknown, although roles in transport of carbon dioxide and bicarbonate across membranes has been proposed.     

A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.     

Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity

Carbonic anhydrases fall into three distinct evolutionary and structural classes: alpha, beta, and gamma. The beta-class carbonic anhydrases (beta-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a beta-CA from Escherichia coli, to a resolution of 2.0 A. In agreement with the structure of the beta-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some beta-CAS: The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a beta-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other beta-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.     

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass. In addition, we identify a unique, noncatalytic binding mode for the substrate bicarbonate that occurs in both the H. influenzae and E. coli enzymes. The kinetic and structural analysis indicates that binding of bicarbonate in this site of the enzyme may modulate its activity by influencing a pH-dependent, cooperative transition between active and inactive forms. We hypothesize that the two structural subclasses of beta-CAs may provide models for the proposed active and inactive forms of the H. influenzae and E. coli enzymes.     

Salicylaldoxime derivatives as new leads for the development of carbonic anhydrase inhibitors

New compounds containing a novel zinc binding group (salicylaldoxime system) were identified as effective inhibitors of carbonic anhydrases (CAs). This structural motif seems to bind the catalytic zinc ion of CAs, revealing itself as a new valid alternative to the sulfonamide group. Computational procedures were used to investigate the binding mode of this class of compounds, within the active site of CAII. This study suggests that the salicylaldoxime moiety binds the zinc ion through the oxime oxygen atom that also forms an H-bond with T199. The results herein obtained will allow the development of new CA-inhibitors bearing the salicylaldoxime moiety.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo,binary, and inhibitory ternary complexes

The human glutathione-dependent formaldehyde dehydrogenase is unique among the structurally studied members of the alcohol dehydrogenase family in that it follows a random bi bi kinetic mechanism. The structures of an apo form of the enzyme, a binary complex with substrate 12-hydroxydodecanoic acid, and a ternary complex with NAD+ and the inhibitor dodecanoic acid were determined at 2.0, 2.3, and 2.3 A resolution by X-ray crystallography using the anomalous diffraction signal of zinc. The structures of the enzyme and its binary complex with the primary alcohol substrate, 12-hydroxydodecanoic acid, and the previously reported binary complex with the coenzyme show that the binding of the first substrate (alcohol or coenzyme) causes only minor changes to the overall structure of the enzyme. This is consistent with the random mechanism of the enzyme where either of the substrates binds to the free enzyme. The catalytic-domain position in these structures is intermediate to the "closed" and "open" conformations observed in class I alcohol dehydrogenases. More importantly, two different tetrahedral coordination environments of the active site zinc are observed in these structures. In the apoenzyme, the active site zinc is coordinated to Cys44, His66 and Cys173, and a water molecule. In the inhibitor complex, the coordination environment involves Glu67 instead of the solvent water molecule. The coordination environment involving Glu67 as the fourth ligand likely represents an intermediate step during ligand exchange at the active site zinc. These observations provide new insight into metal-assisted catalysis and substrate binding in glutathione-dependent formaldehyde dehydrogenase.     

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

Structure of a dimeric fungal α-type carbonic anhydrase

The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7 Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the β class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.Structured summaryAoCA binds to AoCA by molecular sieving (View interaction)AoCA binds to AoCA X-ray crystallography (View interaction)     

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

An archaeal b-type cytochrome containing a nonfunctional carbonic anhydrase-like domain

A new type of cytochrome b was isolated from the cytoplasmatic fraction of the archaeon Acidianus ambivalens, which is the first soluble cytochrome found in this member of the thermoacidophilic order of the Sulfolobales. The protein is a monomeric and monohemic cytochrome b with a molecular mass of 22 kDa. Visible spectroscopy of the as-purified protein shows a Soret peak at 405 nm and a broad band at 625 nm, indicating the presence of a high-spin ferric heme. Upon reduction, the Soret band shifts to 422 nm and a broad band at 560 nm develops, again characteristic of high-spin ferrous heme. The reduced form can bind carbon monoxide, with visible absorption bands arising at 411 and 566 nm. EPR spectroscopy of the oxidized protein shows a spectrum typical of a high-spin heme, with major g values at 6.56 and 5.85. The reduction potential of the heme cofactor was determined to be -16+/-10 mV, at pH 6.5. Analysis of the protein amino acid sequence shows that it consists of a novel arrangement of domains. The first domain, at the N-terminus, has a remarkable similarity towards beta class carbonic anhydrases, whereas the second region comprises a putative cytochrome domain. The latter presumably consists of a novel fold, as it bears no sequence similarities towards other known cytochromes, or towards known domains. Strikingly, the first module contains the C-X (n)-H-X(2)-C motif that accounts for the binding of the catalytic zinc in carbonic anhydrases, but lacks several other critical residues required for substrate binding and proper active site geometry. In agreement with this finding, the isolated cytochrome contains one bound zinc atom, but has no carbonic anhydrase activity. Inspection of the sequences available from the genomic sequencing project of the close relative archaeon Sulfolobus solfataricus shows the presence of an identical protein, suggesting its dissemination among the Sulfolobales. The role of zinc as a key element for the intrinsic thermal stability of these proteins is discussed.