The hyperthermophilic nature of the metallo-oxidase from Aquifex aeolicus

The stability of the Aquifex aeolicus multicopper oxidase (McoA) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. The enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 degrees C. Chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depletion occur before the unfolding of the tertiary structure and McoA is highly prone to aggregate. Indeed, unfolding kinetics measured with the stopped-flow technique quantified the stabilizing effect of copper on McoA (1.5 kcal/mol) and revealed quite an uncommon observation further confirmed by light scattering and gel filtration chromatography: McoA aggregates in the presence of guanidinium hydrochloride, i.e., under unfolding conditions. The aggregation process results from the accumulation of a quasi-native state of McoA that binds to ANS and is the main determinant of the stability curve of McoA. Kinetic partitioning between aggregation and unfolding leads to a very low heat capacity change and determines a flat dependence of stability on temperature.     

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus is a bacterial ortholog of the enzyme from filamentous fungi. (The subunit contains an adenosine 5'-phosphosulfate (APS) kinase-like, C-terminal domain.) The enzyme is highly heat stable with a half-life >1h at 90 degrees C. Steady-state kinetics are consistent with a random A-B, ordered P-Q mechanism where A=MgATP, B=SO4(2-), P=PP(i), and Q=APS. The kinetic constants suggest that the enzyme is optimized to act in the direction of ATP+sulfate formation. Chlorate is competitive with sulfate and with APS. In sulfur chemolithotrophs, ATP sulfurylase provides an efficient route for recycling PP(i) produced by biosynthetic reactions. However, the protein possesses low APS kinase activity. Consequently, it may also function to produce PAPS for sulfate ester formation or sulfate assimilation when hydrogen serves as the energy source and a reduced inorganic sulfur source is unavailable.     

A robust metallo-oxidase from the hyperthermophilic bacterium Aquifex aeolicus

The gene, Aquifex aeolicus AAC07157.1, encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper-resistance determinant, has been cloned, over-expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. The purified enzyme shows spectroscopic and biochemical characteristics typical of the well-characterized multicopper oxidase family of enzymes. McoA presents higher specificity (k(cat)/K(m)) for cuprous and ferrous ions than for aromatic substrates and is therefore designated as a metallo-oxidase. Addition of copper is required for maximal catalytic efficiency. A comparative model structure of McoA has been constructed and a striking structural feature is the presence of a methionine-rich region (residues 321-363), reminiscent of those found in copper homeostasis proteins. The kinetic properties of a mutant enzyme, McoADeltaP321-V363, deleted in the methionine-rich region, provide evidence for the key role of this region in the modulation of the catalytic mechanism. McoA has an optimal temperature of 75 degrees C and presents remarkable heat stability at 80 and 90 degrees C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus.     

A membrane-bound multienzyme, hydrogen-oxidizing, and sulfur-reducing complex from the hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is a hyperthermophilic, chemolithoautotrophic, hydrogen-oxidizing, and microaerophilic bacterium growing at 85 degrees C. We have shown that it can grow on an H2/S degrees medium and produce H2S from sulfur in the later exponential phase. The complex carrying the sulfur reducing activity (electron transport from H2 to S degrees ) has been purified and characterized. It is a membrane-bound multiprotein complex containing a [NiFe] hydrogenase and a sulfur reductase connected via quinones. The sulfur reductase is encoded by an operon annotated dms (dimethyl sulfoxide reductase) that we have renamed sre and is composed of three subunits. Sequence analysis showed that it belongs to the Me2SO reductase molybdoenzyme family and is similar to the sulfur/polysulfide/thiosulfate/tetrathionate reductases. The study of catalytic properties clearly demonstrated that it can reduce tetrathionate, sulfur, and polysulfide, but cannot reduce Me2SO and thiosulfate, and that NADPH increases the sulfur reducing activity. To date, this is the first characterization of a supercomplex from a bacterium that couples hydrogen oxidation and sulfur reduction. The distinctive feature in A. aeolicus is the cytoplasmic localization of the sulfur reduction, which is in accordance with the presence of sulfur globules in the cytoplasm. Association of this sulfur-reducing complex with a hydrogen-oxygen pathway complex (hydrogenase I, bc1 complex) in the membrane suggests that subcomplexes involved in respiratory chains in this bacterium are part of supramolecular organization.     

Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.     

Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Delta19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95 degrees C or higher. Delta19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95 degrees C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80 degrees C. Low concentrations of guanidine-HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutase-prephenate dehydrogenase and other mesophilic homologs.     

Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus

Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.     

Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.     

Sulfide:quinone oxidoreductase in membranes of the hyperthermophilic bacterium Aquifex aeolicus (VF5)

The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 micromol (mg protein)(-1) min(-1) at 20 degrees C. Rates of 220 nmol (mg protein)(-1) min(-1)] for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)(-1) min(-1) for the oxidation of sulfide at 20 C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3+/-0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.     

Biochemical and electron microscopic characterization of the F1F0 ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F(1)F(0) ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F(1) and subunits a and c of F(0). Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F(1) and F(0). In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the delta subunit.     

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.     

New functional sulfide oxidase-oxygen reductase supercomplex in the membrane of the hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H(2)S) to molecular oxygen. H(2)S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc(1) complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c(555) and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba(3)-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O(2) in the presence of the electron donor, H(2)S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.     

The crystal structure of Aq_328 from the hyperthermophilic bacteria Aquifex aeolicus shows an ancestral histone fold

The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 A by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 A over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein's thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper.     

Isolation,characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.     

Biogenesis of the yeast cytochrome bc1 complex

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.     

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

Cytochrome c₅₅₅ from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c₅₅₅) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 3₁₀‐α‐3₁₀ helix containing the heme‐ligating Met61. Herein, we show that AA cyt c₅₅₅ forms dimers by swapping the region containing the extra 3₁₀‐α‐3₁₀ helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cyt c₅₅₅ contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cyt c₅₅₅ dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c₅₅₅ were maintained at pH 2.2–11.0. CN^‐ and CO bound to dimeric AA cyt c₅₅₅ in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN^‐ and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c₅₅₅ changes upon dimerization by domain swapping.