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.     

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.     

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.     

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.     

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.     

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.     

Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5

Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degrees C. The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue. A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da. This endoglucanase was designated cel8Y gene. The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain. The endoglucanase of A. aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8. The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum. The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme. Cel8Y was thermostable, exhibiting maximal activity at 80 degrees C and pH optima of 7.0 and with half-lives of 2 h at 100 degrees C, 4 h at 90 degrees C.     

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

Immobilization of the hyperthermophilic hydrogenase from Aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient H2 oxidation

The electrochemistry of membrane-bound [NiFe] hydrogenase I ([NiFe]-hase I) from the hyperthermophilic bacterium Aquifex aeolicus was investigated at gold and graphite electrodes. Direct and mediated H₂ oxidation were proved to be efficient in a temperature range of 25–70 °C, describing a potential window for H₂ oxidation similar to that of O₂-tolerant hydrogenases. Search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. We report high catalytic currents for H₂ oxidation up to 1 mA cm⁻², 10 times higher than at the bare electrode. Interestingly, high stability of the direct catalytic process was observed when encapsulating A. aeolicus [NiFe]-hase I into a carboxylic functionalized single walled carbon nanotube network. This suggests a peculiar interaction between the enzyme and the electrode material. The parameters that governed the orientation of the enzyme before electron transfer were thus investigated using self-assembled-monolayer gold electrodes. No control of the orientation by the charge or the hydrophobicity of the interface was demonstrated. This behavior was explained on the basis of a structural comparison between A. aeolicus [NiFe]-hase I and Desulfovibrio fructosovorans [NiFe] hydrogenase, which revealed the absence of acidic residues and an additional loop in the environment of the [4Fe–4S] distal cluster in A. aeolicus [NiFe]-hase I. Finally, the effect of inhibitors on the direct oxidation of H₂ by A. aeolicus [NiFe]-hase I encapsulated in a single walled carbon nanotube network was investigated. No inhibition by CO and tolerance toward O₂ were observed. Discussion of the reasons for such tolerance was undertaken on the basis of structural comparison with hydrogenases from aerobic bacteria.     

Protein AQ_1862 from the hyperthermophilic bacterium Aquifex aeolicus is a porin and contains two conductance pathways of different selectivity

The "hypothetical protein" AQ_1862 was isolated from the membrane fraction of Aquifex aeolicus and identified as the major porin. In experiments with one conducting unit (molecule) a conductance of 1.4 nS was observed in 0.1 M KCl at pH 7.5. This stable (basic) conductance was superimposed by conductance fluctuations of approximately 0.25 nS. Because both events were always observed simultaneously, it is suggested that they are caused by the same molecular entity. Nonetheless they show very different properties. The basic conductance is anion selective at neutral pH with a conductance sequence Cl- approximately Br- approximately NO3->F->gluconate approximately acetate approximately propionate and does not saturate up to 0.5 M KCl. At alkaline pH and in the presence of large anions, it becomes unselective and the conductance saturates at low concentrations (Km approximately 20 mM). In contrast the fluctuating component is mainly cation selective with a conductance sequence K+ approximately Rb+>NH4+>Na+ approximately Li+ approximately Cs+. It saturates at low salt concentrations (Km approximately 15 mM) and is not affected by pH. In view of the diverging properties of both conductance components, it seems appropriate to assume that AQ_1862 has two different conducting pathways rather than one with two different open states.     

Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus. 2. Heterologous production of soluble cytochrome c555s and investigation of the role of methionine residues.

The cycB2 gene encoding the soluble cytochrome c555s from Aquifex aeolicus, an hyperthermophilic organism, has been cloned and expressed using Escherichia coli as the host organism. The cytochrome was successfully produced in the periplasm of an E. coli strain coexpressing the ccmABCDEFGH genes involved in the cytochrome c maturation process. Comparison of native and recombinant cytochrome c555s shows that both proteins are indistinguishable in terms of spectroscopic and physicochemical properties. Since two different methionine residues are present in the sequence stretch usually providing the sixth ligand to the heme iron, site-directed mutagenesis has been performed in order to identify the methionine serving as the axial ligand. Two single mutations were introduced, leading to the replacement of each methionine by a histidine residue. Characterization of both mutants, M78H and M84H cytochromes c555s, using biochemical and biophysical techniques has been carried out. The M84H mutant exhibits spectral features identical to those of native cytochrome. Its redox midpoint potential is decreased by 40 mV. By contrast, substitution of methionine 78 by a histidine residue strongly alters the structural and physicochemical properties of the molecule which exhibits characteristics of His/His iron coordination type rather than His/Met. These results allow us to identify methionine 78 as the sixth ligand of cytochrome c555s heme iron. Preliminary results on the thermostability of the native and mutant cytochromes c555 are also reported.