Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag).

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli.

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, ß-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9.35 × 105 s-1 and kcat/Km = 1.1 × 108 M-1 s-1) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s-1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

The first activation study of a bacterial carbonic anhydrase (CA). The thermostable α-CA from Sulfurihydrogenibium yellowstonense YO3AOP1 is highly activated by amino acids and amines.

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its activation with a series of amino acids and amines. D-His, L-Phe, L-Tyr, L- and D-Trp were the most effective SspCA activators, with activation constants in the range of 1-12 nM, whereas L-His, L/D-DOPA, D-Tyr, and several biogenic amines/catecholamines were slightly less effective activators (K(A) in the range of 37 nM-0.97 µM). The least effective SspCA activator was d-Phe (K(A) of 5.13 µM). The thermal stability, robustness and very high catalytic activity of SspCA make this enzyme an ideal candidate for biomimetic CO(2) capture processes.

Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1.

A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO(2) and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.

Anion inhibition studies of an α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1.

The newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 encodes an α-carbonic anhydrases (CAs, EC 4.2.1.1) which is highly catalytically active and thermostable. Here we report the inhibition of this enzyme, denominated SspCA, with inorganic and complex anions and other molecules interacting with zinc proteins. SspCA was inhibited in the micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic and phenylarsonic acid, trithiocarbonate and selenocyanide (K(I)s of 4-70 µM) and in the submillimolar one by iodide, cyanide, (thio)cyanate, hydrogen sulfide, azide, nitrate, nitrite, many complex anions incorporating heavy metal ions and iminodisulfonate (K(I)s of 0.48-0.86 mM). SspCA was not substantially inhibited by bicarbonate and carbonate, hydrogensulfite and peroxidisulfate (K(I)s in the range of 21.1-84.6mM). The exceptional thermostability and lack of strong affinity for hydrogensulfide, bicarbonate, and carbonate make this enzyme an interesting candidate for biotechnological applications of enzymatic CO(2) fixation.

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus.

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.

Protein film voltammetry of arsenite oxidase from the chemolithoautotrophic arsenite-oxidizing bacterium NT-26.

The chemolithoautotrophic bacterium NT-26 (isolated from a gold mine in the Northern Territory of Australia) is unusual in that it acquires energy by oxidizing arsenite to arsenate while most other arsenic-oxidizing organisms perform this reaction as part of a detoxification mechanism against the potentially harmful arsenite [present as As(OH)(3) at neutral pH]. The enzyme that performs this reaction in NT-26 is the molybdoenzyme arsenite oxidase, and it has been previously isolated and characterized. Here we report the direct (unmediated) electrochemistry of NT-26 arsenite oxidase confined to the surface of a pyrolytic graphite working electrode. We have been able to demonstrate that the enzyme functions natively while adsorbed on the electrode where it displays stable and reproducible catalytic electrochemistry in the presence of arsenite. We report a pH dependence of the catalytic electrochemical potential of -33 mV/pH unit that is indicative of proton-coupled electron transfer. We also have performed catalytic voltammetry at a number of temperatures between 5 and 25 degrees C, and the catalytic current (proportional to the turnover number) follows simple Arrhenius behavior.

Expression, purification, and characterization of a new heterotetramer structure of leucyl-tRNA synthetase from Aquifex aeolicus in Escherichia coli.

Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26.

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.

[The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1]. / Fermenty metabolizma ugleroda u termotolerantnoi sul'fobatsilly, shtamm K1.

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.

[Effects of exogenous factors on the activity of enzymes involved in carbon metabolism in thermoacidophilic bacteria of the genus Sulfobacillus]. / Vliianie ékzogennykh faktorov na aktivnost' fermentov metabolizma ugleroda u termoatsidofil'nykh bakterii roda Sulfobacillus.

Aerobic thermoacidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans 1269T and Sulfobacillus thermosulfidooxidans subsp. asporogenes 41 were shown to be resistant to stress factors, including high concentrations of Zn2+ (0.8 M) and H+ (pH 1.2) that exceeded the optimum values. The growth and biomass gain rates decreased, but bacteria retained their functions. The activity of nearly all enzymes involved in carbon metabolism decreased. Glucose was primarily metabolized via the Entner--Doudoroff pathway. The activity tricarboxylic acid cycle enzymes decreased compared to that in cells grown under normal conditions. After saturation of the growth medium with 5 vol % CO2, sulfobacteria utilized glucose by the Embden-Meyerhof and pentose phosphate pathways under mixotrophic conditions.

Multiple copies of ammonia monooxygenase (amo) operons have evolved under biased AT/GC mutational pressure in ammonia-oxidizing autotrophic bacteria.

The recent availability of complete sequences of ammonia monooxygenase (16 amoA, 5 amoB and 5 amoC gene sequences) and particulate methane monooxygenase (2 pmoA, pmoB and pmoC gene sequences each) genes allowed for a detailed analysis of their relatedness. Nucleotide sequence analysis was performed in order to identify the origins of the nearly identical operon copies within a given nitrosofier/methanotroph strain. Our data suggest that amo-homologous gene evolution has occurred in individual strains (orthology) under biased AT/GC pressure rather than by horizontal transfer. The multiple operon copies within individual strains are the result of operon duplication (paralogy). While the near identity of the multiple operon copies makes it impossible to determine whether paralogous gene expansion occurred in the last common ancestor of ammonia oxidizers or after speciation took place, we conclude that the duplication events were not recent events. We propose that the elimination of third basepair degeneracy between copies within one organism is implemented by a rectification mechanism resulting in concerted evolution.

Something from almost nothing: carbon dioxide fixation in chemoautotrophs.

The last decade has seen significant advances in our understanding of the physiology, ecology, and molecular biology of chemoautotrophic bacteria. Many ecosystems are dependent on CO2 fixation by either free-living or symbiotic chemoautotrophs. CO2 fixation in the chemoautotroph occurs via the Calvin-Benson-Bassham cycle. The cycle is characterized by three unique enzymatic activities: ribulose bisphosphate carboxylase/oxygenase, phosphoribulokinase, and sedoheptulose bisphosphatase. Ribulose bisphosphate carboxylase/oxygenase is commonly found in the cytoplasm, but a number of bacteria package much of the enzyme into polyhedral organelles, the carboxysomes. The carboxysome genes are located adjacent to cbb genes, which are often, but not always, clustered in large operons. The availability of carbon and reduced substrates control the expression of cbb genes in concert with the LysR-type transcriptional regulator, CbbR. Additional regulatory proteins may also be involved. All of these, as well as related topics, are discussed in detail in this review.

Pulse radiolysis studies on cytochrome cd1 nitrite reductase from Thiosphaera pantotropha: evidence for a fast intramolecular electron transfer from c-heme to d1-heme.

Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.

Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds.

The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem. The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies. A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes. The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller. The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins. From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure. The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres.

Two enzymes with a common function but different heme ligands in the forms as isolated. Optical and magnetic properties of the heme groups in the oxidized forms of nitrite reductase, cytochrome cd1, from Pseudomonas stutzeri and Thiosphaera pantotropha.

It is shown that, in the oxidized state, heme c of Pseudomonas stutzeri (ZoBell strain) cytochrome cd1 has histidine-methionine ligation as observed for cytochrome cd1 from Pseudomonas aeruginosa [Sutherland, J., Greenwood, C., Peterson, J., and Thomson, A. J. (1986) Biochem. J. 233, 893-898]. However, the X-ray structure of Thiosphaera pantotropha cytochrome cd1 reveals bis-histidine ligation for heme c. It is confirmed by EPR and near-infrared (NIR) MCD measurements that the bis-histidine coordination remains unaltered in the solution phase. Hence, the difference between the heme c ligation states defines two distinct classes of oxidized cytochromes cd1 as isolated. A weak feature in the T. pantotropha NIR MCD at 1900 nm suggests that a small population of heme c has histidine-methionine coordination. The ligation state of heme d1 cannot be defined with the same level of confidence, because the porphyrin-to-Fe(III) charge-transfer (CT) bands are less well characterized for this class of partially reduced porphyrin ring. However, variable temperature absorption and MCD spectra show that, in the T. pantotropha enzyme, heme d1 exists in a thermal low-spin/high-spin mixture with the low-spin as the ground state, whereas in P. stutzeri cytochrome cd1, and d1 heme is low-spin at all temperatures. A weak band, assigned as the heme d1 porphyrin-pi(a1u,a2u)-to-ferric(d) charge-transfer transition has been identified for the first time at 2170 nm. Its magnetic properties show the heme d1 to have an unusual (dxz,yz)4(dxy)1 electronic ground state as is found for low-spin Fe(III) chlorins [Cheesman, M. R., and Walker, F. A. (1996) J. Am. Chem. Soc. 118, 7373-7380]. It is proposed that the localization of the Fe(III) unpaired d-electron in an orbital lying in the heme plane may decrease the affinity of the Fe(III) heme for unsaturated ligands such as NO. Although heme d1 in the enzymes from P. stutzeri and T. pantotropha shows different temperature-dependent spin properties, the positions of the low-spin Fe(III) alpha-absorption band, at approximately 640 nm, are very similar to those observed for cytochromes cd1 from eight other sources, suggesting that all have similar strength fields from the axial ligands and, hence, that all have the same coordination, namely histidine-tyrosine or possibly histidine-hydroxide at the heme.

Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy.

The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two ==O, three -S- and either a fourth -S- or an -O-/-N- as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the -S- ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI)-->Mo(V) reduction is accompanied by conversion of one ==O to -O-. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one ==O, one -O- and four -S-/-Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.

Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not. Implications for c-type cytochrome biogenesis.

Expression from a plasmid of the complete gene, including the codons for the N-terminal periplasmic targeting signal, for cytochrome c550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.

Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification.

Thiosphaera pantotropha, a Gram-negative heterotrophic nitrifying bacterium, expresses a soluble 20 kDa monomeric periplasmic hydroxylamine oxidase that differs markedly from the hydroxylamine oxidase found in autotrophic bacteria. This enzyme can use the periplasmic redox proteins, cytochrome c551 and pseudoazurin as electron acceptors, both of which can also donate electrons to denitrification enzymes. A model of electron transfer is proposed, that suggests a coupling of nitrification and provides a mechanism by which nitrification can play a role in dissipating reductant.