Metagenomic insights into mixotrophic denitrification facilitated nitrogen removal in a full-scale A2/O wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus, an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.

Isotopic Fractionation of Sulfur in Carbonyl Sulfide by Carbonyl Sulfide Hydrolase of Thiobacillus thioparus THI115.

Carbonyl sulfide (COS) is one of the major sources of stratospheric sulfate aerosols, which affect the global radiation balance and ozone depletion. COS-degrading microorganisms are ubiquitous in soil and important for the global flux of COS. We examined the sulfur isotopic fractionation during the enzymatic degradation of COS by carbonyl sulfide hydrolase (COSase) from Thiobacillus thioparus THI115. The isotopic fractionation constant (34ɛ value) was -2.2±0.2‰. Under experimental conditions performed at parts per million by volume level of COS, the 34ɛ value for intact cells of T. thioparus THI115 was -3.6±0.7‰, suggesting that, based on Rees' model, the 34ɛ value mainly depended on COS transport into the cytoplasm. The 34ɛ value for intact cells of T. thioparus THI115 was similar to those for Mycobacterium spp. and Williamsia sp., which are known to involve the conserved region of nucleotide sequences encoding the clade D of ß-class carbonic anhydrase (ß-CA) including COSase. On the other hand, the 34ɛ value was distinct from those for bacteria in the genus Cupriavidus. These results provide an insight into biological COS degradation, which is indispensable for estimating the COS global budget based on the isotope because of the significant contribution of COS degradation by microorganisms harboring ß-CA family enzymes.

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.

Thiosulfate oxidation by Thiomicrospira thermophila: metabolic flexibility in response to ambient geochemistry.

Previous studies of the stoichiometry of thiosulfate oxidation by colorless sulfur bacteria have failed to demonstrate mass balance of sulfur, indicating that unidentified oxidized products must be present. Here the reaction stoichiometry and kinetics under variable pH conditions during the growth of Thiomicrospira thermophila strain EPR85, isolated from diffuse hydrothermal fluids at the East Pacific Rise, is presented. At pH 8.0, thiosulfate was stoichiometrically converted to sulfate. At lower pH, the products of thiosulfate oxidation were extracellular elemental sulfur and sulfate. We were able to replicate previous experiments and identify the missing sulfur as tetrathionate, consistent with previous reports of the activity of thiosulfate dehydrogenase. Tetrathionate was formed under slightly acidic conditions. Genomic DNA from T. thermophila strain EPR85 contains genes homologous to those in the Sox pathway (soxAXYZBCDL), as well as rhodanese and thiosulfate dehydrogenase. No other sulfur oxidizing bacteria containing sox(CD)2 genes have been reported to produce extracellular elemental sulfur. If the apparent modified Sox pathway we observed in T. thermophila is present in marine Thiobacillus and Thiomicrospira species, production of extracellular elemental sulfur may be biogeochemically important in marine sulfur cycling.

Structural insights into the efficient CO2-reducing activity of an NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA.

CO2 fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase from Candida boidinii (CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations, i.e. open (NAD(+)-unbound) and closed (NAD(+)-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2 reduction than in CbFDH.

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.

Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.

Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase.

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt (Co)-containing enzyme that catalyzes the hydrolysis of thiocyanate (SCN⁻), a major component of wastewater from coke oven factories, to carbonyl sulfide and ammonia. Although SCNase exhibits high structural similarities to Co-type nitrile hydratase (NHase), including a unique Co³âº catalytic center with two oxidized Cys ligands, both SCNase and NHase exclusively catalyze only their own substrates. Based on the differences in the substrate-binding pockets of these enzymes, ßArg90 and γArg136 of SCNase, with side chains extending toward the pocket, were separately substituted with Phe and Trp, the corresponding residues, respectively, in Co-type NHase. Both SCNase ßArg90 and SCNase γArg136 mutants showed no SCN⁻ hydrolysis activity but did catalyze the hydration of nitriles. The estimated kcat values (∼2 s⁻¹) corresponded to approximately 0.2% of that of Co-type NHase for nitrile hydration and approximately 3% of that of wild-type SCNase for SCN⁻ hydrolysis. The crystal structure of SCNase γR136W is essentially identical to that of the wild-type, including the Co³âº center having Cys oxidations; the size of the substrate pocket was enlarged because of conformational changes on the side chains of the mutated residue. Discussion of the difference in the environments around the substrate-binding pockets among the wild-type and mutant SCNases and Co-type NHase strongly suggests that ßArg90 and γArg136, positioned at the top of the Co³âº center, predominantly control the substrate selectivity of SCNase.

Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the ß-carbonic anhydrase family enzymes.

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the ß-class carbonic anhydrases (ß-CAs). Although the crystal structure including the catalytic site resembles those of the ß-CAs, CO2 hydration activity of COSase is negligible compared to those of the ß-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the ß-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of ß-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS.

Thiosulphate oxidation by Thiobacillus thioparus and Halothiobacillus neapolitanus strains isolated from the petrochemical industry

Sulphur Oxidizing Bacteria (SOB) is a group of microorganisms widely used for the biofiltration of Total Reduced Sulphur compounds (TRS). TRS are bad smelling compounds with neurotoxic activity which are produced by different industries (cellulose, petrochemical). Thiobacillus thioparus has the capability to oxidize organic TRS, and strains of this bacterium are commonly used for TRS biofiltration technology. In this study, two thiosulphate oxidizing strains were isolated from a petrochemical plant (ENAP BioBio, Chile). They were subjected to molecular analysis by real time PCR using specific primers for T. thioparus. rDNA16S were sequenced using universal primers and their corresponding thiosulphate activities were compared with the reference strain T. thioparus ATCC 10801 in batch standard conditions. Real time PCR and 16S rDNA sequencing showed that one of the isolated strains belonged to the Thiobacillus branch. This strain degrades thiosulphate with a similar activity profile to that shown by the ATCC 10801 strain, but with less growth, making it useful in biofiltration.

Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity.

The Tbd_0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 A resolution X-ray crystal structure reported here revealed a hexameric assembly with D(3) symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

Preliminary X-ray crystallographic analysis of sulfide:quinone oxidoreductase from Acidithiobacillus ferrooxidans.

The gene product of open reading frame AFE_1293 from Acidithiobacillus ferrooxidans ATCC 23270 is annotated as encoding a sulfide:quinone oxidoreductase, an enzyme that catalyses electron transfer from sulfide to quinone. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. The native crystals belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 131.7, c = 208.8 A, and diffracted to 2.3 A resolution. Preliminary crystallographic analysis indicated the presence of a dimer in the asymmetric unit, with an extreme value of the Matthews coefficient (V(M)) of 4.53 A(3) Da(-1) and a solvent content of 72.9%.

Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans.

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had <2% APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

Ferrous iron production mediated by tetrathionate hydrolase in tetrathionate-, sulfur-, and iron-grown Acidithiobacillus ferrooxidans ATCC 23270 cells.

When tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells were incubated with ferric ions and tetrathionate at pH 3.0, ferrous ions were produced enzymatically. Fe(3+)-reductase, which catalyzes Fe(3+) reduction with tetrathionate, was purified to homogeneity not only from tetrathionate-grown, but also from sulfur- and iron-grown A. ferrooxidans ATCC 23270 cells. The results for apparent molecular weight measured by SDS-PAGE (52.3 kD) and the N-terminal amino acid sequences of the purified enzymes from iron-, sulfur, and tetrathionate-grown cells (AVAVPMDSTG) indicate that Fe(3+)-reductase corresponds to tetrathionate hydrolase. The evidence that tetrathionate-grown A. ferrooxidans ATCC 23270 cells have high iron-oxidizing activity at the early log phase, comparable to that of iron-grown ATCC 23270 cells, is supported by our finding that tetrathionate hydrolase produces Fe(2+) from tetrathionate during growth on tetrathionate. This is the first report on ferric reductase activity associated with tetrathionate hydrolase.

Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

Molecular analysis of the distribution and phylogeny of the soxB gene among sulfur-oxidizing bacteria - evolution of the Sox sulfur oxidation enzyme system.

The soxB gene encodes the SoxB component of the periplasmic thiosulfate-oxidizing Sox enzyme complex, which has been proposed to be widespread among the various phylogenetic groups of sulfur-oxidizing bacteria (SOB) that convert thiosulfate to sulfate with and without the formation of sulfur globules as intermediate. Indeed, the comprehensive genetic and genomic analyses presented in the present study identified the soxB gene in 121 phylogenetically and physiologically divergent SOB, including several species for which thiosulfate utilization has not been reported yet. In first support of the previously postulated general involvement of components of the Sox enzyme complex in the thiosulfate oxidation process of sulfur-storing SOB, the soxB gene was detected in all investigated photo- and chemotrophic species that form sulfur globules during thiosulfate oxidation (Chromatiaceae, Chlorobiaceae, Ectothiorhodospiraceae, Thiothrix, Beggiatoa, Thiobacillus, invertebrate symbionts and free-living relatives). The SoxB phylogeny reflected the major 16S rRNA gene-based phylogenetic lineages of the investigated SOB, although topological discrepancies indicated several events of lateral soxB gene transfer among the SOB, e.g. its independent acquisition by the anaerobic anoxygenic phototrophic lineages from different chemotrophic donor lineages. A putative scenario for the proteobacterial origin and evolution of the Sox enzyme system in SOB is presented considering the phylogenetic, genomic (sox gene cluster composition) and geochemical data.

Molecular analysis of the distribution and phylogeny of dissimilatory adenosine-5'-phosphosulfate reductase-encoding genes (aprBA) among sulfur-oxidizing prokaryotes.

Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center.

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.

Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes in the MgCl2-dominated deep hypersaline anoxic basin discovery.

Partial sequences of the form I (cbbL) and form II (cbbM) of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit genes were obtained from the brine and interface of the MgCl2-dominated deep hypersaline anoxic basin Discovery. CbbL and cbbM genes were found in both brine and interface of the Discovery Basin but were absent in the overlying seawater. The diversity of both genes in the brine and interface was low, which might caused by the extreme saline conditions in Discovery of approximately 5 M MgCl2. None of the retrieved sequences were closely related to sequences deposited in the GenBank database. A phylogenetic analysis demonstrated that the cbbL sequences were affiliated with a Thiobacillus sp. or with one of the RuBisCO genes from Hydrogenovibrio marinus. The cbbM sequences clustered with thiobacilli or formed a new group with no close relatives. The results implicate that bacteria with the potential for carbon dioxide fixation and chemoautotrophy are present in the Discovery Basin. This is the first report demonstrating that RuBisCO genes are present under hypersaline conditions of 5 M MgCl2.

Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand.

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.

Intramolecular electron transfer in a bacterial sulfite dehydrogenase.

Sulfite dehydrogenase (SDH) from Starkeya novella, a sulfite-oxidizing molybdenum-containing enzyme, has a novel tightly bound alphabeta-heterodimeric structure in which the Mo cofactor and the c-type heme are located on different subunits. Flash photolysis studies of intramolecular electron transfer (IET) in SDH show that the process is first-order, independent of solution viscosity, and not inhibited by sulfate, which strongly indicates that IET in SDH proceeds directly through the protein medium and does not involve substantial movement of the two subunits relative to each other. The IET results for SDH contrast with those for chicken and human sulfite oxidase (SO) in which the molybdenum domain is linked to a b-type heme domain through a flexible loop, and IET shows a remarkable dependence on sulfate concentration and viscosity that has been ascribed to interdomain docking. The results for SDH provide additional support for the interdomain docking hypothesis in animal SO and clearly demonstrate that dependence of IET on viscosity and sulfate is not an inherent property of all sulfite-oxidizing molybdenum enzymes.