Cofactor-dependent maturation of mammalian sulfite oxidase links two mitochondrial import pathways.

Sulfite oxidase (SO) catalyses the metabolic detoxification of sulfite to sulfate within the intermembrane space of mitochondria. The enzyme follows a complex maturation pathway, including mitochondrial transport and processing, integration of two prosthetic groups, molybdenum cofactor (Moco) and heme, as well as homodimerisation. We have identified the sequential and cofactor-dependent maturation steps of SO. The N-terminal bipartite targeting signal of SO was required but not sufficient for mitochondrial localization. In the absence of Moco, most of the SO, although processed by the inner membrane peptidase of mitochondria, was found in the cytosol. Moco binding was required to induce mitochondrial trapping and retention, thus ensuring unidirectional translocation of SO. In the absence of the N-terminal targeting sequence, SO assembled in the cytosol, suggesting an important function for the leader sequence in preventing premature cofactor binding. In vivo, heme binding and dimerisation did not occur in the absence of Moco and only occurred after Moco integration. In conclusion, the identified molecular hierarchy of SO maturation represents a novel link between the canonical presequence pathway and folding-trap mechanisms of mitochondrial import.

Purification and characterization of sulfite oxidase from goat liver.

Sulfite oxidase (EC 1.8.3.1) catalyzes the physiologically vital oxidation of sulfite to sulfate, the terminal reaction in the degradation of sulfur containing amino acids. Genetic deficiency related to human sulfite oxidase is associated with the severe clinical abnormalities with no effective therapies known, making the enzyme of immense biomedical importance. In the present study, sulfite oxidase was been purified from the goat tissues, a hitherto unexplored source, in particular from the liver, and its physico and biochemical properties were characterized. The liver was chosen as it showed the highest activity, compared to kidney and muscle. The enzyme was purified to homogeneity by salting out, gel filtration and ion-exchange chromatography. It was a dimer (113 kDa) having two identical subunits (56 kDa) and did not contain free sulfhydryl groups. Its spectral analysis showed the presence of heme and molybdenum. circular dichroism (CD) spectra in near and far-UV regions showed the presence of significant amounts of secondary structures (45% alpha helix, 9% beta structure and 26% beta turn and remaining random coil) in the native molecule. The kinetic and hydrodynamic properties of the enzyme were also determined. Results also showed that ferricyanide was 8-times more effective electron acceptor than its physiological acceptor cytochrome c. The limited N-terminal analysis of the enzyme revealed the sequence up to six amino acids Trp-Glu-Pro-Ser-Gly-Ala. Together, these results suggested the liver was a major source of sulfite oxidase in goat and most of its physico-chemical, except secondary structure and amino acid sequence from N-terminal and biological properties were fairly similar to the sulfite oxidase isolated from other mammalian species/organs.

Fed-batch cultivation of the marine bacterium Sulfitobacter pontiacus using immobilized substrate and purification of sulfite oxidase by application of membrane adsorber technology.

Sulfitobacter pontiacus, a gram-negative heterotrophic bacterium isolated from the Black Sea is well known to produce a soluble AMP-independent sulfite oxidase (sulfite: acceptor oxidoreductase) of high activity. Such an enzyme can be of great help in establishing biosensor systems for detection of sulfite in food and beverages considering the high sensitivity of biosensors and the increasing demand for such biosensor devices. For obtaining efficient amounts of the enzyme, an induction of its biosynthesis by supplementing sufficient concentrations of sodium sulfite to the fermentation broth is required. Owing to the fact that a high initial concentration of sodium sulfite decreases dramatically the enzyme expression, different fed-batch strategies can be applied to circumvent such inhibition or repression of the enzyme respectively. By the use of sulfite species immobilized in polyvinyl alcohol gels, an approach to the controlled and continuous feeding of sulfite to the cultivation media could be established to diminish inhibitory concentrations. Furthermore, the purification of the enzyme is described by using membrane adsorber technology.

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli.

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans.

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).