Comparison of the kinetic properties of five recombinant SDO proteins.

<p>The values with different superscripts in the same column are significantly different (p<0.05).</p

Characteristics of the <i>U. unicinctus</i> SDO mutant.

<p>A. SDO specific activities in wild type and mutant. * indicates a significant difference from the wild type (p<0.05). B. GSH affinities in wild type and mutant. C. Oxygen consumption rate versus GSS⁻ concentration in wild type and mutant.</p

Characteristics of the five sub-segment-expressed recombinant SDO proteins.

<p>A. SDO specific activities (mean±SE, n = 3) of five recombinant SDO proteins. B. GSH affinities (mean±SE, n = 3) of the recombinant SDO proteins. Groups containing the same letters on the bar indicate no significant difference while different letters on the bar indicate a significant difference (p<0.05).</p

Multiple sequence alignment (A) and phylogenetic relationships (B) among the SDO sequences from different species.

<p>Identical and similar residues are highlighted in black and gray, respectively. Conserved relevant metal binding sites, GSH binding sites and dimer formation residues are indicated by ▪, ▾ and *, respectively. The β-lactamase signature motif and rhodanese active-site loop are marked with a black box and grey box, respectively. GenBank accession numbers: <i>Afipia broomeae</i> (ZP_11429388.1), <i>Acidithiobacillus caldus</i> (YP_004749948.1), <i>Acidovorax delafieldii</i> (ZP_04761469.1), <i>Apis mellifera</i> (XP_393510.1), <i>Caenorhabditis elegans</i> (NP_501684.1), <i>Capitella teleta</i> (JGI Genome), <i>Ciona intestinalis</i> (XP_002128021.1), <i>Crassostrea gigas</i> (EKC28487.1), <i>Homo sapiens</i> (NP_055112.2), <i>Methylobacter tundripaludum</i> (ZP_08782165.1), <i>Microcystis aeruginosa</i> (ZP_18834377.1), <i>Mus musculus</i> (NP_075643.1), <i>Nitrosococcus watsonii</i> (YP_003760989.1), <i>γ-proteobacterium HTCC2148</i> (ZP_05095460), <i>Salmo salar</i> (ACI68458.1), <i>Takifugu rubripes</i> (XP_003977175.1), <i>Urechis unicinctus</i> (AEV92813.1), <i>Xenopus laevis</i> (NP_001079404.1).</p

The two proposed sulfide oxidation models.

<p>Abbreviations: SQR, sulfide: quinine oxidoreductase; ST, sulfur transferase; SDO, sulfur dioxygenase; GS⁻, glutathione; GSS⁻, glutathione persulfide. Model A: The sulfide can be converted to GSS⁻ by SQR, and the sulfane sulfur of GSS⁻ is oxidized to sulfite using O₂ by SDO in the mitochondrial-matrix; finally, the generated sulfite is converted by ST catalysis to thiosulfate, which is less toxic to the organism. Model B: Thiosulfate biosynthesis occurs in the first step of sulfide oxidation catalyzed by SQR with sulfite as the acceptor of the sulfane sulfur, then thiosulfate can act as the ST substrate to produce GSS⁻ and regenerate the sulfite; finally, the sulfane sulfur of GSS⁻ is oxidized to sulfite using O₂ by SDO. The newly generated sulfite could then enter the cycle again.</p

Mitochondrial location of SDO in <i>U. unicinctus.</i>

<p>A. Midgut total protein; B. Midgut mitochondrial protein.</p

Schematic diagram of SDO sub-segment expression.

<p>Del-1: no structural amino acid site; Del-2: two metal I binding site residues, two metal II binding site residues and no GSH binding site; Del-3: complete metal I binding site, three metal II binding site residues and one GSH binding site; Del-4: complete metal I binding site and metal II binding site as well as three GSH binding sites; complete ORF: complete metal I binding sites and metal II binding sites as well as GSH binding sites. The numbers indicated the expressed sub-segment amino acid length.</p

Primers for the five cDNA sequences of <i>U. unicinctus SDO.</i>

<p>Primers for the five cDNA sequences of <i>U. unicinctus SDO.</i></p

Purified recombinant SDO proteins detected by SDS-PAGE.

<p>Purified recombinant SDO proteins detected by SDS-PAGE.</p

Nucleotide sequence and predicted amino acid sequence of <i>SDO</i> in <i>U. unicinctus.</i>

<p>Start (ATG) and stop (TGA) codons, double lines; RACE gene specific primers (GSP), indicated by an arrow; the AATAAA polyadenylation signal is underlined; Conserved metal binding sites are boxed in black; Conserved GSH binding sites are double boxed.</p