Structural Insights into Sulfite Oxidase Deficiency

Abstract

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.Sulfite oxidase (SO), 3 an enzyme containing the molybdenum cofactor (Moco), catalyzes the oxidation of sulfite to sulfate, the final step in the degradation of sulfur-containing amino acids. It resides in the intermembrane space of mitochondria, where it exists as a homodimer. The crystal structure of chicken sulfite oxidase (CLSO), purified from chicken liver, showed that each subunit contains three domains. A small heme-containing N-terminal cytochrome b 5 domain (residues 3-84) is connected to the rest of the protein via a flexible 10-residue-long loop region. The central domain (residues 92-323) contains the active site of the enzyme. Finally, the C-terminal dimerization domain (residues 324 -466) displays the same topology as the C2 subtype of the immunoglobulin superfamily (1).Upon oxidation of sulfite, Mo(VI) is reduced to Mo(IV) by two electrons. The electrons are subsequently transferred to the heme Fe(III) in the cytochrome b 5 domain in a two-step reaction, which is followed by transfer of the electrons from Fe(II) to cytochrome c (2). The distance between the two metals, molybdenum and iron, in the crystal structure of chicken liver sulfite oxidase (CLSO) is 32 Å, which is much longer than expected for the electron transfer rate observed (1). Two mechanisms were suggested to explain these results; a very efficient electron transfer through main ...

Sulfite-oxidizing enzymes

“…The assignment of residue 6 in the heme domain to glutamine or glutamic acid in the first crystal structure of native chicken SO was incorrect, and has been re-assigned to arginine in the recently reported structure of recombinant chicken SO, based upon sequencing analysis of native chicken SO [24]. The authors could not grow crystals of the recombinant chicken SO encoding the incorrect residue.…”

“…Significant alterations in the substrate-binding pocket were detected in the structure of the chicken SO R138Q mutant, and a comparison between the wild type and mutant proteins revealed that the active site Arg-450 residue adopts different conformations in the presence and absence of bound sulfate [24]. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the Mo.…”

“…Very recently, structures of resting and sulfate-bound recombinant chicken SO proteins (wild type and R138Q) were reported [24]. The assignment of residue 6 in the heme domain to glutamine or glutamic acid in the first crystal structure of native chicken SO was incorrect, and has been re-assigned to arginine in the recently reported structure of recombinant chicken SO, based upon sequencing analysis of native chicken SO [24].…”

“…Very recently, the structures of the recombinant chicken SO R138Q mutant [24] and the S. novella SDH Y236F mutant [41] have provided new structural insights into the impact of point mutations on sulfite-oxidizing activities. Significant alterations in the substrate-binding pocket were detected in the structure of the chicken SO R138Q mutant, and a comparison between the wild type and mutant proteins revealed that the active site Arg-450 residue adopts different conformations in the presence and absence of bound sulfate [24].…”

Synthesis, characterization, and spectroscopy of model molybdopterin complexes

“…From a human perspective, three molybdoenzymes-sulfite oxidase, xanthine oxidase and aldehyde oxidase-are essential for proper health. In the extreme cases where genetic errors prevent the biosynthesis of functional molybdoenzymes, the resultant neurological problems are typically fatal [9][10][11][12].…”