3hbq

Publication Abstract from PubMed

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyrano-pterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in Chicken SO, coordinates the Mo atom in the active site. To better understand the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was solved to 1.9 A resolution, to 2.4 A resolution in the presence of sulfite, and the C185A variant to 2.8 A resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier EXAFS work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue re-localized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand, however Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized MoVI state in both the C185S and C185A variants of chicken SO and show the expected trioxo dithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the Mo=O distances of the complex, however the calculated MoS distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active sites of the C185S and C185A variants are essentially catalytically inactive, the crystal structures of C185S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a conformational change which is of relevance to the reaction mechanism. Additional DFT calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site.

The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site.,Qiu JA, Wilson HL, Pushie MJ, Kisker CF, George GN, Rajagopalan KV Biochemistry. 2010 Mar 31. PMID:20356030^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.