Structural Enzymology of Sulfide Oxidation by Persulfide Dioxygenase and Rhodanese

[摘要] Hydrogen sulfide (H2S) is the third eukaryotic gaseous signaling molecule and evokes a broad range of physiological effects. H2S levels are governed by the rates of its production and clearance. In eukaryotes, sulfide clearance occurs primarily via the mitochondrial sulfide oxidation pathway which consists of the enzymes sulfide: quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese and sulfite oxidase. Mutation of PDO results in ethylmalonic encephalopathy (EE), an autosomal recessive disorder. There are over 20 mutations described in patients with EE. Patients with EE and PDO knockout mice exhibit increased excretion of thiosulfate bringing into question the organization of the sulfur oxidation pathway. Kinetic characterization of several EE patient mutations reported in human PDO: L55P, T136A, C161Y and R163W, was performed to determine the biochemical penalties incurred by these mutations. The mutants displayed 18-, 42-, 65- and 200-fold lower kcat/Km values for R163W, L55P, C161Y and T136A respectively. Similarly, the mutants displayed 3-, 10.5-, 11- and 1.4-fold lower iron content with 1.2-, 1.4-, 1.7- and 1.4-fold lower Tm values, respectively. Recently, bacterial proteins that are fusions between PDO and rhodanese have been identified. Characterization of such fusion proteins will facilitate modeling of interactions between human mitochondrial PDO and rhodanese and aid in elucidating the metabolic intermediates in the sulfide oxidation pathway. We have characterized a bacterial PDO/rhodanese fusion protein from Burkholderia phytofirmans which displays 1.8-fold higher PDO activity and 4.8-fold lower Km for GSSH compared to human PDO, and rhodanese activity comparable to other bacterial rhodaneses. Additionally, the rhodanese domain catalyzed the formation of GSSH and sulfite from thiosulfate and glutathione, but no detectable sulfur transfer activity was observed for the reverse reaction. Combined, these results suggest that the fusion is poised to produce sulfite as its main product. To obtain structural insights into its mechanism, crystal structures of wild-type and the sulfurtransferase inactivating C314S mutation with and without GSH bound were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. Bioinformatic analysis displays that this enzyme may be involved in sulfur assimilation pathways rather than an orthologous sulfide oxidation pathway.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Hydrogen Sulfide Metabolism;Biological Chemistry;Science;Biological Chemistry