Sulfide Oxidation and Transfer Catalyzed by a Bacterial Persulfide Dioxygenase Fused to a Rhodanese

Abstract

Hydrogen sulfide (H2S) is the third eukaryotic gaseous signaling molecule that evokes a broad range of physiological effects. H2S levels are governed by the rates of both production and clearance. In eukaryotes, sulfide clearance occurs primarily via the mitochondrial sulfide oxidation pathway which consists of the enzymes sulfide: quinone oxidoreductase (SQR), persulfide dioxygenase (PDO also known as ETHE1), rhodanese and sulfite oxidase. Recently, bacterial proteins that are fusions between PDO and rhodanese have been identified. Existence of such a fusion suggests that their mitochondrial counterparts in higher organisms may interact and that characterization of such fusion proteins will facilitate the modeling of interactions between human mitochondrial PDO and rhodanese and aid in elucidating the metabolic intermediates in the sulfide oxidation pathway. Here we have characterized a bacterial PDO‐rhodanese fusion (PRF) protein from Burkholderia phytofirmans which displays 1.8‐fold higher PDO activity and 4.8‐fold lower Km for GSSH as 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. Similar observation were made for the stand‐alone rhodanese domain; however, several fold increases in specific activity and Km values for thiosulfate and glutathione were observed for the various sulfur transfer reactions, suggesting that the PDO domain modulates the activity of the rhodanese domain. Combined, these results suggest that the fusion is poised to produce sulfite as its main product. Bioinformatic analysis suggests that this enzyme may be involved in sulfur assimilation pathways rather than in sulfide oxidation as in mammals. Spectral changes were observed with the ferric form of the enzyme in the presence of Na2S. Further analysis of this interaction displayed that Na2S binds with moderate affinity resulting in polysulfide production and suggesting a potentially novel role for non‐heme iron proteins in polysulfide production. To obtain structural insights into its mechanism, the x‐ray crystal structure of PRF co‐crystallized with thiosulfate was determined at 1.79 Å resolution. Notably, in this structure the persulfidated form of the rhodanese domain active site cysteine, C314, was captured.Support or Funding InformationNational Institutes of Health (GM112455 to RB) and National Institute of General Medical Sciences Training Grant support (T32GM008353 to NM)