The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

Abstract

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.

Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases

“…Arg206 acts as a “fork” holding the pathway in place or possibly directly facilitating an ET pathway. Interestingly, arginine residues are often involved in controlling biomolecular ET pathways. − The overall hydroxylation mechanism proposed here has some similarities with 1- H -3-hydroxy-4-oxoquinaldine 2,4-dioxygenase that oxygenates substrates without the need for a cofactor and those proposed for bacterial luciferase involving formation of a radical pair and subsequent ET(s) …”

Bioelectrocatalysis of Sulfite Dehydrogenase from Sinorhizobium meliloti with Its Physiological Cytochrome Electron Partner

“…The combined loss of positive charge and H‐bonding capacity of the residue at position 78 lead to decreased activity of the Mo active site for sulfite oxidation. A parallel study of the effects of these mutations on the solution steady state kinetics of SorT reveals similar trends …”

“…Recombinant wild type Sinorhizobium meliloti sulfite dehydrogenase (SorT WT ) and its variants (SorT R78Q , SorT R78K and SorT R78M ) as well as the c ‐type cytochrome SorU from were purified following expression in E. coli as previously described ,,. Sodium sulfite, chitosan (from shrimp shells, ≥75 % deacetylated) and 3‐mercaptopropionic acid were purchased from Aldrich and were used as received.…”

Biodesulfurization Induces Reprogramming of Sulfur Metabolism in Rhodococcus qingshengii IGTS8: Proteomics and Untargeted Metabolomics

“…It is a membrane-bound molybdoenzyme (five transmembrane helices) bearing resemblance to the well-characterized SoxC subunit of sulfane dehydrogenase from Paracoccus pantotrophus ( 33 ). The most similar structurally characterized proteins are the SorA subunit of Starkeya novella sulfite dehydrogenase ( 34 ), followed by P. pantotrophus SoxC and SorT sulfite dehydrogenase from Sinorhizobium meliloti ( 35 ). Moreover, IGTS8_peg446 encodes a putative membrane protein YeiH with 11 transmembrane helices that is 28% identical to a YeiH family sulfate exporter ( TDL75784 ) from R .…”