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.
We demonstrate electrochemically driven catalytic voltammetry of the Mo‐dependent sulfite dehydrogenase (SorT) from the α‐Proteobacterium Sinorhizobium meliloti with its physiological electron acceptor, the c‐type cytochrome (SorU), with both proteins co‐adsorbed on a chemically modified Au working electrode. Both SorT and SorU were constrained under a perm‐selective dialysis membrane with the biopolymer chitosan as a co‐adsorbate, while the electrode was modified with a 3‐mercaptopropionate self‐assembled monolayer cast on the Au electrode. Cyclic voltammetry of the SorU protein reveals a well‐defined quasireversible FeIII/II redox couple at +130 mV versus NHE in 100 mM phosphate buffer solution (pH 7.0). Introduction of wild‐type sulfite dehydrogenase (SorTWT) and sulfite transforms this transient SorU voltammetric response into a sigmoidal catalytic wave, which increases with sulfite concentration before eventually saturating. In addition to the wild‐type enzyme, the variants SorTR78K, SorTR78M, and SorTR78Q were also examined electrochemically in an effort to better understand the role of amino acid residue Arg78, which is in the vicinity of the Mo active site of SorT.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.