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Mechanistic insights into iron-sulfur clusters and flavin oxidation of a novel xanthine oxidoreductase from Sulfobacillus acidophilus TPY. | LitMetric

AI Article Synopsis

Article Abstract

Xanthine oxidoreductase (XOR) catalyzes the oxidation of purines (hypoxanthine and xanthine) to uric acid. XOR is widely used in various therapeutic and biotechnological applications. In this study, we characterized the biophysical and mechanistic properties of a novel bacterial XOR from Sulfobacillus acidophilus TPY (SaXOR). Our results showed that SaXOR is a heterotrimer consisting of three subunits, namely XoA, XoB, and XoC, which denote the molybdenum cofactor (Moco), 2Fe-2S, and FAD-binding domains, respectively. XoC was found to be stable when co-expressed with XoB, forming an XoBC complex. Furthermore, we prepared a fusion of XoB and XoC via a flexible linker (fusXoBC) and evaluated its function in comparison to that of XoBC. Spectroscopic analysis revealed that XoB harbors two 2Fe-2S clusters, whereas XoC bears a single-bound FAD cofactor. Electron transfer from reduced forms of XoC, XoBC, and fusXoBC to molecular oxygen (O ) during oxidative half-reaction yielded no flavin semiquinones, implying ultrafast single-electron transfer from 2Fe-2S to FAD. In the presence of XoA, XoBC and fusXoBC exhibited comparable XoA affinity and exploited a shared overall mechanism. Nonetheless, the linkage may accelerate the two-step, single-electron transfer cascade from 2Fe-2S to FAD while augmenting protein stability. Collectively, our findings provide novel insights into SaXOR properties and oxidation mechanisms divergent from prior mammalian and bacterial XOR paradigms.

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http://dx.doi.org/10.1111/febs.16987DOI Listing

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