Structural and Spectroscopic Investigations of pH-Dependent\nMo(V) Species in a Bacterial Sulfite-Oxidizing Enzyme

Mono-pyranopterin-containing\nsulfite-oxidizing enzymes (SOEs),\nincluding eukaryotic sulfite oxidases and homologous prokaryotic sulfite\ndehydrogenases (SDHs), are molybdenum enzymes that exist in almost\nall forms of life, where they catalyze the direct oxidation of sulfite\ninto sulfate, playing a key role in protecting cells and organisms\nagainst sulfite-induced damage. To decipher their catalytic mechanism,\nwe have previously provided structural and spectroscopic evidence\nfor direct coordination of HPO<sub>4</sub><sup>2–</sup> to\nthe Mo atom at the active site of the SDH from the hyperthermophilic\nbacterium Thermus thermophilus (<i>Tt</i>SDH), mimicking the proposed sulfate-bound intermediate\nproposed to be formed during catalysis. In this work, by solving the\nX-ray crystallographic structure of the unbound enzyme, we resolve\nthe changes in the hydrogen bonding network in the molybdenum environment\nthat enable the stabilization of the previously characterized phosphate\nadduct. In addition, electron paramagnetic resonance spectroscopic\nstudy of the enzyme over a wide pH range reveals the formation of\npH-dependent Mo(V) species, a characteristic feature of eukaryotic\nSOEs. The combined use of HYSCORE, H<sub>2</sub>O/D<sub>2</sub>O exchange,\nand density functional theory calculations allows the detailed characterization\nof a typical low pH Mo(V) species previously unreported in bacterial\nSOEs, underlining the conservation of the active site properties of\nSOEs irrespective of their source organism.