biostudies-literatureUnknown147(35)Chen HYPathogenic <i>Staphylococcus aureus</i> endures bursts of host-derived reactive nitrogen species, yet the molecular defenses that enable this resilience have remained unclear. We now show that the previously enigmatic di-iron enzyme ScdA functions as a nitrite reductase, converting nitrite to nitric oxide (NO), and we elucidate the structural elements that support this activity. Using an integrative toolkit─X-ray crystallography, solution NMR, AlphaFold modeling, and pulsed EPR/DEER─we solved the full-length homodimeric structure of ScdA and identified a robust di-iron center that forms stable iron-nitrosyl intermediates. Targeted mutagenesis reveals that redox-active cysteines and dimerization state tune catalytic output, whereas steady-state kinetics confirm efficient nitrite-to-NO turnoJournal of the American Chemical Society31558-31569https://www.ebi.ac.uk/biostudies/studies/S-EPMC12412097biostudies-literatureStructure and Nitrite Reductase Activity of the Di-iron Protein ScdA in &lt;i&gt;Staphylococcus aureus&lt;/i&gt;.PMC12412097Tsai RFHu NJFan-Chiang HYWu CYLiao WYSue SCLu YSKuo HWChen HYLo FCCheng YCYang WKChiang YWfalseStructure and Nitrite Reductase Activity of the Di-iron Protein ScdA in &lt;i&gt;Staphylococcus aureus&lt;/i&gt;.Pathogenic <i>Staphylococcus aureus</i> endures bursts of host-derived reactive nitrogen species, yet the molecular defenses that enable this resilience have remained unclear. We now show that the previously enigmatic di-iron enzyme ScdA functions as a nitrite reductase, converting nitrite to nitric oxide (NO), and we elucidate the structural elements that support this activity. Using an integrative toolkit─X-ray crystallography, solution NMR, AlphaFold modeling, and pulsed EPR/DEER─we solved the full-length homodimeric structure of ScdA and identified a robust di-iron center that forms stable iron-nitrosyl intermediates. Targeted mutagenesis reveals that redox-active cysteines and dimerization state tune catalytic output, whereas steady-state kinetics confirm efficient nitrite-to-NO turno2025-01-01T00:00:00Z2025 Sep2026-05-29T21:50:29.355Z2026-04-08T06:08:12.273ZS-EPMC124120974084668210.1021/jacs.5c05573