a, Thermal unfolding of NgTAL wild type and the Lys8Ala and Cys38Ser variants under oxidizing and reducing conditions, as monitored by far-UV CD spectroscopy at 222 nm. Different unfolding temperatures are seen for the oxidized and reduced states in case of the wild-type enzyme and the Lys8Ala variant, whereas the Cys38Ser variant does not exhibit this feature. This suggests an oxidation of Cys38 in the Lys8Ala variant, despite the absence of Lys8. b, Analytical ultracentrifugation analyses of NgTAL wild-type and variants in the oxidized and reduced state shows the predominant formation of the monomeric form in all of the cases we tested. Under oxidizing conditions and high protein concentrations, a small fraction of higher oligomers is observed (presumably resulting from incorrectly linked monomers). c, X-ray crystallographic structure of the NgTAL Lys8Ala variant, showing the allosteric redox switch site with residues Ala8 (mutation site), Cys38, Glu93 and Thr101. For residues Ala8 and Cys38, the corresponding 2mFo − DFc electron density maps are shown in blue at a contour level of 2σ. Inset, peaks in the mFo − DFc difference electron density map (in green, contour level 3σ) around the sulfur atom of residue Cys38 suggest that this atom is oxidized. Owing to the structural flexibility of Cys38, the discrete oxidation state (mono-oxidized and/or dioxidized) cannot be unambiguously assigned. Notwithstanding this ambiguity, this observation supports our proposed mechanism of an initial cysteine oxidation as part of the formation of the NOS bridge. d, X-ray crystallographic structure of the NgTAL Cys38Ser variant, showing the allosteric redox switch site with residues Lys8, Ser38 (mutation site), Glu93 and Thr101. For residues Lys8 and Ser38, the corresponding 2mFo − DFc electron density maps are shown in blue at a contour level of 1.5σ. Lys8 is chemically unmodified, thus ruling out that the covalent linkage between Lys8 and Cys38 seen in the wild-type enzyme results from the addition of CO2 or formaldehyde potentially establishing an NCS linkage18.