Fig. 7. Spectroscopic analyses in solution support structural dynamics as a consequence of O₂ binding.

(A) ¹⁹F NMR spectra from anaerobic solutions of IPNS* ± Fe²⁺ ± ACV ± NO support α3 dynamics after NO binding. (i) IPNS* (¹⁹F-labeled IPNS); (ii) IPNS* + Fe²⁺; (iii) IPNS* + Fe²⁺+ACV; (iv) IPNS* + Fe²⁺ACV + NO; and (v) IPNS* + Fe²⁺+NO. (B) ¹⁹F NMR of IPNS* ± Cd²⁺ ± ACV ± NO. (i) IPNS* + Cd²⁺; (ii) IPNS* + Cd²⁺+ACV; (iii) IPNS* + Cd²⁺ACV + NO; and (iv) IPNS* + Cd²⁺+NO (fig. S8). (C) Comparison of the structural elements of α3/β11 in IPNS:Fe:ACV:O₂ (PDB: 6ZAP) and IPNS*:Fe:ACV (cryo-cooled single-crystal analysis, PDB: 6ZAM, 1.55-Å resolution, used for ¹⁹F NMR, showing keto and gem diol forms of fluorine label). (D) Overview of the IPNS* complex with α3 and β11 in red and presence of the fluorine label on α3. (E) View of the active site and α3 of the IPNS*:Fe:ACV complex, showing the distance from the ACV valinyl carboxylate to the alkylated Cys⁵⁵ (16.5 Å). Changes in the ACV valinyl carboxylate conformation as a function of O₂ binding are proposed to induce structural rearrangement of α3/β11. Cys⁵⁵ was alkylated with a CH₂COCF₃ group to study conformational changes in solution by ¹⁹F NMR. Tryptic digestion confirmed the presence of alkylated Cys⁵⁵ (fig. S10). (F) Views of the active site of the aerobically grown IPNS:Cd:ACV complex (PDB: 6ZW8, 1.22-Å resolution) obtained by single-crystal cryogenic data collection. A Polder omit map of the active site of IPNS:Cd:ACV after omitting Cd and ACV (3.0 σ contour level, green, modeled occupancy of 90% for Cd). Unmodeled electron density was observed next to Trp²¹³ on the protein surface. Note that exposure of IPNS:Cd:ACV crystals to NO reveals no NO binding to the metal and no evidence for α3/β11 rearrangements, even after multiple NO exposures with 10 crystals, consistent with the NMR analysis (B).