FIGURE 3.

Characterization of 1-NO state of sGC. The 1-NO state of sGC is achieved by titration of ferrous sGC with PROLI NONOate (A), and the concentration of ferrous nitrosyl (Fe²⁺-NO) sGC is estimated by deconvolution of the final spectrum (red; 55% Fe²⁺-NO) to ferrous (blue) and Fe²⁺-NO (green) sGC spectra. The stability of 1-NO sGC was monitored by UV-visible spectroscopy and activity (B). sGC activity was tested immediately after the formation of Fe²⁺-NO (O) and after a 1-h incubation (B, inset). C, contribution of Fe²⁺ (basal) and Fe²⁺-NO (1-NO) sGC to the final activity observed in 1-NO samples. ATP inhibition of sGC in the 1-NO state for WT (D) and DM (E). Activity data are replotted as double reciprocal plots for WT (F) and DM (G). Ferrous nitrosyl sGC (100–200 nm) wild type or variant was assayed in 50 mm HEPES, pH 7.4, 6 mm MgCl₂, and 1 mm tris(2-carboxyethyl)phosphine. Each curve was fit to mixed type inhibition with two binding sites (Equations 1–3). Error bars, S.E.