Model for activation of sGC. In the conversion of the 6C species to the 5C NO–heme complex (k₃), NO acts as a catalyst in the reaction such that it is not consumed. Simulations (vide infra) are consistent with this. The step represented by k₅ does not involve NO and shows NO bound on the distal and proximal sides of the heme.

STS model for sGC activation. This model is a subset of that shown in Scheme . It excludes k₅ and makes k₃ independent of NO.

Data and simulations for the reaction of sGC with NO. (A) Spectra are shown of the ferrous sGC recorded before the reaction with NO (λ_Soret at 431 nm) of the 6C sGC–NO complex immediately after mixing sGC with NO (λ_Soret at 420 nm) and of 5C sGC–NO recorded 5 min after initiating the reaction (λ_Soret at 399 nm). A spectrum of a mixture of 6C and 5C NO complexes during conversion to the 5C NO complex helps to define the isosbestic point at 407 nm. The sGC concentration was 0.6 μM, and the NO concentration was 0.57 μM. Spectra were recorded at 200 nm/s. (B) The effect of NO concentration on the overall reaction was examined as follows: sGC (0.47 μM) was mixed anaerobically with 0.59, 1.53, 6.6, and 500 μM NO at 4°C, and absorbance changes at 431 nm are shown. (C) Simulations of the reactions in B according to Scheme . The values used are ɛ_sGC = 124 mM⁻¹⋅cm⁻¹, ɛ_6C = 66 mM⁻¹⋅cm⁻¹, and ɛ_5C = 46 mM⁻¹⋅cm⁻¹; k₁ = 1.55 × 10⁸ M⁻¹⋅s⁻¹, k₂ = 1 × 10⁻² s⁻¹, k₃ = 3.3 × 10⁵ M⁻¹⋅s⁻¹, k₄ = 1 × 10⁻³ s⁻¹, and k₅ = 5 × 10⁻⁴ s⁻¹. NO concentrations are indicated as solid circles (0.47 μM), open circles (1.53 μM), open squares (6.6 μM), and a solid line (500 μM). At the low concentration, the best fit was obtained with 0.47 μM NO. The instability of NO and the experimental design contributed to this loss, which is most pronounced at this low concentration. (D) Simulations of the STS model with the same extinction coefficients as in C and with the rate constants used by Bellamy et al. (): k₁ = 8 × 10⁷ M⁻¹⋅s⁻¹, k₂ = 200 s⁻¹, k₃ = 1.3 s⁻¹, and k₄ = 6.6 × 10⁻² s⁻¹. Symbols are the same as in C. Note that in D the starting absorbance of the second phase varies with NO concentration, whereas in B (experimental data) and C (simulation based on our model) the second phase has the same starting absorbance when NO concentrations are in the 0.57–6.6 μM range. This indicates that the equilibrium between the 6C sGC–NO complex and ferrous sGC required by the Bellamy model lies almost fully toward 6C sGC–NO. [A and B are reproduced with permission from Zhao et al. () (Copyright 1999, Natl. Acad. Sci.).]

Time course after mixing NO with sGC of various species and of production of cGMP. (A) sGC [1 μM in 50 mM Hepes (pH 7.4) and 50 mM NaCl, 15 μl] was mixed rapidly with 15 μl of buffer containing GTP (3 mM), Mg²⁺ (10 mM), and NO in a Kintek rapid quench-flow device at 4°C and were quenched at appropriate times with 40 mM HCl. Data for NO concentrations after mixing at 0.75 μM (closed circles) or at 10 μM (open circles) are shown. The sGC heme concentration was 0.5 μM after mixing. Each point is the mean of the cGMP values determined for three measurements. (B) Simulation of the reaction of 0.5 μM sGC with 10 μM NO. (C) simulation of the reaction of 0.5 μM sGC with 0.75 μM NO. In B and C: sGC, closed circles; 6C sGC–NO, open circles; and 5C sGC–NO, open squares. [A is reproduced with permission from Zhao et al. () (Copyright 1999, Natl. Acad. Sci.).]

Simulation of the return to free sGC after the removal of free NO from solution. 5C sGC–NO (0.47 μM) was used as the starting condition. The reaction was simulated according to Scheme and the rate constants in Fig. C, assuming that all free NO was removed from solution.