A molecular basis for nitric oxide sensing by soluble guanylate cyclase

Abstract

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (k(on) > 1.4 x 10(8) M(-1).s(-1) at 4 degrees C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k = 2.4 x 10(5) M(-1).s(-1) at 4 degrees C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k = 7.1 +/- 2 x 10(8) M(-1).s(-1) at 4 degrees C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.

Figure 1

Nitric oxide signaling in the vascular system. NO biosynthesis in the vascular endothelium is regulated by Ca²⁺ and calmodulin in response to external signals such as bradykinin. Upon receiving such a signal, intracellular Ca²⁺ is released and complexes with calmodulin leading to transient activation of NOS. The newly synthesized NO is subject to several possible fates. As indicated by the bold red arrows, free diffusion of NO into the vascular smooth muscle activates sGC. The kinetic results reported here demonstrate the feasibility of this path. Diffusion into the lumen of the blood vessel and into erythrocytes will lead to reactions with Hb and HbO₂. Some evidence suggests that a nitrosylated Hb might function to deliver NO to specific tissue sites (dashed arrow) (21). It has also been suggested that small molecule carriers of NO (e.g., S-nitrosoglutathione or metal complexes) might serve as transporters. Termination of the signal via various decomposition reactions and in the smooth muscle itself via reactions with myoglobin are also shown. Based on the findings reported here, the activation of sGC is shown to proceed through two steps, with activation after step 2. Once activated, the cGMP synthesized activates a cascade of events as illustrated, leading to smooth muscle relaxation. MLCK, myosin light chain kinase; cGK, cGMP-dependent kinase; PDE, phosphodiesterase.

Figure 2

Stopped-flow analysis of NO binding to heterodimeric sGC. (A) All concentrations are postmixing. sGC (0.6 μM) and NO (0.57 μM) were mixed anaerobically in a stopped-flow spectrophotometer (Hi-Tech Scientific, Salisbury, U.K.) at 4°C. Absorbance changes at 399, 420, and 431 nm are shown. (B) Spectra are shown of the ferrous sGC recorded before the reaction with NO (λ_Soret at 431 nm), of the 6-coordinate NO complex intermediate immediately after mixing sGC with NO (λ_Soret at 420 nm), and of the 5-coordinate NO complex recorded 5 min after initiating the reaction (λ_Soret at 399 nm). A spectrum of a mixture of 6- and 5-coordinate NO complexes during conversion to the 5-coordinate NO complex is shown and also shows the isosbestic point at 406 nm. The sGC concentration was 0.6 μM, and the NO concentration was 0.57 μM. Spectra were recorded at 200 nm/s. (C) 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, 2.28 (data not shown for clarity), 6.6, and 500 μM NO at 4°C, and absorbance changes at 431 nm are shown. (D) The data in C were fit to three consecutive exponential processes. k_obs obtained from the first phase (k₁), which represents NO binding to sGC heme (filled circles), and that for the third phase (k₃), which represents conversion of the 6- to the 5-coordinate ferrous-NO complex (open circles), are plotted against the NO concentration. For the k₃ points, the NO concentrations were corrected by the amount that is bound to the heme in the first phase: i.e., the sGC concentration.

Figure 3

Determination of the activity of the 6-coordinate sGC-NO complex intermediate. (A) sGC [1 μM, in 50 mM Hepes (pH 7.4) and 50 mM NaCl, 15 μl) was rapidly mixed 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 (filled 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 ± SD. (B) The dependence of lag time on NO concentration. The lag time is the time before which no cGMP was detectable (limit, 0.15 pmol). Each point is the average value from two independent experiments. The P values determined for the lag times using ANOVA were <0.04 for each nonadjacent pair of reciprocal NO concentrations.

Figure 4

Stopped-flow analysis of NO binding to β1(1–385). (A) All concentrations are postmixing. β1(1–385) and NO were mixed anaerobically in a stopped-flow (Hi-Tech Scientific) at 4°C. After each experiment, a final spectrum was recorded to measure the total absorbance change at either 397 nm or 434 nm. The stopped-flow traces shown were obtained at 434 and 397 nm when both β1(1–385) and NO were 0.93 μM. (B) Nitrosyl complex formation at different NO concentrations (0.5, 0.93, 1.7 μM), β1(1–385) = 0.93 μM. Stopped-flow traces were simulated according to the second order process A + B → C, where A = β1(1–385), B = NO, and C = β1(1–385)-NO. From this analysis, k_on is estimated to be 7.1 ± 2 × 10⁸ M⁻¹⋅s⁻¹ at 4°C. It should be noted that >60% of these reactions have occurred in the dead-time of the stopped-flow instrument.