Soluble guanylate cyclase is activated differently by excess NO and by YC-1: resonance Raman spectroscopic evidence

Abstract

Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low-level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to that of the xsNO state. In this study, the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains five-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents undergo changes in intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO stretching frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO.

Figure 1

(a) Domain structure of sGC (adapted from (4)). (b) Structure of YC-1.

Figure 2

Homology model of sGC β1 H-NOX domain (based on Tt H-NOX structure, pdB: 1U55).

Figure 3

UV-visible spectra of ligand-free (black), NO bound (blue) and NO + YC-1 bound (red) full-length WT sGC. Effect of 1-NO (top panel) and xsNO (bottom panel) binding are shown. Absorption at 326 nm is due to YC-1.

Figure 4

400 nm – excited RR spectra of full-length WT sGC containing one NO (1-NO), excess NO (xsNO), and the ¹⁴NO-¹⁵NO difference bands (in black), and with added YC-1 (in red). Band assignments and frequencies are indicated. Full spectra are given in Figure S1.

Figure 5

400 nm – excited RR spectra of WT β1(1-194) with excess NO (xsNO) and of the P118A and I145Y variants. The ¹⁴NO-¹⁵NO difference bands are shown.

Figure 6

Labeling scheme for iron protoporphyrin-IX (heme). Pyrrole C_α and C_β positions, as well as porphyrin C_m positions are shown in green, while vinyl C_a, C_b, and propionate C_c, C_d positions are shown in blue.

Figure 7

Computed frequencies for (NO)Fe(II)P when the FeNO angle was constrained at the indicated values. The equilibrium angle is 142°. The inset shows computed energies associated with the angle constraints.

Figure 8

νFeN/νNO data for Fe(II)NO adducts of full-length (●) and truncated (○) wild-type sGC. The solid line is the correlation obtained from a series of 5-coordinate (NO)Fe(II)TPP-X complexes (50).

Figure 9

Structural model for activation-linked transitions of sGC-NO. Binding of NO to the heme breaks the proximal Fe-His bond, and induces porphyrin distortion via protein contacts. sGC exhibits a low level of activity in this state. Effector binding (YC-1 or BAY 41-2272) is suggested to induce an activating rotation of the two halves of the H-NOX domain, which alters the heme-protein contacts. In the altered conformation the Fe-NO angle is opened and pyrrole rings are rotated, producing net planarization of the heme. Binding of additional NO to the 1-NO adduct also induces high activity via an alternative mechanism that does not affect the heme-NO structure.