Characterization of Two Different Five-Coordinate Soluble Guanylate Cyclase Ferrous–Nitrosyl Complexes

Abstract

Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a five-coordinate ferrous-nitrosyl complex and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, and g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, and g 3 = 2.010. The truncated heme domain constructs beta1(1-194) and beta2(1-217) were compared to the full-length enzyme. The EPR spectrum of the beta2(1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, and g 3 = 2.010, indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the spectrum of the beta1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the beta1(1-194)-NO and beta2(1-217)-NO complexes show that the Fe-NO stretching vibration of the beta2(1-217)-NO complex (535 cm (-1)) is significantly different from that of the beta1(1-194)-NO complex (527 cm (-1)). This shows that sGC can adopt different five-coordinate ferrous nitrosyl conformations and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state.

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

“…Previously reported resonance Raman studies demonstrated a difference between sGC-NO complexes obtained in excess or stoichiometric NO (26) and subtle changes in the EPR spectrum due to varying g 2 values (45). The addition of GTP induced a much more pronounced difference in the EPR spectra of the sGC-NO complex (45). Corroborating these findings, our EPR measurements also showed a changed line shape in the presence of GTP (supplemental Fig.…”

How Do Heme-Protein Sensors Exclude Oxygen? Lessons Learned from Cytochrome c′,Nostoc puntiformeHeme Nitric Oxide/Oxygen-Binding Domain, and Soluble Guanylyl Cyclase

“…Consequently, a high affinity 5c NO-heme complex can be formed by displacing the proximal histidine. This idea was first proposed by Traylor, Sharma, and colleagues in the 1980-1990s (106,107,119), and is supported by the multi-step NO binding scheme proposed for cyt c¢ and by multiple electron paramagnetic resonance (EPR) and resonance Raman (rR) studies, which show that at equilibrium, the sGC-NO complex is pentacoordinate (25,28,64), as represented in Figure 8.…”

Structural insights into the role of iron–histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins

“…Further detailed in vitro investigations into the involvement of additional equivalents of NO in the activation of sGC provided evidence that two distinct, spectrally identical, NO-bound states of the enzyme exist. Importantly, these states were hypothesized to be a result of either (i) two five-coordinate NO-bound species, with NO located in either the distal or proximal pocket, or (ii) a five-coordinate NO-bound heme with the additional NO equivalent modifying a cysteine residue to generate an NO-addition complex (4,(31)(32)(33)(34). The Fe II -NO structure presented here provides crystallographic evidence for the formation of proximal NO in H-NOX proteins.…”