Entry of water into the distal heme pocket of soluble guanylate cyclase β1 H-NOX domain alters the ligated CO structure: a resonance Raman and in silico simulation study

Abstract

Resonance Raman spectra (rRS) of the CO-Ns H-NOX (heme nitric oxide and oxygen binding) complex, a bacterial model for the mammalian CO–sGC (soluble Guanylyl Cyclase) complex, are reported. The CO-Ns H-NOX complex exhibited two Fe–CO stretching modes (ν_Fe–CO) with an intense band at 470 cm⁻¹ and a weaker band at 494 cm⁻¹, which are very similar to those observed for a CO-ligated full-length bovine lung sGC. However, the rRS of truncated sGC β1_(1–194) H-NOX exhibited comparable bands, but with a reversed intensity ratio, the ν_Fe–CO at 492 cm⁻¹ was dominant. This difference is similar to that observed for six-coordinate CO–sGC's in the absence and presence of YC-1, an effector. Molecular dynamic (MD) simulations of truncated sGC β1_(1–194) H-NOX demonstrate that water may enter the distal heme pocket to form a hydrogen bond with ligated CO; whereas it hardly happens to Ns H-NOX. By employing density functional theory (DFT) analysis, we calculated the C–O and Fe–CO stretching frequencies of the isolated imidazole (ImH)- and porphyrin (P) containing complex, (ImH)FeP(CO), in the presence or absence of water molecules. The calculated rRS exhibited bands at a higher ν_Fe–CO frequency in the presence of water, in agreement with observations for truncated sGC, further supporting the hypothesis that water access to the distal heme pocket influences the ligation of CO, although the proximal effects remain to be elucidated.