Volume 5 Supplement 1
Resonance raman investigation of effects of YC-1 and GTP on structure of CO-bound heme of soluble guanylate cyclase
© BioMed Central Ltd 2005
Published: 16 June 2005
Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been categorized into two types; sGC1 and sGC2. Here we demonstrate that the RR spectra of sGC isolated from bovine lung contains only sGC2 while both species are observed in the spectra of CO-bound form (CO-sGC). The relative populations of the two forms altered from an initial composition in which the CO-sGC2 form predominated, with the Fe-CO (νFe-CO) and C-O stretching modes (νCO) at 472 and 1985 cm-1, respectively, to a composition dominated by the CO-sGC1 form with νFe-CO and νCO at 488 and 1969 cm-1, following the addition of xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a similar but significantly smaller effect, while a substrate analogue, GTP-γ-S had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced.
In the absence of any effectors, the Fe-CO stretching and the heme vinyl bending modes appeared at 473 and 424 cm-1, respectively. There was no band around 372 cm-1. However, in the presence of YC-1 and GTP, the Fe-CO stretching mode was shifted to 489 cm-1 and the vinyl mode appeared at 400 cm-1. The propionate mode appeared at 372 cm-1 as a sharp band. The vinyl modes at 424 and 400 cm-1 are assigned to the vinyl-2 and vinyl-4 Cβ - Ca = Cb bending vibrations that are strongly coupled with ring modes of pyrroles-I and II, respectively, while the propionate mode is coupled with ring modes of pyrroles-III and IV. In addition, new CO-isotope sensitive bands were observed at 521, 363, and 227 cm-1. The 521 cm-1 band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron-protoporphyrin IX in CTAB micelles. Distinct from the 472 cm-1 species, both the 488-cm-1 and 521-cm-1 species were apparently unphotodissociated when an ordinary Raman spinning cell was used with a CW laser source for Raman excitation, indicating rapid recombination of photodissociated CO in the presence of YC-1 and GTP. On the basis of these findings, binding of YC-1 and GTP to the heme pocket is proposed.