Resonance Raman Evidence for the Presence of Two Heme Pocket Conformations with Varied Activities in CO-Bound Bovine Soluble Guanylate Cyclase and Their Conversion

Abstract

Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC(1) and sGC(2). Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC(2) while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC(2) form predominated, with the Fe-CO (nu(Fe)(-)(CO)) and C-O stretching modes (nu(CO)) at 472 and 1985 cm(-)(1), respectively, to a composition dominated by the CO-sGC(1) form with nu(Fe)(-)(CO) and nu(CO) at 488 and 1969 cm(-)(1), respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-gamma-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. New CO isotope-sensitive bands were observed at 521, 488, 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 and 521 cm(-)(1) species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed.