Regeneration of the ferrous heme of soluble guanylate cyclase from the nitric oxide complex: Acceleration by thiols and oxyhemoglobin

Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP to cGMP and is activated several hundred-fold by binding of nitric oxide (. NO) to the heme prosthetic group. We have examined the stability of the nitrosyl-heme complex of sGC (. NO-sGC) at 37 degrees C in order to determine whether si mple dissociation of . NO from sGC could account for the observed in vivo d eactivation time. Recombinant sGC was purified from Sf9 cells coinfected wi th baculoviruses containing the cDNAs for the alpha 1 and beta 1 subunits o f rat lung sGC. The purified protein contained a stoichiometric equivalent of ferrous high-spin heme. Characterization of the purified protein found i t to be essentially identical to that purified from bovine lung. Ferrous-ni trosyl sGC prepared anaerobically and exchanged into aerobic buffer contain ing no reducing agents was essentially stable on ice and had a half-life of approximately 90 min at 37 degrees C. In the presence of thiols [DTT, glut athione (GSH), or L-cysteine], . NO was rapidly lost from sGC regenerating the ferrous high-spin form of the heme. The half-life of . NO-sGC in the pr esence of 1 mM GSH at 37 degrees C was 6.3 min. In the presence of oxyhemog lobin, the half-life was further reduced to 2.9 min. Although these rates a re not fast enough to account for that observed in vivo, and thus probably involve additional agent(s), these data do imply a role for low molecular w eight thiols, such as GSH, and oxyferrohemoproteins, such as oxymyoglobin, in the deactivation of sGC.