title: Regeneration of the ferrous heme of soluble guanylate cyclase from the nitric oxide complex: acceleration by thiols and oxyhemoglobin.
abstract: 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 degreesC in order to determine whether simple dissociation of *NO from sGC could account for the observed in vivo deactivation time. Recombinant sGC was purified from Sf9 cells coinfected with baculoviruses containing the cDNAs for the alpha1 and beta1 subunits of rat lung sGC. The purified protein contained a stoichiometric equivalent of ferrous high-spin heme. Characterization of the purified protein found it to be essentially identical to that purified from bovine lung. Ferrous-nitrosyl sGC prepared anaerobically and exchanged into aerobic buffer containing no reducing agents was essentially stable on ice and had a half-life of approximately 90 min at 37 degreesC. In the presence of thiols [DTT, glutathione (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 presence of 1 mM GSH at 37 degreesC was 6.3 min. In the presence of oxyhemoglobin, the half-life was further reduced to 2.9 min. Although these rates are 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 weight thiols, such as GSH, and oxyferrohemoproteins, such as oxymyoglobin, in the deactivation of sGC.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-1065, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Brandish
authors.forename: W
authors.initials: W
authors.lastname: Buechler
authors.forename: M A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation; D005296:Ferrous Compounds / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008956:Models, Chemical; D009569:Nitric Oxide / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D018411:Spodoptera
chemical_list: D005296:Ferrous Compounds; D006420:Hemeproteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D007501:Iron; D006162:Guanylate Cyclase