Regeneration of the Ferrous Heme of Soluble Guanylate Cyclase from the Nitric Oxide Complex: Acceleration by Thiols and Oxyhemoglobin

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• 作者：Philip E. Brandish ; Wolfgang Buechler ; and Michael A. Marletta
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：December 1, 1998
• 年：1998
• 卷：37
• 期：48
• 页码：16898 - 16907
• 全文大小：173K
• 年卷期：v.37,no.48(December 1, 1998)
• ISSN：1520-4995

文摘

Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP to cGMP and is activatedseveral hundred-fold by binding of nitric oxide (NO) to the heme prosthetic group. We have examinedthe stability of the nitrosyl-heme complex of sGC (NO-sGC) at 37 C in order to determine whethersimple 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 forthe rs/alpha.gif" BORDER=0>1 and rs/beta2.gif" BORDER=0 ALIGN="middle">1 subunits of rat lung sGC. The purified protein contained a stoichiometric equivalent offerrous high-spin heme. Characterization of the purified protein found it to be essentially identical tothat purified from bovine lung. Ferrous-nitrosyl sGC prepared anaerobically and exchanged into aerobicbuffer containing no reducing agents was essentially stable on ice and had a half-life of approximately 90min at 37 C. In the presence of thiols [DTT, glutathione (GSH), or L-cysteine], NO was rapidly lostfrom sGC regenerating the ferrous high-spin form of the heme. The half-life of NO-sGC in the presenceof 1 mM GSH at 37 C was 6.3 min. In the presence of oxyhemoglobin, the half-life was further reducedto 2.9 min. Although these rates are not fast enough to account for that observed in vivo, and thusprobably involve additional agent(s), these data do imply a role for low molecular weight thiols, such asGSH, and oxyferrohemoproteins, such as oxymyoglobin, in the deactivation of sGC.