Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?
Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.
Ding
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Department of Biochemistry, University of Rochester School of Medicine, NY 14642, USA.
McCoubrey
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WK
Maines
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MD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006418:Heme / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism* / Q000494:pharmacology; D009599:Nitroprusside / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009599:Nitroprusside; D009569:Nitric Oxide; D006418:Heme; C002385:linsidomine; D008981:Molsidomine; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2