Interaction of heme oxygenase‐2 with nitric oxide donors
- 15 September 1999
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 264 (3), 854-861
- https://doi.org/10.1046/j.1432-1327.1999.00677.x
Abstract
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 (≈ 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.Keywords
This publication has 69 references indexed in Scilit:
- Heme Oxygenase-2 Is a Hemoprotein and Binds Heme through Heme Regulatory Motifs That Are Not Involved in Heme CatalysisJournal of Biological Chemistry, 1997
- THE HEME OXYGENASE SYSTEM:A Regulator of Second Messenger GasesAnnual Review of Pharmacology and Toxicology, 1997
- Heme Oxygenase-1, Intermediates in Verdoheme Formation and the Requirement for Reduction EquivalentsJournal of Biological Chemistry, 1997
- The mechanism of nitric oxide formation from S-nitrosothiols (thionitrites)Chemical Communications, 1996
- Distribution of constitutive (HO-2) and heat-inducible (HO-1) heme oxygenase isozymes in rat testes: HO-2 displays stage-specific expression in germ cellsEndocrinology, 1995
- Spectroscopic and Kinetic Studies on Reaction of Cytochrome P450nor with Nitric OxideJournal of Biological Chemistry, 1995
- Site‐directed mutagenesis of cysteine residues in biliverdin reductaseEuropean Journal of Biochemistry, 1994
- Regulation by Heme of Mitochondrial Protein Transport Through a Conserved Amino Acid MotifScience, 1993
- Biosynthesis and Metabolism of Endothelium-Derived Nitric OxideAnnual Review of Pharmacology and Toxicology, 1990
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970