Interaction of heme oxygenase‐2 with nitric oxide donors

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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.