Heme oxygenase-2 interaction with metalloporphyrins: Function of heme regulatory motifs

Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)l to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys(264)-Pro(265) and Cys(281)-Pro(282). We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP not PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each FIRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys(264)-Pro(265) result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.