Abstract
The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 --> Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 --> Ala mutation inactivates HO-2, Cys264 --> Ala and Cys281 --> Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 --> Ala or Pro282 --> Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell.
Highlights
A great deal remains to be learned about the biochemical and physiological functions of heme oxygenase (HO)1 isozymes, HO-1 and HO-2 [1, 2], which have been traditionally viewed only in terms of heme catabolism
In the course of this investigation we have found that HO-2 is a hemoprotein and provide good evidence to suggest that heme regulatory motifs (HRMs) are involved in binding of heme but not in heme catalysis
We report that HO-2 is a constitutive hemoprotein, and, as indicated by site-directed mutagenesis experiments, the hemoprotein nature appears linked to the HRMs of the protein; these motifs are not involved in the catalytic activity of the protein
Summary
A great deal remains to be learned about the biochemical and physiological functions of heme oxygenase (HO) isozymes, HO-1 and HO-2 [1, 2], which have been traditionally viewed only in terms of heme catabolism. Aside from the overall differences in amino acid composition of HO-1 and HO-2, a major difference is the presence of two cysteines in all HO-2s and the absence of this residue in all HO-1s (11, 14, 15, 19, 24 –28) This residue is the axial ligand for the heme prosthetic moiety in various hemoproteins, including all cytochrome P450s and nitric-oxide synthase isozymes (29 –33). The Cys-Pro dipeptide, often flanked downstream by a hydrophobic residue, phenylalanine, is the absolutely conserved core of the recently identified motif called the heme regulatory element (HRM) [34]. Given the fact that in HO-2, two copies of the core HRM are present, we undertook the present study to examine whether, in intact HO-2 protein, the HRMs are involved in heme binding and to investigate their function in heme catalysis. We confirm that the His151 in the 24-residue heme pocket is essential for HO-2 activity
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