The C-terminal heme regulatory motifs of heme oxygenase-2 are redox-regulated heme binding sites.
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Heme oxygenase-2 (HO2), an enzyme that catalyzes the conversion of heme to biliverdin, contains three heme regulatory motifs (HRMs) centered at Cys127, Cys265, and Cys282. Previous studies using the soluble form of human HO2 spanning residues 1-288 (HO2sol) have shown that a disulfide bond forms between Cys265 and Cys282 and that, in this oxidized state, heme binds to the catalytic site of HO2sol via His45. However, various mutational and spectroscopic studies have confirmed the involvement of cysteine in Fe(3+)-heme binding upon reduction of the disulfide bond. In an effort to understand how the HRMs are involved in binding of heme to disulfide-reduced HO2sol, in the work described here, we further investigated the properties of Fe(3+)-heme bound to HO2. Specifically, we investigated binding of Fe(3+)-heme to a truncated form of soluble HO2 (residues 213-288; HO2tail) that spans the C-terminal HRMs of HO2 but lacks the catalytic core. We found that HO2tail in the disulfide-reduced state binds Fe(3+)-heme and accounts for the spectral features observed upon binding of heme to the disulfide-reduced form of HO2sol that cannot be attributed to heme binding at the catalytic site. Further analysis revealed that while HO2sol binds one Fe(3+)-heme per monomer of protein under oxidizing conditions, disulfide-reduced HO2sol binds slightly more than two. Both Cys265 and Cys282 were identified as Fe(3+)-heme ligands, and His256 also acts as a ligand to the Cys265-ligated heme. Additionally, Fe(3+)-heme binds with a much weaker affinity to Cys282 than to Cys265, which has an affinity much weaker than that of the His45 binding site in the catalytic core. In summary, disulfide-reduced HO2 has multiple binding sites with varying affinities for Fe(3+)-heme.
author list (cited authors)
Fleischhacker, A. S., Sharma, A., Choi, M., Spencer, A. M., Bagai, I., Hoffman, B. M., & Ragsdale, S. W.
complete list of authors
Fleischhacker, Angela S||Sharma, Ajay||Choi, Michelle||Spencer, Andrea M||Bagai, Ireena||Hoffman, Brian M||Ragsdale, Stephen W