Identification of Histidine 45 as the Axial Heme Iron Ligand of Heme Oxygenase-2

Abstract

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (⌬HHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (⌬H45A) and His-152 to Ala (⌬H152A) mutants have been prepared using this expression system. ⌬H45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-⌬H45A complex. The ⌬H152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified ⌬H152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

“…Theoretically, the Lys residue at this position in AtHO2 could substitute for His in this reaction given its similar charge. Animal HOs contain a C-terminal hydrophobic extension that is thought to anchor the enzyme to the microsomal membrane (44). Consistent with their behavior as soluble enzymes in the chloroplast stroma (15,17), neither AtHO1 nor 2 contains a similar hydrophobic extension (Fig.…”

“…In preliminary studies using a full-length recombinant AtHO1 protein (including the predicted transit sequence) expressed from E. coli (S.J.D., S. Beale, and R.D.V., unpublished data), both crude extracts and purified AtHO1 failed to generate meso-BV from meso-heme by a standard in vitro assay (30). This was not completely surprising given the difficulties that have been reported by others in their attempts to generate active human and Synechocystis HOs by recombinant methods (30,44). Moreover, it is possible that plant HOs function as a complex with other factors in synthesizing P⌽B and may be unstable or inactive in their absence (20).…”

“…One cluster contains a positionally conserved His (His-198 in AtHO1) shown to be important for structural stability in human HOs (43). A second cluster near the N terminus of human HOs (residues 76-96 in AtHO1) is required for the oxidative cleavage and employs a His to bind the heme substrate (44). This His is present within AtHO1 (His-86), but is notably absent from AtHO2 (Fig.…”

Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the hemO Gene

“…The His-25 residue (corresponds to His-45 of HO-2) and a water molecule have been identified as heme ligands of human heme oxygenases (11). This conserved histidine residue was also identified in neisserial HemO proteins (data not shown).…”

Evidence That the Heme Regulatory Motifs in Heme Oxygenase-2 Serve as a Thiol/Disulfide Redox Switch Regulating Heme Binding

“…HO-2 Assay-The HO-2 enzymatic assay was slightly modified from that described previously (26). The 1-ml reaction mixture contained 0.1 M potassium phosphate buffer, pH 7.4, 20 M freshly prepared hemin (from a 2 mM stock containing 0.1 M NaOH and 5% Me 2 SO), 0.1 mg/ml bovine serum albumin, 30 g of NADPH-cytochrome P450 reductase, 60 g of purified human biliverdin reductase, and 30 -100 g of HO-2.…”