Assignment of the Heme Axial Ligand(s) for the Ferric Myoglobin (H93G) and Heme Oxygenase (H25A) Cavity Mutants as Oxygen Donors Using Magnetic Circular Dichroism

Abstract

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4°C. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known fivecoordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (Fe III PPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of Fe III PPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.Heme proteins with protein-derived oxygen donor proximal ligands are relatively rare in nature. The best known example of such ligation is that of bovine liver catalase which contains a tyrosine phenolate proximal heme iron ligand (1).In addition, a series of naturally occurring hemoglobin mutants having distal or proximal histidines replaced by tyrosine or glutamate (the M hemoglobins) have been established by X-ray crystallography (2-4) and resonance Raman spectroscopy (5, 6) to have phenolate or carboxylate ligation. Other proteins with tyrosinate and glutamate oxygen donor ligation have been recently produced by site-directed mutagenesis of various myoglobins (7-10) and cytochrome c peroxidase (11).The use of site-directed mutagenesis techniques has become invaluable in identifying catalytically and structurally important protein residues within a protein system. A relatively new type of mutation, based on altering the size of the amino acid at the point of mutation, substitutes a glycine or alanine for the larger original residue and leaves a cavity within the protein. Termed cavity mutants, these mutated proteins demonstrate the ability to employ exogenous ligands to reconstitute their wild-type activity. This rescue of activity has been seen for the cavity mutants of azurin (12), carbonic anhydrase (13), hexose-1-phosphate uridyltransferase (14), and various heme proteins (15-18).

The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

“…This possibility is supported by the similarity of the MmpL3-E1 and MmpL11-E1 absorption spectra to that of H25Y human heme oxygenase (61) and H93Y Mb (Table 2) (62). Both of these proteins bind a single fivecoordinate, Tyr-ligated heme.…”

The proximal and distal pockets of the H93G myoglobin cavity mutant bind identical ligands with different affinities: Quantitative analysis of imidazole and pyridine binding

“…Complete oxidation of the heme iron is accomplished by addition of a few crystals of potassium ferricyanide (Fluka) followed by gel-filtration column chromatography. Im can be completely removed from the proximal cavity by means of heme extraction followed by reconstitution with hemin as previously reported [18].…”

“…A molar absorptivity (ε) value of 112 mM −1 cm −1 for the Soret absorption peak (405-406 nm) for exogenous ligand-free ferric H93G Mb at pH 7.0 [18] was routinely used as a reference standard value. All proximal and distal pocket ligand binding studies were carried out at pH 7.0 in 0.1 M potassium phosphate buffer at 4 • C. Im stock solutions were prepared by dissolving commercial solid Im (99.9%) in 0.1 M potassium phosphate buffer and adjusting pH to 7.0 by adding HCl.…”

Comparison of the crystal structure and function to wild-type and His25Ala mutant human heme oxygenase-1

“…The histidine (His) residue at position 25 (His25) plays an important role in forming a unique HO-1 complex with fivecoordinated ferric heme molecules (25,26). Replacement of histidine with alanine (His25Ala) upregulates the activity of catalase and the level of intracellular glutathione, thereby enhancing the resistance to organic or inorganic peroxides.…”