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).
One of the difficulties in preparing accurate ambient-temperature model complexes for heme proteins, particularly in the ferric state, has been the generation of mixed-ligand adducts: complexes with different ligands on either side of the heme. The difference in the accessibility of the two sides of the heme in the H93G cavity mutant of myoglobin (Mb) provides a potential general solution to this problem. To demonstrate the versatility of H93G Mb for the preparation of heme protein models, numerous mixed-ligand adducts of ferrous, ferric, and ferryl imidazole-ligated H93G (H93G(Im) Mb) have been prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analogous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spectroscopically resembles wild-type ferric Mb as expected for a complex containing a single imidazole in the proximal cavity and water bound on the distal side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge neutral (imidazole) or anionic (cyanide and azide), results in formation of six-coordinate low-spin complexes with MCD characteristics similar to those of parallel derivatives of wild-type ferric Mb. Reduction of ferric H93G(Im) Mb and subsequent exposure to either CO, NO, or O2 produces ferrous complexes (deoxy, CO, NO, and O2) that consistently exhibit MCD spectra similar to the analogous ferrous species of wild-type ferrous Mb. Most interestingly, reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are essentially identical to those of oxoferryl wild-type Mb. The generation of such a wide array of mixed-ligand heme complexes demonstrates the efficacy of the H93G Mb cavity mutant as a template for the preparation of heme protein model complexes.
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