The H93G myoglobin cavity mutant as a versatile template for modeling hemeproteins: Ferrous, ferric, and ferryl mixed-ligand complexes with imidazole in the cavity

One of the difficulties in preparing accurate ambient-temperature model com plexes for heme proteins, particularly in the ferric state, has been the ge neration of mixed-ligand adducts: complexes with different ligands on eithe r 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 be en prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analog ous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spec troscopically resembles wild-type ferric Mb as expected for a complex conta ining a single imidazole in the proximal cavity and water bound on the dist al side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge n eutral (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(I m) Mb and subsequent exposure to either CO, NO, or O-2 produces ferrous com plexes (deoxy, CO, NO, and O-2) that consistently exhibit MCD spectra simil ar to the analogous ferrous species of wild-type ferrous Mb. Most interesti ngly; reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are es sentially identical to those of oxoferryl wild-type Mb. The generation of s uch a wide array of mixed-ligand heme complexes demonstrates the efficacy o f the H93G Mb cavity mutant as a template for the preparation of heme prote in model complexes.