The role of the distal and proximal protein environments in controlling the ferric spin state and in stabilizing thiolate ligation in heme systems: Thiolate adducts of the myoglobin H93G cavity mutant

Recently, heme protein cavity mutants have been engineered in which the pro ximal coordinating amino acid has been replaced by a smaller, noncoordinati ng residue leaving a cavity that can be filled by exogenous axial ligands. This approach was pioneered by Barrick (Biochemistry 1994, 33, 6546-6554) w ith H93G sperm whale myoglobin whew the coordinating histidine is replaced by glycine and the proximal cavity is filled with imidazole. in the present study, models for cysteine thiolate-ligated ferric cytochrome P450 have be en prepared using H93G myoglobin containing thiolate ligands in the cavity. Despite the availability of water to serve as a distal ligand as occurs in both ferric wild type myoglobin and ferric H93G myoglobin with imidazole i n the cavity, the ferric H93G thiolate complexes spectroscopically resemble five-coordinate high-spin substrate-bound ferric P450, which contains a th iolate proximal ligand and lacks a water distal ligand. Thus, the distal pr otein environment plays a crucial role in controlling whether a five-coordi nate thiolate-ligated ferric heme binds water or not. Two advantages pertai n to the present thiolate-ligated heme protein model relative to purely syn thetic thiolate-ligated ferric porphyrins: (a) aliphatic thiols can form co mplexes without reduction of the ferric iron and (b) mixed ligand complexes that are stable at ambient temperatures can be prepared with a neutral lig and such as imidazole trans to thiolate. However, when anionic Ligands are added to the ferric thiolate adduct in an attempt to prepare mixed ligand c omplexes with two anionic ligands, the thiolate Ligand is displaced (or los t) without formation of a stable mixed Ligand derivative. Further, reductio n of the ferric-thiolate complex leads to loss of the thiolate Ligand even in the presence of GO. The data presented for the thiolate adducts of ferri c H93G myoglobin are analyzed in the context of the spectrally related H93C myoglobin mutants. The inability of the thiolate adducts of H93G myoglobin to accommodate a second anionic ligand in the ferric state or to remain th iolate-ligated in the ferrous state is likely due to the lack of (a) correc tly positioned hydrogen bond donating groups and (b) a properly oriented he lix dipole to stabilize the thiolate Ligand as occurs in the proximal prote in environment of P450. The present results illustrate the important role o f the distal and proximal heme environments in controlling the ferric spin state and in stabilizing thiolate ligation in heme systems, respectively.