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Recently, heme protein cavity mutants have been engineered in which the proximal coordinating amino acid has been replaced by a smaller, noncoordinating residue leaving a cavity that can be filled by exogenous axial ligands. This approach was pioneered by Barrick (Biochemistry 1994, 33, 6546-6554) with H93G sperm whale myoglobin where 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 been 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 in the cavity, the ferric H93G thiolate complexes spectroscopically resemble five-coordinate highspin substrate-bound ferric P450, which contains a thiolate proximal ligand and lacks a water distal ligand. Thus, the distal protein environment plays a crucial role in controlling whether a five-coordinate thiolateligated ferric heme binds water or not. Two advantages pertain to the present thiolate-ligated heme protein model relative to purely synthetic thiolate-ligated ferric porphyrins: (a) aliphatic thiols can form complexes without reduction of the ferric iron and (b) mixed ligand complexes that are stable at ambient temperatures can be prepared with a neutral ligand such as imidazole trans to thiolate. However, when anionic ligands are added to the ferric thiolate adduct in an attempt to prepare mixed ligand complexes with two anionic ligands, the thiolate ligand is displaced (or lost) without formation of a stable mixed ligand derivative. Further, reduction of the ferric-thiolate complex leads to loss of the thiolate ligand even in the presence of CO. The data presented for the thiolate adducts of ferric 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 thiolate-ligated in the ferrous state is likely due to the lack of (a) correctly positioned hydrogen bond donating groups and (b) a properly oriented helix dipole to stabilize the thiolate ligand as occurs in the proximal protein environment of P450. The present results illustrate the important role of the distal and proximal heme environments in controlling the ferric spin state and in stabilizing thiolate ligation in heme systems, respectively.