Part I: Spectroscopic investigations of the heme active site coordination structure of the exogenous ligand-free myoglobin cavity mutant H93G and investigations of H93G-thiolate adducts as structural and mechanistic model systems for cytochrome P450. Part

Part I. Recently, protein engineering efforts have produced a new class of heme proteins called "cavity mutants" through replacement of the protein-derived proximal heme iron ligand with smaller, non-coordinating amino acid residues. Cavity mutants are now being studied by several research groups investigating structure-function relationships within heme-containing proteins and enzymes. The heme iron structure of the histidine 93 to glycine (H93G) mutant of sperm whale myoglobin is the subject of this work. H93G is capable of binding a wide variety of exogenous ligands in place of the original histidine ligand.;Spectroscopic investigations of the exogenous ligand free form of ferric H93G by magnetic circular dichroism spectroscopy indicate that its heme is a five-coordinate aquo complex at neutral pH and five-coordinate hydroxo complex at alkaline pH. Ferric thiolate adducts of H93G spectroscopically resemble the five-coordinate high-spin form of cytochrome P450-CAM. This P450 model system does not extend to ferrous thiolate adducts because the thiolate is displaced upon reduction of the ferric heme. These results are discussed in context of spectroscopic data for cytochrome P450 and proximal cysteine mutants of myoglobin (H93C).;A series of aromatic thiolate adducts of H93G have been investigated. The position of the Soret absorption band has been found to correlate with the Hammett parameter ;Part II. Two novel heme-containing peroxidases, one able to incorporate halogens into aromatic substrates and the other able to remove them, have recently been isolated from marine sources. These two enzymes have been examined with magnetic circular dichroism and UV-visible absorption spectroscopy to identify the histidine as their proximal ligands. Resonance Raman investigations indicate that the unusual catalytic activities of the two enzymes are not a result of unusually strong hydrogen bonding of their proximal histidine ligands or unusual distal pocket polarity.