Generation and study of intermediates in hemeprotein catalysis

A wide variety of reactivities are associated with highly oxidized intermediates in heme-based catalysis. Attachment of photoactive metal complexes to myoglobin and cytochrome c peroxidase provides a means of initiating and following redox transformations. Oxidative flash/quench experiments are used to generate high oxidation states in ruthenium-modified myoglobin and cytochrome c peroxidase. One-electron oxidation is shown to generate protein radicals observable by electron paramagnetic resonance spectroscopy.; Attachment of a ruthenium complex to the heme domain of P450bm3 is discussed. Reductive flash/quench experiments are used to investigate the electron transfer pathway from flavin to heme. Presence of the enzyme substrate effects the rates of electron transfer.; Heme cdl nitrite reductase is an unusual bifunctional catalyst. An oxochlorin derivative of mesoporphyrin IX is synthesized and used as a model for heme cd₁ nitrite reductases. The oxochlorin is reconstituted into myoglobin (MpMb) and cytochrome c peroxidase (MpCcP). MpMb is found to act as an effective model of nitrite reductase activity. Electrochemical reduction of MpMb generates the ferrous-nitrosyl adduct. A stable intermediate, nitroxyl MpMb is observed during the reduction of nitrite and nitric oxide. MpCcP is found to have enhanced small-molecule peroxidase activity in comparison to the native enzyme. A crystal structure of MpCcP reveals that structural features of MpCcP resemble that of the enzyme intermediate of WTCcP with hydrogen peroxide rather than the resting state of the enzyme.; Cytochrome P450 CYP119 in surfactant films is shown to reduce nitrite and nitric oxide catalytically. The nitrosyl/nitroxyl redox couple is observed at fast scan rates in the presence of nitrite. The reduction of nitric oxide is proposed to model the catalytic activity of cytochrome P450nor.; The reaction of ferrous myoglobin and Mp myoglobin with nitrites is investigated. The reaction of myoglobin with nitrite produces equal amounts of ferric and nitrosyl myoglobin. In Mpmyoglobin, the ferric nitrosyl adduct is the exclusive product. The reactions are rate-limited by formation of nitrous acid.; The nature of bonding in nitroxyl myoglobin is investigated by X-ray absorbance and resonance raman spectroscopies. It is proposed that nitroxyl is a good σ-donating and π-backbonding ligand. The nitroxyl adducts of hemoglobin, leghemoglobin, and Mp myoglobin are also discussed.