Generation of high-oxidation states of myoglobin in the nanosecond time-scale by laser photoionization.

The 248 nm laser flash photolysis of myoglobin in various redox states (oxy, met and ferryl) in neutral aqueous solution yielded hydrated electrons with concurrent changes in the visible absorption spectrum of the heme. The results could be ascribed to the photoionization of both the peptide and the heme group, in approximately equal yields. The ionization of met- and ferrylmyoglobin was biphotonic, but that of oxymyoglobin was a mixture of mono- and biphotonic processes. Using appropriate electron and radical scavengers, the changes in the heme absorption could be investigated at times > or = 100 ns and were shown to be associated with a +1 increase of the formal oxidation state of the heme. Using this method, the formal iron (V) state of native myoglobin could be spectroscopically characterized for the first time. Its absorption, blue-shifted and less intense relative to the ferryl state, is reminiscent of that of the compound I of peroxidases, which contains a ferryl-oxo (iron[IV]) group and a porphyrin radical cation. On this basis, the same structure is proposed for the formal iron(V) state of native myoglobin. The transition from oxy- to metmyoglobin took approximately 5 microsecond, which may reflect the kinetics of exchange of oxygen with water as ligand. The transitions from the met to the ferryl state, and from ferryl to iron(V) states were faster (approximately 250 ns), consistent with processes that involve proton or electron movements but no changes in the iron coordination state.