Resonance Raman characterization of a high-spin six-coordinate iron(III) intermediate in metmyoglobin-azido complex formation trapped by microsecond freeze-hyperquenching (MHQ)

The reaction of metmyoglobin with azide was used to characterize a novel freeze-quench instrument with a mixing and freezing time resolution in the microsecond time range. Samples quenched within 95 and 245 μs after mixing of metmyoglobin with 1M azide were characterized by low-temperature UV-visible and resonance Raman spectroscopy with excitation into the Soret band. Comparison of these data with control samples where azide is absent or metmyoglobin is preincubated with azide demonstrates the formation of an intermediate complex in the first 95 μs after mixing that has fully decayed after 245 μs. Porphyrin skeletal modes displayed by this transient species identify it as a six-coordinate high-spin species. Minor low-spin components are also observed and suggest that the hexacoordinated intermediate exists as a spin equilibrium. This preliminary study demonstrates the feasibility of this approach to detect new intermediates and to characterize early reaction intermediates in other metalloproteins.