Crystallographic and Single-Crystal Spectral Analysis of the Peroxidase Ferryl Intermediate

The ferryl [Fe(IV)O] intermediate is important in many heme enzymes, and thus, the precise nature of the Fe(IV)−O bond is critical in understanding enzymatic mechanisms. The 1.40 Å crystal structure of cytochrome c peroxidase Compound I has been determined as a function of X-ray dose while the visible spectrum was being monitored. The Fe−O bond increases in length from 1.73 Å in the low-X-ray dose structure to 1.90 Å in the high-dose structure. The low-dose structure correlates well with an Fe(IV)═O bond, while we postulate that the high-dose structure is the cryo-trapped Fe(III)−OH species previously thought to be an Fe(IV)−OH species.