Direct Detection of Fe(IV)=O Intermediates in the Cytochrome aa3 Oxidase from Paracoccus denitrificans/H2O2 Reaction*

We report the first evidence for the formation of the “607- and 580-nm forms” in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607–580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin π-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm–1. The former two are assigned to the Fe(IV)=O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm–1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607–580-nm transition. We suggest that the 804–790 cm–1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm–1 mode has been found for the first time to accumulate with the 804 cm–1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.