UC Irvine

Our current understanding of biological C-H bond activation in heme enzymes points to an important role of basic ferryl intermediates in how Nature supports this chemistry. Among heme proteins, only the thiolate-ligated enzymes have been demonstrated to react with unactivated C-H bonds. Strong electron donation from the proximal thiolate ligand results in a basic Compound II species, an iron(IV) hydroxide. This is important for balancing the reduction potential of Compound I and avoiding deleterious autoxidation of the protein superstructure. However, recent neutron crystallography experiments point to a basic ferryl species present in a histidine-ligated heme enzyme as well, which have been previously shown to only support unprotonated oxos. Our work demonstrates that ascorbate peroxidase, the histidine-ligated heme enzyme, really has an unprotonated iron(IV) oxo Compound II species (APX-II). We further examine the iron(III) hydroxide state of ascorbate peroxidase in order to better understand the reported crystal structure and what it represents. We conclude with a vibrational spectroscopic method to observe the Fe-O bond in APX-II and assign it to an Fe(IV) to a doubly bound oxygen atom, split between multiple vibrational modes resulting in apparent stretching frequencies of 770 and 732 cm-1 , or 1.67 and 1.69 Å, respectively. This is well within range of what is expected for an iron(IV) oxo