Two-state reactivity, electromerism, tautomerism, and "surprise" isomers in the formation of compound II of the enzyme horseradish peroxidase from the principal species, compound I

QM and QM/MM calculations on Compound II, the enigmatic species in the catalytic cycle of the horseradish peroxidase enzyme, reveal six low-lying isomers. The principal isomer is the triplet oxoferryl form (PorFe ^IV=O) that yields the hydroxo-ferryl isomer (PorFe ^IV-OH⁺). These are the only forms observed in experimental studies. Theory shows, however, that these are the least stable isomers of Compound II. The two most stable forms are the singlet and triplet states of the Por^+.Fe^III-OH electromer. In addition, theory reveals species never considered in heme enzymes: the singlet and triplet states of the Por^+.Fe^III-OH₂ electromer. The computational results reproduce the experimental features of the known isomers and enable us to draw relationships and make predictions regarding the missing ones. For example, while the "surprise" species, singlet and triplet Por ^+.Fe^III-OH₂, have never been considered in heme chemistry, the calculated Fe-O bond lengths indicate that these isomers may have, in fact, been observed in one of the two opposing EXAFS studies reported previously. Furthermore, these ferric-aqua complexes could be responsible for the reported ¹⁸O exchange with bulk water. It is clear, therefore, that the role of Compound II in the HRP cycle is considerably more multi-faceted than has been revealed so far. Our suggested multi-state reactivity scheme provides a paradigm for Compound II species. The calculated Mössbauer parameters may be helpful toward eventual characterization of these missing isomers of Compound II.