Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an Fe-IV=O or Fe-IV-OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated Fe-IV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 angstrom and 1.50 angstrom crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 angstrom) is notably shorter than in APX (1.87 angstrom). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.
This work was supported by BBSRC grants BB/N015940/1 and BB/S020586/1 and travel grants from Diamond Light Source to P.C.E.M. and E.L.R. EPR experiments were carried out in the EPSRC National EPR Research Facility & Service (NS/A000055/1). We acknowledge access to SACLA (experiments (2018A8009, 2019B8009, 2020A8030) and JSPS for support (grant JP20H05452 to H.S. and JP19H05783 to M.Y.).
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