XFEL Crystal Structures of Peroxidase Compound II.

Hanna Kwon,Hiroshi Sugimoto,Mahdi Hussain,Natalia Stefanou,Jaswir Basran,Hazel A Sparkes,Kensuke Tono,Hideo Ago,Alistair J Fielding,Kunio Hirata,Keitaro Yamashita,Yoshitsugu Shiro,Samuel L Freeman,Hitomi Sawai,Go Ueno,Chinar Pathak,Masaki Yamamoto,Takehiko Tosha,Hideo Murakami ,E.l Raven ,A Bailey ,Peter C.e Moody

doi:10.1002/anie.202103010

Abstract

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 FeIV=O or FeIV‐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 FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å 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 Å) is notably shorter than in APX (1.87 Å). 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.

Highlights

A large number of heme enzymes use ferryl heme during catalysis
The majority of the early spectroscopy on these intermediates was carried out on one of the more experimentally amenable peroxidases, or on myoglobin, each of which contain a histidine as proximal ligand
We present X-ray free electron lasers (XFEL) structures of Compound II in two different peroxidases—cytochrome c peroxidase and ascorbate peroxidase

Summary

Introduction

A large number of heme enzymes use ferryl heme during catalysis. Ferryl heme refers broadly to a highly oxidized form of the heme in which the iron is in an oxidation state that is either one equivalent (FeIV, known as Compound II) or two equivalents (formally FeV, known as Compound I) above the resting oxidation state (FeIII). The second oxidizing equivalent in Compound I resides on the porphyrin ring or, in some cases, on a protein radical.[1] These Compound I and Compound II species were identified in the late 1930s[2] and were later given their names.[3] Because these intermediates are used in such a wide range of catalytic enzymes, they continue to attract attention these many years later.[4] The chemical nature of the ferryl species has been a matter of intense interest. The discussion has often focussed on whether the ferryl species is best formulated as an unprotonated FeIV=O or a protonated FeIV-OH species. This matters because the protonation state controls the reactivity. The functional implications of this finding, in terms of proton delivery to the heme, are discussed

Results

Discussion

Conflict of interest