Structural Insights into the Mechanism of Cytochrome c Peroxidase from Nitrosomonas europaea

Abstract

Cytochrome c peroxidase from Nitrosomonas europaea (NeCcP), a diheme bacterial peroxidase, differs from most bacterial cytochrome c peroxidases (bCcPs). Most bCcPs require an initial pre-reduction of the electron transfer site heme to become active. The reduction of the electron-transfer heme triggers a conformational change to open up the active site for these bCcPs. In contrast, NeCcP’s active site is open without any pre-reduction of the electron-transfer heme. Additionally, NeCcP appears to use a radical based mechanism that is similar to eukaryotic monoheme CcPs as opposed to most bCcPs that use a mechanism that does include the formation of a radical. However, the active site of NeCcP is conserved with other bCcPs and is not conserved with monoheme CcPs. bCcP active sites contain a conserved glutamate that is proposed to serve as the acid-base catalytic residue. In this study we validated E102 as the acid-base catalytic residue in NeCcP using X-ray crystallographic structures. These structural snapshots also revealed an ordered water that participates in proton transfer from the proximal peroxide oxygen to E102 and ultimately to the distal peroxide oxygen. Our structures also confirmed that the active site heme is likely a His-hydroxide ligated 6C heme rather than the His ligated 5C heme seen in previous structures of NeCcP. Finally, our structure revealed an increase in the distortion from planarity, for both the active site and electron-transfer heme, in the E102Q mutant. The increase in distortion was primarily due to an increase in the ruffling normal mode. Our structures also revealed key residues that may be involved in “tuning” the distortion of the hemes and communicating changes in distortion between the hemes.