Rapid Decay of the Native Intermediate in the Metallooxidase Fet3p Enables Controlled Fe^II Oxidation for Efficient Metabolism

Abstract

The multicopper oxidases (MCOs) couple four 1e – oxidations of substrate to the 4e – reduction of O2 to H2O. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s–1 and those that oxidize metal ions with low TOFs, ∼1 s–1 or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the native intermediate (NI), which only slowly decays to the resting form. Here, we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development

“…Despite a similar configuration of the Fe 2+ -binding and electron-transfer sites near the T1 Cu center, the topology of the Fe 2+ -binding pocket greatly differs between LPR1 and yeast Fet3p ( Figures S7 B and S7C). 48

Figure 6 Land plant progenitors acquired LPR1-type ferroxidase from soil bacteria (A) Maximum-likelihood midpoint-rooted phylogenetic tree of 188 annotated multicopper oxidase (MCO) proteins (UniProt Knowledge Database). Group I: Fet3p-related ferroxidases and fungal laccases (clade Ia, gray); plant laccases and ascorbate oxidases (clade Ib, green).

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“… 34 , 61 Guided by the high homology of the LPR1 model with the experimental structure of Bacillus CotA laccase, a 3d-MCO, we focused our cladistic analysis on 3d-MCO proteins, the largest group in the MCO superfamily, 32 and conducted structure-function studies ( Figures 1 and S3 ). Although LPR1 and Fet3p share similar catalytic parameters and an equivalent Fe 2+ -binding and adjoining T1 Cu site, 48 LPR1 typifies an ancient ferroxidase cohort that emerged early during bacterial land colonization ( Figure 7 A). The hallmark of LPR1-type ferroxidases, which are only distantly related to yeast ferroxidases and plant laccases ( Figure 6 E), is a distinctively configured Fe 2+ -binding site that possibly gave rise to the organic substrate-binding pocket in CotA-like MCOs ( Figure 6 C; Data S3 ).…”

Self-Luminescent Lanthanide Metal–Organic Frameworks as Signal Probes in Electrochemiluminescence Immunoassay

“…We also found from the HRTEM images that iron tended to aggregate into nanoparticles and adhered to the surface of ultrathin nickel nanosheets when the ratio of Ni/Fe was 1:1. 19,22,23 The iron nanoparticles owned almost uniform and small particle sizes, as shown in Figure 1D. The HRTEM image in Figure 1E expressly showed the structure state of iron particles attached to the ultrathin nickel nanosheet, and the regular lattice (0.1 nm) structure could be clearly discerned.…”

Bacterial-type plant ferroxidases tune local phosphate sensing in root development

“…Metal oxidases including ferroxidases related to Fet3p (limited to Fungi) or ceruloplasmin (limited to Animalia) form a major group within the ancient MCO superfamily whose diverse members are widely distributed in all domains of life (Janusz et al, 2020). Although LPR1 and Fet3p share similar catalytic parameters as well as an analogous architecture of the Fe 2+ -binding and adjoining T1 Cu site (Jones et al, 2020), our study suggests that LPR1-type MCOs displaying ferroxidase activity emerged very early during bacterial land colonization. LPR1-type ferroxidases (or their MCO progenitors) possibly crossed bacterial phyla multiple times to diversify by lateral gene transfer, which is widespread among soil bacteria (Ochman et al, 2000; Klumper et al, 2015).…”