Biochemical evidence of both copper chelation and oxygenase activity at the histidine brace

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.

The model structure of the copper-dependent ammonia monooxygenase

Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract

A copper site is required for iron transport by the periplasmic proteins P19 and FetP

An atypical five-coordinate copper site linked to an adjacent iron site is required for iron-dependent bacterial growth.

Biochemistry of Copper Site Assembly in Heme-Copper Oxidases: A Theme with Variations

Copper is an essential cofactor for aerobic respiration, since it is required as a redox cofactor in Cytochrome c Oxidase (COX). This ancient and highly conserved enzymatic complex from the family of heme-copper oxidase possesses two copper sites: CuA and CuB. Biosynthesis of the oxidase is a complex, stepwise process that requires a high number of assembly factors. In this review, we summarize the state-of-the-art in the assembly of COX, with special emphasis in the assembly of copper sites. Assembly of the CuA site is better understood, being at the same time highly variable among organisms. We also discuss the current challenges that prevent the full comprehension of the mechanisms of assembly and the pending issues in the field.