PCu_AC domains from methane-oxidizing bacteria use a histidine brace to bind copper

Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain periplasmic copper-A chaperone (PCu_AC) domains. Using bioinformatics analyses, we identified three distinct classes of PCu_AC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCu_AC domains from other types of bacteria bind a single Cu(I) ion via an HX_nMX_21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCu_AC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCu_AC domain. Crystal structures of the PmoF1 and PmoF2 PCu_AC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX₁₀H motif. This binding site is distinct from those of previously characterized PCu_AC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCu_AC family reveals previously unappreciated diversity, including sequences that contain both the HX_nMX_21/22HXM and HX₁₀H motifs, and sequences that lack either set of copper-binding ligands. These findings provide the first characterization of an additional class of copper proteins from methanotrophs, further expand the PCu_AC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.