PCuC 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 eriplasmic opper-haperone (PCuC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuC domains from other types of bacteria bind a single Cu(I) ion via an HMHM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuC domain. Crystal structures of the PmoF1 and PmoF2 PCuC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HH motif. This binding site is distinct from those of previously characterized PCuC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuC family reveals previously unappreciated diversity, including sequences that contain both the HMHM and HH 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 PCuC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.