Abstract
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-Achaperone (PCuAC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuAC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3. PCuAC domains from other types of bacteria bind a single Cu(I) ion via an HXnMX21/22HXM motif, which is also present in PmoF3, but PmoF1 and PmoF2 lack this motif entirely. Instead, the PCuAC domains of PmoF1 and PmoF2 bind only Cu(II), and PmoF1 binds additional Cu(II) ions in a His-rich extension to its PCuAC domain. Crystal structures of the PmoF1 and PmoF2 PCuAC 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 PCuAC domains but resembles copper centers in CopC proteins and lytic polysaccharide monooxygenase (LPMO) enzymes. Bioinformatics analysis of the entire PCuAC family reveals previously unappreciated diversity, including sequences that contain both the HXnMX21/22HXM 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 PCuAC family, and afford new insight into the biological significance of histidine brace-mediated copper coordination.
Highlights
Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase, is copper-dependent
Crystal structures of the PmoF1 and PmoF2 PCuAC domains reveal that Cu(II) is coordinated by an N-terminal histidine brace HX10H motif
To identify PCuAC domain-containing proteins in methanotrophs, we searched all methanotroph genomes deposited in the IMG-JGI database for genes encoding proteins containing a PCuAC domain
Summary
To identify PCuAC domain-containing proteins in methanotrophs, we searched all methanotroph genomes deposited in the IMG-JGI database for genes encoding proteins containing a PCuAC (pfam04314) domain. The addition of 0 –5 Cu(II) equivalents indicates PmoF1-⌬C binds 0.75 Ϯ 0.3 Cu(II) ions (Fig. 2A) Together, these data suggest that the His-rich extension, which in Methylocystis sp. Rockwell PmoF1 includes 7 histidine residues (Fig. S2), contains additional copper-binding sites. It is possible that the His-rich extension in PmoF1-FL binds periplasmic copper and delivers it to the PCuAC domain or to other protein targets. PmoF2 homologs contain a C-terminal extension to the PCuAC domain Many of these extensions include two histidine residues in the equivalent part of the sequence to the PmoF1 His-rich region (Fig. S1D). Data collection Wavelength (Å) Space group Cell dimensions a, b, c (Å) ␣, , ␥ (degrees)
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