Insight into Metal Removal from Peptides that Sequester Copper for Methane Oxidation

Abstract

Methanobactins (Mbns) are modified peptides that sequester copper (Cu) methanotrophs use to oxidize methane. Limited structural information is available for this class of natural products, as is an understanding of how cells are able to utilize Mbn‐bound Cu. The crystal structure of Methylosinus sporium NR3K CuI–Mbn provides further information about the structural diversity of Mbns and the first insight into their Cu‐release mechanism. Nitrogen ligands from oxazolone and pyrazinediol rings chelate CuI along with adjacent coordinating sulfurs from thioamides. In vitro solution data are consistent with a CuI–Mbn monomer as found for previously characterized Mbns. In the crystal structure, the N‐terminal region has undergone a conformational change allowing the formation of a CuI 2–Mbn2 dimer with CuI sites bound by chelating units from adjacent chains. Such a structural alteration will facilitate CuI release from Mbns.

The Coordination Chemistry of Copper Uptake and Storage for Methane Oxidation

“…Methanobactins have a strong preference for Cu I , and the addition of Cu II to an apo‐Mbn results in the rapid formation of the Cu I form, although the mechanism of reduction is not known. For those Mbns whose Cu I affinities have been measured, values in the 10 20 –10 21 m −1 range are found, some of the highest known for biological Cu sites . Due to their susceptibility to reduction, the stability constants of Cu II ‐Mbns have been calculated from the reduction potential ( E m value) of a Cu‐Mbn, along with its Cu I affinity.…”

Bacterial copper storage proteins

“…Related Mbn operons are present in some non-methanotrophic bacteria (17,51,59). Work in our lab has found that Mbns bind Cu(I) with affinities in the 10 21 M -1 range, and have Cu(II) affinities that are ~ 6-10 orders of magnitude weaker (52,54,60). We suggested oxidation could assist removal of the metal ion in cells (52), although a conformational change at the Nterminus of the peptide now appears to be the most likely mechanism to promote release (60).…”

“…However, the observation that Csp3 is present alone in 22 methanotrophs (Csp1 is rarely present on its own) could indicate the function of this protein is independent of Csp1 and Mbn (see section about the functions of Csps). Much attention has been paid to the role of Mbn in Cu acquisition and utilization by methanotrophs (17,(50)(51)(52)(54)(55)(56)(57)(58)(59)(60). However, the currently identified Mbn operon is only found in just over 10% of sequenced methanotroph genomes, suggesting other systems are important for these processes.…”

“…Work in our lab has found that Mbns bind Cu(I) with affinities in the 10 21 M -1 range, and have Cu(II) affinities that are ~ 6-10 orders of magnitude weaker (52,54,60). We suggested oxidation could assist removal of the metal ion in cells (52), although a conformational change at the Nterminus of the peptide now appears to be the most likely mechanism to promote release (60). Having characterized a range of Mbns isolated from spent media in which methanotrophs were grown at low Cu concentrations (52,54,60), including determination of their high-resolution crystal structures (Fig.…”

“…We suggested oxidation could assist removal of the metal ion in cells (52), although a conformational change at the Nterminus of the peptide now appears to be the most likely mechanism to promote release (60). Having characterized a range of Mbns isolated from spent media in which methanotrophs were grown at low Cu concentrations (52,54,60), including determination of their high-resolution crystal structures (Fig. 1, A and B) and analysis of the Cu(I)-Mbn uptake process (Fig.…”