Copper-Binding Properties and Structures of Methanobactins from Methylosinus trichosporium OB3b

Abstract

Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ∼9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (∼ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.

Methanobactin reverses acute liver failure in a rat model of Wilson disease

“…MB is a posttranslationally modified peptide from the methanotrophic proteobacterium Methylosinus trichosporium OB3b, with a MW of 1,154 Da (metal free) and an exceptionally high copper affinity (23)(24)(25). MB (Supplemental Figure 2A; supplemental material available online with this article; doi:10.1172/JCI85226DS1) Figure 1A).…”

Bacterial copper storage proteins

“…Comparative Cu(I) removal studies have also been carried out with well-characterized chromophoric Cu(I) chelating molecules such as bicinchoninic acid, and particularly bathocuproine disulfonate (BCS). These ligands have routinely been used to measure how tightly Cu(I) binds to a range of proteins (26,(68)(69)(70)(71), including determination of the Cu(I) affinities of Mbns (52,54) and the average values for the Csps (35,36). They have also been implemented as model acceptors for investigating Cu(I) removal from homeostasis proteins (68,72).…”

“…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.…”

“…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).…”

“…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.…”