Identification and characterisation of a novel family of copper storage proteins from Methylosinus trichosporium OB3b

Abstract

Methane oxidizing bacteria (MOB) use methane as their main source of carbon and energy. The main methane oxidizing enzyme in MOB is the copper-containing particulate methane monooxygenase (pMMO), a rare example of cytoplasmic copper enzyme. Some ‘switchover’ strains are capable of differentially expressing pMMO as well as a soluble iron-containing form (sMMO), and the switchover is regulated by copper. MOB secrete methanobactin (mb) which mediates copper uptake and is internalized in the cytoplasm. Despite this pathway for copper import, as well as copper regulating components such as CopA, CopZ and CueR being present in MOB, little is known on how these bacteria handle the large amounts of copper required for methane oxidation by pMMO. Through metalloproteomic analysis of soluble extracts from the switchover MOB M. trichosporium OB3b a large number of soluble copper pools were visualized and a novel copper protein, Csp1, was identified. Two more homologues, Csp2 and Csp3, were identified in M. trichosporium OB3b through bioinformatics. In vitro characterization of Csp1 and the homologue Csp3 showed these proteins are tetramers of 4-helix bundles that bind 13 and 18 Cu(I) ions per monomer, respectively, all of which are stored inside the core of the 4-helix bundle and are coordinated mostly by Cys residues. Csp1 binds tightly at least 10 Cu(I) ions whereas Csp3 has an average Cu(I) affinity at the order of 1017 M-1. Csp1 and Csp3 do not remove Cu(I) from Cu(I)-mb, however it is likely that apo-mb, which removes Cu(I) from these proteins, notably at very different rates, transports Cu(I) to pMMO. Csp1 is thought to be exported from the cytosol potentially to the intra-cytoplasmic membranes, where pMMO is localised, to store copper for the enzyme. Csp3 is thought to be cytosolic and either sequesters copper to prevent copper-induced toxicity or, more likely, supplies copper to unknown cytosolic copper enzymes, consistent with the large number of soluble copper pools visualized in the organism. Csp1 protein homologues are present in other bacteria, including MOB, while homologues of the cytosolic Csp3 are widely distributed in members of all major bacterial phyla. The presence of Csp3 in the bacterial cytosol implies either a function as a defense mechanism against copper-induced toxicity or more likely copper storage for supply to cytosolic copper enzymes, yet to be identified. The latter possibility challenges the present model according to which bacteria do not have a cytosolic requirement for copper.