Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).
Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO)1,2. Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper3,4. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock5,6. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location.
The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.
Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.
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.
Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10 21 M −1. The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the ironcontaining soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methaneoxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms. C opper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1,(3)(4)(5)(6)(7)(8). Although copper uptake by eukaryotes is well defined (1, 4, 9), acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10-18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper (19), primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane (19), an important greenhouse gas. A subclass of "switchover" organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copperdeficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio (15, 21).Methanobactin production has been examined in a number of methane-oxidizing bacteria (22-24), but mbtins from only two organisms have been characterized (13,18). The mbtin (two forms) from Methylosinus trichosporium OB3b (a switchover organism) is the most extensively studi...
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