MbnH is a diheme MauG-like protein associated with microbial copper homeostasis

Abstract

Edited by Ruma Banerjee Methanobactins (Mbns) are ribosomally-produced, post-translationally modified peptidic copper-binding natural products produced under conditions of copper limitation. Genes encoding Mbn biosynthetic and transport proteins have been identified in a wide variety of bacteria, indicating a broader role for Mbns in bacterial metal homeostasis. Many of the genes in the Mbn operons have been assigned functions, but two genes usually present, mbnP and mbnH, encode uncharacterized proteins predicted to reside in the periplasm. MbnH belongs to the bacterial diheme cytochrome c peroxidase (bCcP)/MauG protein family, and MbnP contains no domains of known function. Here, we performed a detailed bioinformatic analysis of both proteins and have biochemically characterized MbnH from Methylosinus (Ms.) trichosporium OB3b. We note that the mbnH and mbnP genes typically co-occur and are located proximal to genes associated with microbial copper homeostasis. Our bioinformatics analysis also revealed that the bCcP/MauG family is significantly more diverse than originally appreciated, and that MbnH is most closely related to the MauG subfamily. A 2.6 Å resolution structure of Ms. trichosporium OB3b MbnH combined with spectroscopic data and peroxidase activity assays provided evidence that MbnH indeed more closely resembles MauG than bCcPs, although its redox properties are significantly different from those of MauG. The overall similarity of MbnH to MauG suggests that MbnH could post-translationally modify a macromolecule, such as internalized CuMbn or its uncharacterized partner protein, MbnP. Our results indicate that MbnH is a MauG-like diheme protein that is likely involved in microbial copper homeostasis and represents a new family within the bCcP/MauG superfamily. Natural products that sequester and import vital and toxic metal ions play important roles in maintaining metal homeostasis in many species. Most well-studied are bacterial small molecules that bind ferric iron and are known as siderophores (iron "carriers") (1). In recent years, similar molecules that bind other metals have been discovered and characterized (2). One of these is a family of copper-binding compounds called methanobactins (Mbn) 5 (3, 4), which are produced from ribosomally synthesized peptides. All Mbns contain post-translational modifications that include nitrogen-containing heterocycles (oxazolones and pyrazinedione/diols) and neighboring thioamide/enethiol groups, and many have other less widespread modifications including "N-terminal" carbonyl groups (5, 6), intramolecular disulfide bonds (3, 6), and sulfonated threonines (7, 8). Of these functional groups, the N-heterocycles and thioamides provide ligands that chelate a copper ion. Mbns bind both Cu I and Cu II with high affinity (binding constants of 10 19-21 M Ϫ1 for Cu I and 10 11-14 M Ϫ1 for Cu II) (7-9); upon binding, the latter is quickly reduced to Cu I via an unknown mechanism. Under copper-limited conditions, some methanotrophs, bacteria that metabolize methane under...

Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

“…1B). These mbnPH pairs are not only present in Mbn operons but also found in proximity to genes that encode a range of copper-binding proteins and membrane transporters, including CopC, CopD, and PCu A C. Three mbnPH pairs are found in the methanotroph Methylosinus (Ms.) trichosporium OB3b, one in the Mbn operon, and two in additional operons encoding MbnT, MbnP, and MbnH; all are regulated by copper (7,10,16,20).…”

“…MbnH is a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG family (PF03150) and belongs to a distinct and divergent group within a broader subfamily that encompasses MauG-like diheme enzymes (16). In MauG, dual heme groups, one high-spin and one low-spin, generate a highly reactive bis-Fe IV catalytic intermediate that oxidizes two tryptophan residues in the methylamine dehydrogenase (MADH) precursor protein (preMADH) to form a tryptophan tryptophylquinone cofactor (17).…”

“…In MauG, rapid electron transfer between the two heme groups through an intervening tryptophan residue stabilizes the bis-Fe IV species, a phenomenon called CR stabilization (17)(18)(19). In addition to CR stabilization, MbnH may also be similarly capable of forming a bis-Fe IV intermediate upon oxidation with peroxide (16), but the reactivity and potential substrate of this intermediate have not been established.…”

“…In contrast to MbnH, MbnP does not belong to a known protein family and contains no recognizable domains (16). Sequence analysis predicts that MbnP proteins are periplasmic, with six highly conserved cysteines, two highly conserved histidines, and a highly conserved WxW motif (Fig.…”

“…Sequence analysis predicts that MbnP proteins are periplasmic, with six highly conserved cysteines, two highly conserved histidines, and a highly conserved WxW motif (Fig. 1 A and D) (16). There is a strong genetic association of MbnP and MbnH, with ∼88.1% of…”