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

Abstract

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

Posttranscriptional Regulation by Copper with a New Upstream Open Reading Frame

“…Other genes frequently found in operons with bp2923 homologs code for putative proteins involved in Cu transport, binding, or homeostasis, including the copper chaperones ScoC and Pcu A C that participate in the assembly of heme-copper subunits of respiratory complexes ( 39 , 40 ), and the Cu-binding proteins YcnI and CopC ( 41 , 42 ). These operons also comprise MbnPH-like genes notably found in biosynthesis operons of copper-binding methanobactin-type molecules ( 43 , 44 ) and AhpC_TSA genes, whose products detoxify peroxides ( 45 ). Long intergenic distances between bp2923 homologs and the following genes are generally observed, indicating a potential role in regulation.…”

Post-transcriptional regulation by copper with a new upstream Open Reading Frame

“…Other genes frequently found in operons with bp2923 homologues code for proteins involved in Cu transport, binding or homeostasis, including the chaperones ScoC and Pcu A C that participate in the assembly of heme-copper subunits of respiratory complexes (Serventi et al, 2012, Trasnea et al, 2016), and the Cu-binding proteins YcnI and CopC (Damle et al, 2021, Lawton et al, 2016). Some of these operons also comprise MbnPH-like genes notably found in biosynthetic operons of Cu-binding methanobactin-type molecules (Kenney and Rosenzweig, 2018, Manesis et al, 2021), and AhpC_TSA genes, whose products detoxify peroxides (Dubbs and Mongkolsuk, 2007). Long intergenic distances between cruR homologues and the following genes are generally observed.…”

Capturing a bis-Fe(IV) State in Methylosinus trichosporium OB3b MbnH

“…Thus, the predicted ET rate for the hopping mechanism is again ∼10 6 times that for the single step tunneling reaction, suggesting that electron hopping is dominant in the MbnPH complex. Previous studies have shown that replacement of Trp176 with tyrosine does not affect the modification of Trp174, 23 suggesting that tyrosine can also support this electron/hole hopping pathway. Thus, MbnH uses tryptophan residues as hopping stations both between its two hemes and between heme 1 and the target of modification, Trp 174, in its substrate, MbnP.…”

“…The predicted pathway for oxidation includes MbnP Trp 176, a strictly conserved residue (Figure S18). 23 For a multistep hopping pathway, H AB for the first segment from Trp 174 to Trp 176 has a calculated value of 7.8 × 10 −4 with a β of 0.9 Å −1 . The second segment from Trp 176 to heme 1 has a calculated H AB of 1.9 × 10 −5 and a β of 1.24 Å −1 .…”

“…Further studies of MbnH, BthA (which uses a different ET pathway for delocalization), 27 and other family members should elucidate additional factors governing the formation and stabilization of bis-Fe(IV) intermediates. Moreover, MbnH is the only bis-Fe(IV)-forming enzyme besides MauG that has a confirmed oxidation target a distant tryptophan in MbnP 23 and, as such, provides an excellent platform for investigating long-range ET linked to complex chemical transformations.…”