Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Chacón, Kelly N.; Perkins, Jonathan C; Mathe, Zachary; Alwan, Katherine B.; Ho, Ethan N.; Ucisik, Melek N.; Merz, Kenneth M.; Blackburn, Ninian J.

doi:10.1038/s42003-018-0181-9

Cited by 22 publications

(28 citation statements)

References 50 publications

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“…In that system, the small periplasmic protein CusF acts as a metallochaperone, delivering Cu + or Ag + to CusB 46,47 . The mechanism involves the nearly diffusion limited formation of a CusB/CusF complex with shared ligation to the copper ion 48,49 . Whether AztD and AztC form a similar intermediate with shared ligation to the zinc is uncertain from our data.…”

Section: Discussionmentioning

confidence: 99%

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins

Neupane¹,

Fullam²,

Chacón³

et al. 2019

Commun Biol

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Zinc acquisition from limited environments is critical for bacterial survival and pathogenesis. AztD has been identified as a periplasmic or cell surface zinc-binding protein in numerous bacterial species. In Paracoccus denitrificans , AztD can transfer zinc directly to AztC, the solute binding protein for a zinc-specific ATP-binding cassette transporter system, suggesting a role in zinc acquisition and homeostasis. Here, we present the first cry stal structures of AztD from P. denitrificans and tbe human pathogen Citrobacter koseri , revealing a beta-propeller fold and two high-affinity zinc-binding sites that are highly conserved among AztD homologs. These structures combined with transfer assays using WT and mutant proteins provide rare insight into the mechanism of direct zinc transfer from one protein to another. Given the importance of zinc import to bacterial pathogenesis, these insights may prove valuable to the development of zinc transfer inhibitors as antibiotics.

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Section: Discussionmentioning

confidence: 99%

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins

Neupane¹,

Fullam²,

Chacón³

et al. 2019

Commun Biol

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“…Rather, the HO2 model appears to be more mechanistically and functionally similar to the transfer of copper from CusF to CusB, components of the Escherichia coli efflux pump. In that system, the rate of complex formation between CusF and CusB is fast and transfer of copper is slow, proceeding at a rate of 0.015 s -1 (44). When copper is transferred from CusF to CusB, the efflux pump is activated; when copper is transferred back to CusF from CusB as metal concentrations decrease, the efflux pump is deactivated (45).…”

Section: Core-ho2mentioning

confidence: 99%

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Fleischhacker

Gunawan

Kochert

et al. 2020

Journal of Biological Chemistry

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Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

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“…Spectral simulation was carried out by least-squares curve fitting, using full curved wave calculations as formulated by the program EXCURVE 9.2 as previously described. 15,58,59 Fourier-Transform Infrared Spectroscopy.-Purified CusF was concentrated to approximately 1 mM and transferred to an airtight conical vial in an anaerobic COY chamber. Samples were purged with a gentle stream of CO for 5 min and allowed to incubate for 10 min.…”

Section: Spectroscopic Data Collection and Processingmentioning

confidence: 99%

Rational Design of a Histidine–Methionine Site Modeling the M-Center of Copper Monooxygenases in a Small Metallochaperone Scaffold

et al. 2019

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Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine βmonooxygenase (DBM) catalyze the hydroxylation of high energy C-H bonds utilizing a pair of chemically distinct copper sites (CuH and CuM) separated by 11 Å. In earlier work, we constructed single-site PHM variants that were designed to allow study of the M-and H-centers independently in order to place their reactivity sequentially along the catalytic pathway. More recent crystallographic studies suggest that these single-site variants may not be truly representative of the individual active sites. In this work we describe an alternative approach that uses rational design to construct an artificial PHM model in a small metallochaperone scaffold. Using site-directed mutagenesis, we constructed variants that provide a His 2 Met copper-binding ligand set that mimics the M-center of PHM. The results show that the model accurately reproduces the chemical and spectroscopic properties of the M-center, including details of the methionine coordination, and the properties of Cu(I) and Cu(II) states in the presence of endogenous ligands such as CO and azide. The rate of reduction of the Cu(II) form of the model by the chromophoric reductant N,N'-dimethyl phenylenediamine (DMPD) has been compared with that of the PHM M-center, and the reaction chemistry of the Cu(I) forms with molecular oxygen has also been explored, revealing an unusually low reactivity towards molecular oxygen. This latter finding emphasizes the importance of substrate triggering of oxygen reactivity, and implies that the His 2 Met ligand set while necessary, is insufficient on its own to activate oxygen in these enzyme systems.

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Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Cited by 22 publications

References 50 publications

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Rational Design of a Histidine–Methionine Site Modeling the M-Center of Copper Monooxygenases in a Small Metallochaperone Scaffold

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