Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches

da Silva, Ronivaldo Rodrigues; Adedoyin, Victoria; DuBois, Jennifer L.

doi:10.1007/978-1-0716-4043-2_7

Methods in Molecular Biology

2024

DOI: 10.1007/978-1-0716-4043-2_7

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Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches

Ronivaldo Rodrigues da Silva,

Victoria Adedoyin,

Jennifer L. DuBois

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Cited by 1 publication

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How the microbiome liberates iron from heme

Nath,

da Silva,

Gauvin

et al. 2024

Preprint

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The microbiome is a hidden organ system with diverse metabolic roles. Using Bacteroides thetaiotaomicron (B theta) as a model, we asked how microbiome species process iron, the quintessential biometal supporting respiration and life as we know it, when iron is provided in complex with protoporphyrin IX (PPIX) as heme. Canonical pathways for reclaiming heme-iron from host cells or the diet require O2, which is unavailable in the GI tract and many pathological microenvironments. HmuS, a homolog of cobalamine- (vitamin B12) and chlorophyll-building chelatases, is widespread in anaerobic microbial ecosystems. In this work, we provide direct physiological, biochemical, and structural evidence for the anaerobic removal of iron from heme by HmuS, a de-chelatase that deconstructs heme to PPIX and Fe(II). We show that heme can be used as a sole iron source by B theta, yielding PPIX, and that hmuS inactivation under these conditions is lethal. Iron removal from heme depends on the B theta membrane fraction, NADH, and O2 exclusion. Absorbance spectra indicate that heterologously expressed HmuS is isolated with a non-substrate heme bound and can accommodate multiple heme equivalents under saturating conditions. Solution of the cryoEM structure reveals heme and two cations at sites that are conserved across the HmuS family and the chelatase superfamily, respectively. The proposed structure-based mechanism for iron removal further links biosynthetic and biodegradative pathways for heme, chlorophyll, and vitamin B12, 3 ancient and biologically crucial metallocofactors.

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How the microbiome liberates iron from heme

Nath,

da Silva,

Gauvin

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

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Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches

Cited by 1 publication

References 13 publications

How the microbiome liberates iron from heme

How the microbiome liberates iron from heme

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