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Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins

DOI: 10.1074/jbc.RA120.014502 DOI Help

Authors: Cecilia Piergentili (Newcastle University) , Jennifer Ross (University of Edinburgh) , Didi He (University of Edinburgh) , Kelly J. Gallagher (University of Edinburgh) , Will A. Stanley (Newcastle University) , Laurène Adam (Newcastle University) , C. Logan Mackay (University of Edinburgh) , Arnaud Basle (Newcastle University) , Kevin J. Waldron (Newcastle University) , David J. Clarke (Newcastle University) , Jon Marles-wright (Newcastle University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: September 2020
Diamond Proposal Number(s): 9487

Abstract: Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and neighboring secondary metal binding sites. We have previously identified a putative metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Journal Keywords: Encapsulated ferritin; encapsulin; ferroxisase; native mass spectrometry; iron mass spectrometry (MS); X-ray crystallography; ferritin; iron metabolism

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography