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Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.ORCID iD: 0000-0003-3686-3062
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-5574-9383
Number of Authors: 42019 (English)In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 2, p. 211-221Article in journal (Refereed) Published
Abstract [en]

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O-2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O-2, but prefers manganese in the presence of O-2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O-2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O-2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.

Place, publisher, year, edition, pages
2019. Vol. 24, no 2, p. 211-221
Keywords [en]
Di-metal carboxylate protein, Ferritin, Ribonucleotide reductase, R2-like ligand-binding oxidase, X-ray crystallography
National Category
Biological Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-167623DOI: 10.1007/s00775-019-01639-4ISI: 000460591600006PubMedID: 30689052OAI: oai:DiVA.org:su-167623DiVA, id: diva2:1304382
Available from: 2019-04-12 Created: 2019-04-12 Last updated: 2019-04-12Bibliographically approved

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Griese, Julia J.Högbom, Martin
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