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Publication Abstract from PubMed

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) 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 O2, but prefers manganese in the presence of O2, 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 O2, 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 O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase.,Griese JJ, Kositzki R, Haumann M, Hogbom M J Biol Inorg Chem. 2019 Mar;24(2):211-221. doi: 10.1007/s00775-019-01639-4. Epub , 2019 Jan 28. PMID:30689052^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.