Abstract
Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.
Highlights
R2-like ligand-binding oxidases (R2lox) can assemble a Mn/Fe or diiron cofactor
We have recently shown that the R2lox protein itself can select for manganese in site 1 and iron in site 2, thereby assembling the heterodinuclear Mn/Fe cofactor [22], but it is not clear whether the coordination geometry in R2lox depends on metal identity
A Mixture of Hydroxylated Long-chain Fatty Acids Copurifies with R2lox—A striking feature of R2lox proteins is the hydrophobic tunnel extending from the active site to the protein surface in which a long-chain fatty acid is bound that copurifies with the protein from the heterologous expression host [21, 22]
Summary
R2-like ligand-binding oxidases (R2lox) can assemble a Mn/Fe or diiron cofactor. Results: The metal centers are structurally similar and activate oxygen, resulting in redox-coupled structural changes. Whereas class Ia and Ib R2 proteins carry the stable radical on a tyrosine residue close to the metal cluster [10, 11, 13, 18, 19], this tyrosine residue is replaced by a redox inert phenylalanine in class Ic [20], and the radical equivalent is instead stored in form of the MnIV/FeIII state of the cofactor [14] Even more recently, another group of ferritin-like proteins utilizing a Mn/Fe cofactor was discovered in which the R2 scaffold is remodeled to house a hydrophobic ligand-binding channel [21,22,23]. Structural models for the reduced and oxidized homo- and heterodinuclear metal centers are derived that provide insight into the oxygen activation mechanism
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