The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron

Kristīne Grāve,Martin Högbom,Matthew D Bennett,Julia J Griese,Gustav Berggren

doi:10.1007/s00775-020-01782-3

Kristīne Grāve, Martin Högbom + Show 3 more

Open Access

https://doi.org/10.1007/s00775-020-01782-3

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Abstract

Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving–Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with MnII and FeII in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving–Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.Graphical

Highlights

Achieving correct metallation in the complex environment of the cell is of paramount importance for the function of metalloproteins
While some systems compartmentalize metalloproteins with their required metals or employ specialized metallo-chaperones, divalent metal ion affinities to protein ligands are commonly described by the Irving–Williams series (MnII < FeII < CoII < NiII < CuII > ZnII) [1, 2]
During recent years it has been shown that protein scaffolds of R2-like ligand-binding oxidases (R2lox) and the radical-generating subunit in class Ic ribonucleotide reductases (R2c), both belonging to the ferritin superfamily, directly influence metal specificity by selectively forming a heterodinuclear Mn/Fe cofactor instead of an Fe/Fe metal center, apparently defying the Irving–Williams series [4, 5]

Summary

Introduction

Achieving correct metallation in the complex environment of the cell is of paramount importance for the function of metalloproteins. While some systems compartmentalize metalloproteins with their required metals or employ specialized metallo-chaperones, divalent metal ion affinities to protein ligands are commonly described by the Irving–Williams series (MnII < FeII < CoII < NiII < CuII > ZnII) [1, 2]. This order describes relative metal–ligand complex stability and is often an indirect indication of metal–ligand affinity. The protein scaffold can potentially control metallation by both steric effects and by influencing reactivity [4, 6]

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