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

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 Mn II and Fe II 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 AbstractElectronic supplementary material The online version of this article (https ://doi.

Metalation: nature’s challenge in bioinorganic chemistry

“…In this issue [1], Grāve, Högbom and colleagues address a tremendously important challenge: How do proteins acquire the correct metals? This is important because almost a half of enzymes are estimated to require metals [2,3].…”

“…In response to oxidative stress, a sensor triggers expression of manganese import, manganese availability rises and nascent SOD becomes correctly populated with manganese [8][9][10]. The question addressed by Högbom and colleagues [1], concerning discernment between manganese and iron, is especially pertinent.…”

“…An inherent specificity of a class Ib ribonucleotide reductase (RNR) for manganese over iron is intriguing, because, in the first instance, this seemingly defies the Irving-Williams series [1]. In bacteria, two classes of RNR show divergent functional requirements for metal ions: class Ia enzymes are functionally dependent on iron while class Ib enzymes are manganese-dependent [11].…”

“…Given the above understanding of how specificity arises in class Ia and Ib RNRs through oxidation and the involvement of accessory factors in that process [13,14], the finding that class Ib RNRs can exhibit inherent selectivity for Mn(II) without any accessory factors is intriguing, although oxidation to Mn(III) likely still plays a part [1]. The inherent flexibility of amino acid side chains is a hurdle to generating binding sites that show steric preference for a single metal ion.…”

“…Ligating side chains and water molecules are shown for bound Mn 2+ (pink spheres) and Fe 2+ (orange spheres) along with green dashes to indicate bonding interactions (< 2.3 Å). Note that the coordination geometry for Mn 2+ is hexacoordinate in each site, while for iron the same set of residues contribute to tetra-and pentacoordinate geometries (PDB 3N37 and 3N38 using PyMOL, Schrödinger, LLC) for example, introduce steric selection and changes in oxidation state for residually challenging combinations of metals such as iron and manganese [1]. The thermodynamic framework in Fig.…”