Time-Resolved Investigations of Heterobimetallic Cofactor Assembly in R2lox Reveal Distinct Mn/Fe Intermediates

Miller, Effie K.; Trivelas, Nicholas E.; Maugeri, Pearson T.; Blaesi, Elizabeth J.; Shafaat, Hannah S.

doi:10.1021/acs.biochem.7b00403

Cited by 19 publications

(66 citation statements)

References 47 publications

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“…The different electronic structures and redox potentials of the two cofactors are expected to impact their catalytic potential and lead to different reactivities. Observations on the closely related cofactor of R2c and quantum chemical calculations also suggest that the Mn/Fe cofactor is more stable in the high-valent IV/IV redox state than a diiron cluster [22, 23, 28, 38–44]. Thus, the Mn/Fe cofactor may be more efficient in initiating cross-link formation, as corroborated by the data presented here.…”

Section: Resultssupporting

confidence: 83%

“…Primary or secondary carbon atoms, however, cannot be oxidized by the Mn/Fe cofactor of R2lox, likely because the resulting alkyl radical would be too unstable [23, 28, 38, 39]. …”

Section: Resultsmentioning

confidence: 99%

“…Cross-link formation has been proposed to be initiated from the Mn IV /Fe IV state of the cofactor formed by oxygen reduction by abstraction of an electron from the tyrosine, with the radical subsequently transferred to the valine [23]. Alternatively, electron transfer may occur directly from the valine [28]. A second electron transfer to the metal center results in a tertiary valine carbocation, and the cross-link is formed by nucleophilic attack of the phenolic oxygen of Y162, leaving the cofactor in the observed stable resting Mn III /Fe III state [23, 28].…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, electron transfer may occur directly from the valine [28]. A second electron transfer to the metal center results in a tertiary valine carbocation, and the cross-link is formed by nucleophilic attack of the phenolic oxygen of Y162, leaving the cofactor in the observed stable resting Mn III /Fe III state [23, 28].

Fig.…”

Section: Introductionmentioning

confidence: 99%

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Ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2018

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R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine–valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.Electronic supplementary materialThe online version of this article (10.1007/s00775-018-1583-3) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 83%

“…Primary or secondary carbon atoms, however, cannot be oxidized by the Mn/Fe cofactor of R2lox, likely because the resulting alkyl radical would be too unstable [23, 28, 38, 39]. …”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fig.…”

Section: Introductionmentioning

confidence: 99%

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Ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2018

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“…In contrast to R2c, R2lox performs two-electron redox chemistry, forming a tyrosine-valine ether cross-link in the vicinity of its metal binding site during cofactor maturation [4,23]. Cofactor maturation in R2c and R2lox proteins appears to follow a common pathway [4,[24][25][26][27][28][29], but the assembly pathways of the metal sites differ [4,5,30]. The physiological role of R2lox proteins is yet to be discovered.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

et al. 2022

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R2‐like ligand‐binding oxidase (R2lox) is a ferritin‐like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross‐link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry‐based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

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Time-Resolved Investigations of Heterobimetallic Cofactor Assembly in R2lox Reveal Distinct Mn/Fe Intermediates

Cited by 19 publications

References 47 publications

Ether cross-link formation in the R2-like ligand-binding oxidase

Ether cross-link formation in the R2-like ligand-binding oxidase

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

Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

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