Dataset Information

LCMS quantification of internal crosslink in WT and G68L mutant R2lox proteins from Geobacillus kaustophilus.

ABSTRACT: A heterobimetallic Mn/Fe cofactor is found in the R2 subunit of class Ic ribonucleotide reductases (R2c) and R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (μ-oxo/μ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (μ-hydroxo) and a fatty acid ligand. Here we identify a second coordination sphere residue which directs the divergent reactivity of the protein scaffold. The residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group, but not in R2c. Mutation of the glycine to leucine converts the resting state R2lox cofactor into an R2c-like cofactor, a µ-oxo/µ-hydroxo bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in wild-type R2lox, demonstrating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine mutant no longer functions as a two-electron oxidant. Specifically the mass spectrometry data here deposited tracks the abundance of the cross-linked peptide AVIRAATVYNMIVE-AVTLD, which was used as surrogate marker for the abundance of the internal V72-Y162 ether cross-link, which is directly impacted by the metalation state of the protein as well as the residue present in position 68 (G or L). Taken together, our data demonstrate that the residue preceding the N-terminal metal ligand directs the cofactor’s reactivity towards one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Geobacillus Kaustophilus

SUBMITTER: Rui Branca

LAB HEAD: Janne Lehtiö

PROVIDER: PXD015549 | Pride | 2019-10-31

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Griese_20181031_R2lox_S01_r1.msf	Msf
	Griese_20181031_R2lox_S01_r1.raw	Raw
	Griese_20181031_R2lox_S01_r2.msf	Msf
	Griese_20181031_R2lox_S01_r2.raw	Raw
	Griese_20181031_R2lox_S01_r3.msf	Msf

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Publications

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

Kutin Yury Y Kositzki Ramona R Branca Rui M M RMM Srinivas Vivek V Lundin Daniel D Haumann Michael M Högbom Martin M Cox Nicholas N Griese Julia J JJ

The Journal of biological chemistry 20191007 48

A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (μ-oxo/μ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single ...[more]

PMID: 31591267

Publication: 1/2

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