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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 O 2 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., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apo-protein exhibiting greater Mn II affinity than Fe II affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

Oxygen (O 2 ) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O 2 cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only 57 Fe(II) or Mn(II) plus 57 Fe(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including 16/18 O and H/D exchange. 57 Fe-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mössbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H 2 O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (μOH - ) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas 18 O from 18 O 2 cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O 2 reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O 2 cleavage route in an enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 oxygen bridge (μ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a μ-oxo/μ-hydroxo-bridged Mn III /Fe III dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating 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-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward 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., (© 2019 Kutin et al.)

Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

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 Mn II and Fe II 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.

The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine cross-link near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Φ = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligand-to-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn II and Fe II . Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn II promotes heterobimetallic cofactor assembly. In the absence of Fe II , R2c cooperatively binds Mn II at both metal sites, whereas R2lox does not readily bind Mn II at either site. Heterometallic cofactor assembly is favored at substoichiometric Fe II concentrations in R2lox. Fe II and Mn II likely bind to the protein in a stepwise fashion, with Fe II binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn II by Fe II at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn II /Fe II concentrations in the host organisms from which they were isolated., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.