The assembly mechanism of the Mn/Fe ligand-binding oxidases (R2lox), a family of proteins that are homologous to the nonheme diiron carboxylate enzymes, has been investigated using time-resolved techniques. Multiple heterobimetallic intermediates are observed through optical and magnetic resonance spectroscopies that exhibit unique spectral features, including visible absorption bands and exceptionally broad EPR signatures. On the basis of comparison to known diiron species and model compounds, the spectra have been attributed to (μ-peroxo)-MnIII/FeIII and high-valent Mn/Fe species. Global spectral analysis coupled with isotopic substitution and kinetic modeling reveals elementary rate constants for the assembly of Mn/Fe R2lox under aerobic conditions. A complete reaction mechanism for cofactor maturation that is consistent with experimental data has been developed. These results suggest that the Mn/Fe cofactor can perform direct C–H bond abstraction, demonstrating the potential for potent chemical reactivity that remains unexplored.
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.
In order to sustain life, nature accomplishes a diverse array of chemical reactions; one such way this diversity is achieved is through the use of metalloenzymes. In Mycobacterium tuberculosis, a unique Mn/Fe heterobimetallic cofactor has recently been discovered in the R2‐like ligand binding oxidase metalloprotein, R2lox.1 While the function of this protein remains elusive, the cofactor is similar to that of the ribonucleotide reductase Ic subclass, which holds the one‐electron oxidizing equivalent necessary for enzymatic function in the form of a Mn(IV)/Fe(III) metal cofactor.2 R2lox, following oxygen activation, forms an unprecedented tyrosine‐valine ether crosslink in its active site, indicative of two‐electron chemistry, ultimately giving a stable Mn(III)/Fe(III) resting state.1–3 However, the means by which these virtually identical metal cofactors accomplish these different types of chemistry is unknown. In addition, while R2lox can also incorporate a Fe/Fe cofactor when incubated with exclusively Fe(II), the Mn/Fe cofactor is proposed to be the biologically relevant cofactor, despite going against the well‐established Irving‐Williams series.4 To begin to probe potential reactivity in this system, R2lox was investigated using electrochemistry techniques. Both the Fe/Fe and Mn/Fe cofactors were studied in three different R2lox variants. For all three variants tested, only the Mn/Fe cofactor displayed redox activity under the conditions tested with an oxidation process at biologically accessible potentials. Following this initial characterization, a pH dependence was conducted to investigate potential proposed states.5 This study represents the first electrochemical characterization of R2lox and illuminates the potential reactivity of this novel cofactor. As electrons are a form of chemical currency, the reduction potentials reported here provide a handle on the accessible energy available for catalysis.Support or Funding InformationOhio State University Graduate School Presidential Fellowship; NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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