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

ABSTRACT: 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 that exhibit unique spectral features, including visible absorption bands and exceptionally broad electron paramagnetic resonance signatures, are observed through optical and magnetic resonance spectroscopies. On the basis of comparison to known diiron species and model compounds, the spectra have been attributed to (?-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.

SUBMITTER: Miller EK

PROVIDER: S-EPMC5680038 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Miller Effie K EK Trivelas Nicholas E NE Maugeri Pearson T PT Blaesi Elizabeth J EJ Shafaat Hannah S HS

Biochemistry 20170616 26

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 that exhibit unique spectral features, including visible absorption bands and exceptionally broad electron paramagnetic resonance signatures, are observed through optical and magnetic resonance spectroscopies. On the basis of comparison to known diiron ...[more]

PMID: 28574263

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Project description:Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Dataset Information

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

Publications

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

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