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Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

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Author

Summary, in English

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase (RNR1). Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by X-ray photoreduction (XPR). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis RNR using X-ray absorption spectroscopy. Rapid and biphasic XPR kinetics at 20 K and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of Fe and Mn sites. Comparing with typical X-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordinations and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2-activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free-electron-laser protein crystallography techniques.

Department/s

Publishing year

2013-02-11

Language

English

Publication/Series

Journal of Biological Chemistry

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Physical Sciences
  • Natural Sciences

Status

Published

ISBN/ISSN/Other

  • ISSN: 1083-351X