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Combined Mössbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase

Abstract

Spore photoproduct lyase (SPL), a member of the radical S-adenosyl-l-methionine (SAM) superfamily, catalyzes the direct reversal of the spore photoproduct, a thymine dimer specific to bacterial spores, to two thymines. SPL requires SAM and a redox-active [4Fe–4S] cluster for catalysis. Mössbauer analysis of anaerobically purified SPL indicates the presence of a mixture of cluster states with the majority (40 %) as [2Fe–2S]2+ clusters and a smaller amount (15 %) as [4Fe–4S]2+ clusters. On reduction, the cluster content changes to primarily (60 %) [4Fe–4S]+. The speciation information from Mössbauer data allowed us to deconvolute iron and sulfur K-edge X-ray absorption spectra to uncover electronic (X-ray absorption near-edge structure, XANES) and geometric (extended X-ray absorption fine structure, EXAFS) structural features of the Fe–S clusters, and their interactions with SAM. The iron K-edge EXAFS data provide evidence for elongation of a [2Fe–2S] rhomb of the [4Fe–4S] cluster on binding SAM on the basis of an Fe···Fe scatterer at 3.0 Å. The XANES spectra of reduced SPL in the absence and presence of SAM overlay one another, indicating that SAM is not undergoing reductive cleavage. The X-ray absorption spectroscopy data for SPL samples and data for model complexes from the literature allowed the deconvolution of contributions from [2Fe–2S] and [4Fe–4S] clusters to the sulfur K-edge XANES spectra. The analysis of pre-edge features revealed electronic changes in the Fe–S clusters as a function of the presence of SAM. The spectroscopic findings were further corroborated by density functional theory calculations that provided insights into structural and electronic perturbations that can be correlated by considering the role of SAM as a catalyst or substrate.

Graphical abstract

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Acknowledgments

This work has been supported by the National Institutes of Health (GM67804 and GM54608 to J.B.B.) and the National Science Foundation (MBS0744820 to R.K.S.). Portions of this research were conducted at SSRL, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.

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Correspondence to Joan B. Broderick.

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Silver, S.C., Gardenghi, D.J., Naik, S.G. et al. Combined Mössbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase. J Biol Inorg Chem 19, 465–483 (2014). https://doi.org/10.1007/s00775-014-1104-y

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Keywords

  • Spore photoproduct lyase
  • Radical S-adenosyl-l-methionine
  • Iron–sulfur cluster
  • Mössbauer spectroscopy
  • X-ray absorption spectroscopy