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

• Type of Material: Article
• Authors: Sunshine C. Silver, Emory University; David J. Gardenghi, Emory University; Sunil Naik, Emory University; Eric M. Shepard, Emory University; B H Vincent Huynh, Emory University; Robert K. Szilagyi, Montana State University; Joan B. Broderick, Montana State University
• Language: English
• Date: 2014-03
• Publisher: Springer Verlag (Germany)
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: SBIC 2014
• Final Published Version (URL): http://dx.doi.org/10.1007/s00775-014-1104-y
• Title of Journal or Parent Work: Journal of Biological Inorganic Chemistry
• ISSN: 0949-8257
• Volume: 19
• Issue: 3
• Start Page: 465
• End Page: 483
• Grant/Funding Information: Portions of this research were conducted at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the U.S. 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.; The Astrobiology Biogeocatalysis Research Center is funded by the NASA Astrobiology Institute grant NNA08C-N85A.; This work has been supported by the NIH (GM67804 and GM54608 to JBB) and NSF (0744820 to RKS).
• Keywords: Spore photoproduct lyase; iron-sulfar cluster; density functional theory; radical SAM; X-ray absorption spectroscopy; Mossbauer spectroscopy
• Research Categories: Chemistry, Organic

Abstract

Spore photoproduct lyase (SPL), a member of the radical SAM superfamily, catalyzes the direct reversal of the spore photoproduct (SP), a thymine dimer specific to bacterial spores, to two thymines. SPL requires S-adenosyl-L-methionine (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+ and a smaller amount (15%) as [4Fe-4S]2+ clusters. Upon 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 (XANES) and geometric (EXAFS) structural features of the Fe-S clusters, and their interactions with SAM. The Fe K-edge EXAFS provide evidence for elongation of a [2Fe-2S] rhomb of the [4Fe-4S] cluster upon 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, indicating that SAM is not undergoing reductive cleavage. The XAS data for SPL samples and data for model complexes from literature allowed for 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 SAM presence. 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.