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Combined Mossbauer 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.

SUBMITTER: Silver SC

PROVIDER: S-EPMC4089880 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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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.

Silver Sunshine C SC Gardenghi David J DJ Naik Sunil G SG Shepard Eric M EM Huynh Boi Hanh BH Szilagyi Robert K RK Broderick Joan B JB

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20140216 3

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 red ...[more]

PMID: 24532333

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Project description:5-Thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5'-dA• generated by the SAM cleavage reaction to abstract the H(6proR) atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by an iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO(2)(-) and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for TpTSO(2)(-) formation. Should any other protein residue serve as the H donor, its presence would result in TpT being the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, because of the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton-coupled electron transfer) process, contrasting to the H atom transfer mechanism in the wild-type (WT) SPL reaction. The C141A mutant repairs SP at a rate that is ~3-fold slower than that of the WT enzyme. Formation of TpTSO(2)(-) and TpT exhibits a V(max) deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2, which is smaller than the (D)V(max) KIE of 2.8 ± 0.3 determined for the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process during enzyme catalysis. As expected, the prereduced C141A mutant supports only ~0.4 turnover, which is in sharp contrast to the >5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.

Dataset Information

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

Publications

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

Similar Datasets

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