H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes

Communicaon

Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes

Francesca Camponeschi y, Riccardo Muzzioli y, ‡, Simone Ciofi-Baffoni, Mario Piccioli and Lucia Banci

Magnetic Resonance Center CERM, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy

Correspondence to : [email protected] https://doi.org/10.1016/j.jmb.2019.08.018

Abstract

Ironesulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate molecules with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes. © 2019 Elsevier Ltd. All rights reserved.

Introduction Cys 141 and Cys 144 in LIAS) and the fourth iron ion, termed as catalytic iron ion, is exposed for the Lipoyl synthase (LIAS in humans) is a member of binding of SAM (Fig. 1) [12,13]. The FeSaux cluster is 0 the radical S-(5 -Adenosyl)-L-Methionine (SAM) bound through a conserved CX4CX5C motif (Cys superfamily of enzymes and uses two [4Fe-4S] 106, Cys 111 and Cys 117 in LIAS) and a serine (Ser clusters to catalyze the final step of the biosynthesis 345 in LIAS) (Fig. 1) [12,13]. The complete turnover of the lipoyl cofactor [1e7]. The mechanism of lipoyl of lipoyl synthase requires two equivalents of SAM synthase consists of a two-step reaction where a (one per sulfur insertion) and two sulfur atoms from protein-bound octanoyl chain is converted into lipoic the auxiliary cluster. Therefore, lipoyl synthase acid by a consecutive insertion of two sulfur atoms at typically catalyzes no more than one turnover in in C6 and C8 positions of the octanoyl chain [8,9]. The vitro reactions as a consequence of the FeSaux two [4Fe-4S] clusters are both involved in the cluster disruption [1,14]. Recently, it was shown that catalytic mechanism [9,10]. One of them (Fig. 1), the Fe-S cluster carrier protein NfuA from Escher- typical of all radical SAM enzymes (hereafter named ichia coli can regenerate the auxiliary cluster of E. FeSRS), performs a reductive cleavage of a SAM coli lipoyl synthase (LipA) after each turnover. In 0 molecule to obtain methionine and a 5 -deoxyade- such a way, although the consumption of the FeSaux nosyl radical (50-dA) [1]. The 50-dA radical serves cluster after each turnover, LipA can act catalytically to generate a radical on the octanoyl chain. The upon the continuos supply of [4Fe-4S] clusters by other cluster (usually defined as auxiliary cluster and NfuA [15]. On the other hand, this mechanism of named FeSaux hereafter, Fig. 1) provides the action, i.e. the FeSaux cluster disruption, implies the inorganic sulfides to the formed octanoyl chain release of free iron and inorganic sulfide ions and it radical [5,9e11]. The FeSRS cluster is bound to a would seem to present a problem for the cell CX3CX2C motif, i.e. three iron ions are covalently considering the high iron and sulfide toxicity. Thus, bound to three Cys residues of the motif (Cys 137, it is reasonable that several important chemical

Cys 137 tion (data not shown). Therefore, the chemically reconstituted procedure was avoided for all WT LIAS samples used in the following spectroscopic characterization. The paramagnetic 1H NMR spectrum of WT LIAS Cys 141 Cys 144 (Fig. 3a) shows two intense (a and b) and one weak FeSRS (c) hyperfine shifted signals in the 20e13 ppm spectral region. Their size and linewidths are typical of bCH2 and aCH of cysteine/serine bound to a [4Fe- FeS Cys 111 þ aux 4S]2 cluster [20e22]. The temperature depen- Cys 106 dence of hyperfine shifted NMR signals provides information on the electronic state of the paramag- 2þ Cys 117 Ser 345 netic center [23]. In the case of [4Fe-4S] clusters, the antiferromagnetic couplings among the four Fig. 1. Structure of lipoyl synthase from Mycobac- equivalent iron ions (each of them being formally þ terium tuberculosis in ribbon representation (PDB ID Fe2.5 ) give rise to a S ¼ 0 diamagnetic ground 5EXJ). The FeSRS and FeSaux clusters and the iron state; the paramagentism arises from the excited ligands, numbered according to LIAS sequence, are levels of the energy diagram, that become more shown in CPK mode and sticks, respectively. populated as temperature increases [24,25]. The hyperfine shifts due to the contribution of the excited states therefore increase when increasing tempera- aspects remain to be answered in order to have a ture, giving rise to the so-called antiCurie tempera- complete description of the various steps of the ture dependence. Conversely, in the [4Fe-4S]þ enzymatic cycle [16]. The most enigmatic questions clusters which contain two Fe2.5þ ions and two Fe3þ concerns how the second sulfur atom is inserted in ions, magnetic couplings produce a paramagnetic the substrate and the ultimate fate of the degraded S ¼ 1/2 ground state. Here, paramagentism auxiliary cluster. decreases at increasing temperature and the che- This study reports for the first time that 1H mical shifts follow the classical Curie law. As a paramagnetic NMR spectroscopy is a valuable tool consequence, the temperature dependence of 1H investigating the binding of SAM and substrate NMR spectrum shown in Fig. 3d allowed us to obtain molecules to the two clusters of LIAS, in such a information about the oxidation state of the [4Fe-4S] way resulting instrumental to contribute answering clusters of LIAS. Signal a experiences an anti-Curie the still open questions of the enzymatic mechanism. temperature dependence and the weak signal c To achieve this, we have exploited the hyperfine- exhibits a Curie temperature dependence (Fig. 3d). 1 shifted H NMR signals of iron ligands of FeSRS and Temperature dependence of signal b suggests that FeSaux clusters and follow their changes upon at least two protons, having respectively Curie and cluster interaction with SAM, with compounds anti-Curie temperature dependence, are overlapped mimicking SAM (S-(50-Adenosyl)-L-Homocysteine, in this signal (Fig. 3d). Longitudinal relaxation times 0 SAH) and the breakdown product of SAM (5 -deoxy- (T1) of signals a-d have been measured in WT LIAS ’ 5 -MethylThioAdenosine, MTA), and with a synthetic in D2O. T1 values are in the 1e3 ms range (signal a, octanoyl-peptide substrate (Glu-Ser-Val-(N6-octa- 1.2 ± 0.2 ms; signal b,2.1ms± 0.4; signal c, noyl)Lys-Ala-Ala-Ser-Glu, named octanoyl-peptide 2.5 ± 0.2 ms; signal d 1.6 ± 0.6 ms). They are hereafter) (Fig. S1). slightly smaller than previous findings in electron Wild-type LIAS lacking the N-terminal mitochon- transfer Fe-S proteins [26e28] and account for drial targeting sequence was expressed and pur- electronic correlation time of iron ions around ified from E. coli cells (WT LIAS, hereafter). The 1 1011 s. protein was obtained highly pure and monomeric in We used site-directed mutagenesis to obtain a solution (Fig. 2a) with a characteristic brown color, cluster specific assignment of these NMR signals. In displaying, in the UVevisible absorption spectrum, a LIAS variant, the three cysteine residues that binds a weak shoulder at 330 nm and a broad peak at the FeSRS cluster (i.e. Cys 137, Cys 141 and Cys 400 nm, in addition to the peak at 280 nm due to 144) were mutated into alanines; in another LIAS aromatic amino acids (Fig. 2b). The peak at 400 nm variant, the same type of mutations were performed þ is characteristic of [4Fe-4S]2 clusters with a on the FeS cluster Cys ligands (i.e. Cys 106, Cys þ þ aux delocalized redox state, i.e. with two Fe2.5 -Fe2.5 111 and Cys 117). The triple C137/C141/C144A and pairs [17e19].TheUVevisible spectrum of WT C106/C111/C117A LIAS variants can thus bind only LIAS does not significantly change upon its chemi- the FeSaux or the FeSRS cluster, respectively. The cal reconstitution; consistently the signals in the UVevisible spectra of both variants are similar to paramagnetic 1H NMR spectra of WT LIAS remain that of WT LIAS, possessing the same distinctive unperturbed before and after chemical reconstitu- features at 330 and 400 nm characteristic of [4Fe- 4516 Catalytic Mechanism of Radical SAM Enzymes

Fig. 2. Analytical gel filtration and UVevisible absorption spectra of LIAS.(a) Analytical gel filtration chromatogram (protein concentration 500 mM) in 50 mM potassium phosphate, pH 7.0, with 5 mM DTT. The protein eluted at a volume corresponding to a molecular mass of 41 kDa. Right inset: Standard calibration curve for the Superdex 200 10/300 increase column. Left inset: SDS-PAGE of the last purification step of WT LIAS, 1. proteins obtained after the first HiTrap chelating column; 2. protein marker in kDa; 3. protein obtained after TEV protease cleavage and a second passage on HiTrap chelating column. (b)UVevisible absorption spectra of WT LIAS (blue line), chemically reconstituted C137/C141/ C144A LIAS variant (green line), as purified C106/C111/C117A LIAS variant (black line). The buffer was 50 mM potassium phosphate, pH 7.0, with 5 mM DTT, and the sample concentration was 25 mM. The spectra were recorded in 1 cm cuvette.

4S]2þ clusters and not displaying features that are for the mutated [4Fe-4S]2þ HIPIP with respect to indicative of [2Fe-2S]2þ clusters (Fig. 2b). The those of wild-type [4Fe-4S]2þ HIPIP. This compar- paramagnetic 1H NMR spectrum of the C137/ ison thus suggest that Ser 345 is a ligand in the C141/C144A LIAS variant shows two signals with auxiliary cluster of human LIAS, in agreement with chemical shift values very similar to those of the what observed in the crystal structures of bacterial signals a and b observed in WT LIAS (Fig. 3b), both LIAS homologs, in which the same serine was found having an anti-Curie temperature dependence (Fig. to bind the auxiliary cluster [12,13]. Moreover, signal 3d). The chemical shifts and the temperature a has a T1 value shorter than what previously found 2þ dependence of these two signals are typical of for bCH2 of cysteines bond to [4Fe-4S] cluster 2þ bCH2 of Cys or Ser residues bound to a [4Fe-4S] [26,27], and this could be interpreted as a further cluster with an S ¼ 0 electronic ground state. As indirect evidence of serine coordination, because of previously reported for [4Fe-4S] and [2Fe-2S] the shorter Fe-O vs. Fe-S distance. In conclusion, all clusters [22,29], a Cys cluster ligand mutation to the NMR data on C137/C141/C144A LIAS variant 1 Ser affects the overall electronic distribution within allowed us to assign the H signals due to the bCH2 the cluster but does not change dramatically the of Cys/Ser residues of the FeSaux cluster in WT LIAS 1 features of the H NMR spectrum. Signals of serine and to determine that the FeSaux cluster is in an 2þ 2þ bCH2 will experience similar hyperfine shift as oxidized [4Fe-4S] state ([4Fe-4S]aux, hereafter). 1 cysteine bCH2 signals. The shorter metal-to-proton The paramagnetic H NMR spectrum of the C106/ distance of the oxygen-bound serine, compared to C111/C117A LIAS variant contains two contiguous the sulfur-bound cysteine, may result in a faster intense signals with very close chemical shifts that nuclear relaxation of serine bCH2; however, metal-to are similar to that of signal b observed in WT LIAS proton distances are affected also by Fe-S(O)-C-H spectrum (Fig. 3c). As a result, the paramagnetic 1H dihedral angles. Altogether we can expect that the NMR spectra of both variants indicated that signal b chemical shifts observed for the beta protons of the in WT LIAS spectrum arises from three proton cluster ligands bound to the auxiliary cluster of LIAS resonances and that one of these protons is due to can be attributed to either cysteine or serine ligands. a cluster ligand bound to the FeSaux cluster and the However, comparing the paramagnetic 1H NMR other two protons are due to cluster ligands bound to spectrum of the C137/C141/C144A LIAS variant, the FeSRS cluster. Both WT and C106/C111/C117A containing exclusively the auxiliary cluster, with LIAS when dissolved in 100% D2O buffer, which those of wild-type [4Fe-4S]2þ HIPIP and a stable determines the disappearance of exchangeable variant [4Fe-4S]2þ HiPIP in which a cysteine ligand signals, showed the presence of a signal (d)(Fig. had been replaced by serine ligand [22,30,31],it 3c), which is hidden in the spectrum acquired in H2O results that the chemical shift values observed for buffer by the sharp NH signal at 12.9 ppm (Fig. 3a). the beta protons of the cluster ligands bound to the The anti-Curie temperature dependence of this auxiliary cluster are more similar to those reported signal (Fig. 3d) and its chemical shift are typical of Catalytic Mechanism of Radical SAM Enzymes 4517

Fig. 3. Paramagnetic 1H NMR spectra of WT LIAS and of the triple LIAS variants and temperature dependence of hyperfine-shifted signals of cluster ligands. 1H NMR spectra acquired at 400 MHz of: (a) WT LIAS in the 30e10 ppm spectral region and in the far-shifted 90e30 ppm region (inset), in degassed 50 mM phosphate buffer pH 7.0, and 5 mM DTT; (b) chemically reconstituted C137/C141/C144A LIAS variant in degassed 50 mM phosphate buffer pH 7.0, and 5 mM DTT; (c) as purified C106/C111/C117A LIAS variant in degassed 50 mM phosphate 100% D2O buffer pH 7.0, and 5 mM DTT; (d) Temperature dependence of hyperfine-shifted signals of cysteine/serine ligands in WT LIAS (black dots) and triple C137/C141/C144A (red dots) and C106/C111/C117A (green dots) LIAS variants.

a bCH2 of a cysteine residue bound to an oxidized WT LIAS, and indicated that the FeSRS cluster in WT þ þ [4Fe-4S]2 cluster. A weak shoulder signal with the LIAS is mainly present in an oxidized [4Fe-4S]2 2þ same chemical shift and Curie temperature depen- state ([4Fe-4S]RS hereafter), and only a small dence as those of the weak signal c observed in WT fraction of the FeSRS cluster is in a reduced [4Fe- 1 þ þ LIAS is also present in the paramagnetic H NMR 4S] state ([4Fe-4S]RS hereafter). spectrum of the C106/C111/C117A LIAS variant The NMR data described in the last paragraph on (Fig. 3c). The detection of this Curie temperature thevariantswereacquiredi)onaC106/C111/ dependent signal in the 1H NMR spectra of both WT C117A LIAS variant that was not chemically and C106/C111/C117A LIAS suggest that a small reconstituted since this variant is characterized by fraction of the FeSRS cluster is in the reduced state. a low protein stability and largely precipitates upon By comparing the chemical shift of this signal with chemical reconstitution, and ii) on a C137/C141/ known paramagnetic 1H NMR spectra of small C144A LIAS variant that was, on the contrary, electron transfer reduced [4Fe-4S]þ ferredoxins chemically reconstituted to form a [4Fe-4S]2þ [32], it appears that signal c may arise from a aCH cluster, since the paramagnetic 1HNMRspectrum proton of a Cys residue bound to a reduced [4Fe- of the as purified C137/C141/C144A LIAS variant þ 4S] cluster. According to this interpretation, bCH2 shows signals of bCH2/aCH cysteine ligands typical protons of cysteines bound to a [4Fe-4S]þ cluster of a mixture of [4Fe-4S]2þ a [3Fe-4S]þ clusters should be observed through a paramagnetic tailored [35,36] with a 30/70 intensity ratio for the two experiment performed over a wide spectral window species (Fig. S2). Overall, the NMR spectra on the using fast repetition rates [33,34]. Three very weak two variants showed that a structural cooperativity signals with Curie temperature dependence have is present between the two clusters. Indeed, the been, indeed, detected, in the WT protein, in the absence of the FeSRS cluster exposes the FeSaux 90e30 ppm region (inset of Fig. 3a). Overall, the cluster to oxidative conversion from [4Fe-4S]2þ to þ NMR data on the C106/C111/C117A LIAS variant [3Fe-4S] , and when FeSRS cluster is the only 1 allowed us to identify the H signals due to the bCH2 cluster present in the enzyme, the protein stability is protons of the Cys residues of the FeSRS cluster in greatly reduced. 4518 Catalytic Mechanism of Radical SAM Enzymes

Fig. 4. Paramagnetic 1H NMR spectra of WT LIAS in the presence of SAM and the octanoyl-peptide substrate. 1H NMR spectra acquired at 400 MHz of: (a) WT LIAS in the absence (black) and in the presence of one (red) and two (blue) equivalents of SAM; (b) WT LIAS in the absence (black) and in the presence of one equivalent of octanoyl-peptide substrate (violet); (c) WT LIAS in the presence of one equivalents of SAM (red), and one equivalent of SAM and one equivalent of octanoyl-peptide substrate (green). Sample conditions are degassed 50 mM phosphate buffer pH 7.0, and 5 mM DTT. Catalytic Mechanism of Radical SAM Enzymes 4519

The cluster-specific assignment of the paramag- 50-dAdo is reasonably quenched upon production netic 1H NMR signals of WT LIAS allowed us to follow to generate 50-deoxyadenosine (50-dA). Bearing in 0 the interactions of SAM and substrate molecules with mind that 5 -dA is expected to go close the FeSaux the two [4Fe-4S] clusters of WT LIAS. We have first cluster in order to form the radical on the octanoyl investigated the interaction of WT LIAS with SAM chain of the substrate, we can suggest that the z and (Fig. S1). Stepwise additions of SAM to WT LIAS (up z’ signals originate from the bCH2 of the cysteines 0 to two equivalents) give rise to a new set of intense bound to the 5 -dA-interacting FeSaux cluster spe- peaks in the 22e13 ppm region (labeled with cies. According to this interpretation, the low symbols a-d in Fig. 4a), at decrement of signals b intensity of the z and z’ signals with respect to the and c, which contain resonances from the FeSRS a-d signals is in agreement with the low percentage þ cluster ligands, while signal a of the FeSaux cluster of the reduced [4Fe-4S]RS cluster present in the remains essentially unaffected by the SAM-LIAS protein that can promote SAM cleavage and thus interaction. All the new a-d signals have chemical form the 50-dA-interacting species. To validate this shift values and anti-Curie temperature dependences model, the interaction between WT LIAS and a consistent with a new species containing a [4Fe- compound mimicking the 50-dA (MTA, Fig. S1) was 4S]2þ cluster (Fig. S3). All these data indicated the investigated. Unlike what we observed following the formation of a SAM-LIAS adduct in which SAM additions of SAM, an excess of MTA (up to 2.5 2þ closely interacts with the [4Fe-4S]RS cluster, while it is equivalents) was required to observe some spectral not interacting with FeSaux cluster. This is in agree- changes in WT LIAS. The formation of new signals ment with previous ENDOR studies showing that with chemical shifts similar to those of a-d signals SAM binds to radical SAM Fe-S cluster via coordina- was, however, not observed upon MTA additions tion of the amino and carboxylate of SAM to the (Fig. S4), thus indicating that MTA does not interact 2þ catalytic iron ion of the [4Fe-4S]RS cluster [37,38], with FeSRS cluster. However, two new weak signals and with the crystal structure of LipA complexed with at 26.8 and 23.7 ppm, with very similar chemical shift the SAM-mimic compound SAH (Fig. S1), which to the z and z’ signals observed in the SAM-WT LIAS showed that SAH is located close to the FeSRS complex, appeared (Fig. S4). These new signals cluster while being far from the FeSaux cluster [13]. have chemical shift values and anti-Curie tempera- Consistent with this analysis, the same spectral ture dependences consistent with a [4Fe-4S]2þ changes were observed when monitoring the inter- state, as the two signals z and z’ have (Fig. S3). action between WT LIAS and the SAM-mimic Considering that MTA is proximal to one iron ion of compound SAH, i.e. signals with chemical shifts the FeSaux cluster in the LipA crystal structure similar to those of the a-d signals, all having anti- complexed with MTA [13], we conclude that these Curie temperature dependence, were observed (data two signals originate from the presence of a species 2þ not shown). Moreover, the stepwise disappearance formed by the [4Fe-4S]aux cluster interacting with of signal c in Fig. 4a indicate that the small fraction of MTA in the 1:2.5 WT LIAS-MTA mixture. This þ reduced [4Fe-4S]RS cluster gets oxidized upon observation confirms the interpretation described þ 0 addition of SAM, as expected by the [4Fe-4S]RS- above, i.e. 5 -dA and methionine are formed by SAM induced reductive cleavage of SAM. No signals of cleavage by the small fraction of reduced FeSRS 0 bCH2 of cysteines bound to the small fraction of cluster present in WT LIAS and the formed 5 -dA þ reduced [4Fe-4S]RS cluster were indeed observed in interacts with the FeSaux cluster. the 70e30 ppm region in the final mixture, in The interaction between WT LIAS and the agreement with the FeSRS cluster oxidation. octanoyl-peptide substrate mimicking the amino In the interaction between WT LIAS and SAM, two acid sequence of the glycine cleavage system H signals z and z’ at 26.9 and 23.5 ppm, having lower protein was followed by acquiring paramagnetic 1H intensity than a-d signals, appear by SAM additions. NMR spectra on a 1:1 mixture of WT LIAS and The chemical shift values and the anti-Curie octanoyl-peptide, and on a 1:1:1 mixture of WT LIAS, temperature dependence of these two signals (Fig. octanoyl-peptide and SAM (sequential or simulta- S3) are consistent with the formation of a [4Fe-4S]2þ neous addition of SAM and octanoyl-peptide pro- cluster species. The observed oxidation of the small duce the same final 1H NMR spectrum). The þ 1 fraction of [4Fe-4S]RS cluster induced by SAM paramagnetic H NMR spectrum of the 1:1 mixture addition produces the formation of methionine and of WT LIAS and octanoyl-peptide is fully super- of the 50-dA radical. This radical is a transient and imposable with that of WT LIAS, indicating that the higly reactive intermediate and was only very octanoyl-peptide does not interact with the protein in recently observed at low temperature using a novel the absence of SAM (Fig. 4b). On the contrary, the approach involving cryogenic photoinduced electron paramagnetic 1H NMR spectrum of the 1:1:1 mixture transfer [38e40]. It is thus impossible that it would of WT LIAS, octanoyl-peptide and SAM shows survive in these experiments long enough to several spectral changes. Specifically, two new produce interactions observable by NMR. Therefore, intense signals at 28.8 and 24.1 ppm appear, signal 4520 Catalytic Mechanism of Radical SAM Enzymes b increases while signal a decreases in intensity ESFRI project, and specifically CERM/CIRMMP (Fig. 4c). The two new signals have chemical shift Italy Centre. This article is based upon work from values and anti-Curie temperature dependences COST Action CA15133, supported by COST (Fig. S3) consistent with the presence of [4Fe- (European Co-operation in Science and Technol- 4S]2þ clusters in the mixture. These spectral ogy). Financial support of the Fondazione Cassa di changes involve signals of the FeSaux cluster Risparmio di Firenze (CRF20160985) is gratefully cysteines. Indeed, signal a is exclusively due to acknowledged. bCH2 of cysteine/serine of the FeSaux cluster, signal b includes bCH2 of a cysteine/serine of the FeSaux cluster, and the signals at 28.8 ppm and 24.1 ppm Competing interests have chemical shift values similar to those of z and z’ The authors declare no competing financial signals, which characterize the FeSaux cluster interactions, as discussed above. Therefore, the interests. observed spectral changes monitor the interaction between the octanoyl-peptide and the FeSaux cluster. On the contrary, the signals a-d assigned Author contributions to the SAM-bound FeS cluster remains essentially RS Conceptualization: L.B. and S.C.B.; Methodology: unperturbed (Fig. 4c), indicating that SAM remains M.P.; Validation: F.C.; Investigation: M.P., R.M., F.C. bound to the cluster. Overall, the SAM:peptide:WT and S.C.B.; Writing e Original Draft: L.B., S.C.B. and LIAS interaction studies indicate that, only once M.P. Writing e Review & Editing: M.P., R.M., F.C., SAM is bound to the protein, the octanoyl-peptide S.C.B and L.B.; Visualization: R.M. and F.C. Super- substrate can recognize its binding site. This is in vision: M.P., S.C.B and L.B.; Funding Acquisition: agreement with what found in the radical SAM M.P. and L.B. enzyme biotin synthase, where dethiobiotin substrate binding occurs only in the presence of SAM and the substrate binding is highly cooperative [41]. Appendix A. Supplementary data In conclusion, the data here presented showed 1 that H NMR spectroscopy tailored to reveal Supplementary data to this article can be found hyperfine-shifted signals of metal-ligands [42] is a online at https://doi.org/10.1016/j.jmb.2019.08.018. powerful tool to investigate the sequential binding of SAM and of the octanoyl-peptide substrate to the Received 9 July 2019; [4Fe-4S] clusters of human lipoyl synthase. We can Received in revised form 30 August 2019; specifically monitor the interaction between SAM Accepted 30 August 2019 and the FeSRS cluster, the redox changes of the Available online 4 September 2019 FeSRS cluster upon SAM interaction, follow the reductive cleavage of SAM, and monitor the sub- Keywords: strate binding at the FeSaux cluster. These informa- Lipoyl synthase; tion can be exploited to characterize the catalytic Ironesulfur proteins; mechanism of the radical SAM superfamily of Enzyme mechanism; enzymes. In particular, we believe that, while X- Electron transfer; ray, EPR and ENDOR spectroscopies offer a more Metallo enzyme detailed but also more static view by trapping the intermediates of the catalytic mechanism and freez- y These authors contributed equally to this work. ing transient interactions, paramagnetic NMR provides a picture at room temperature of the kinetics of z Present address: Department of Cancer Systems substrate uptake and its transformation, as well as Imaging, The University of Texas MD Anderson the dynamics of site specific interactions of SAM and Cancer Center, 1881 East Road, 3SCR4.3600, the substrate occuring at both clusters, thus being Unit 1907, Houston, TX 77054. essential to obtain a comprehensive view of the catalytic mechanism. Abbreviations used: SAM, S-adenosylmethionine; 50-dA, 50-deoxyadenosyl radical; LIAS, Human lipoyl synthase; LipA, E. coli lipoyl synthase; SAH, S-(50-Adenosyl)-L- Homocysteine; MTA, 50-deoxy-5’-MethylThioAde- Acknowledgement nosine; SDS-PAGE, Sodium Dodecyl Sulphate The authors acknowledge the support and the PolyAcrylamide Gel Electrophoresis. use of resources of Instruct-ERIC, a Landmark Catalytic Mechanism of Radical SAM Enzymes 4521

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