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. Author manuscript; available in PMC: 2022 Feb 11.
Published in final edited form as: J Biol Inorg Chem. 2019 Sep 7;24(6):769–776. doi: 10.1007/s00775-019-01706-w

Radical SAM Enzymes: Surprises along the Path to Understanding Mechanism

William E Broderick 1, Joan B Broderick 1
PMCID: PMC8837180  NIHMSID: NIHMS1571834  PMID: 31494759

Abstract

As the field of radical SAM enzymology has grown from a few examples in the 1990s to hundreds of thousands today, a fundamental question has remained: how does Nature use S-adenosyl-L-methionine to initiate radical reactions? This was a driving question when we first began studying pyruvate formate-lyase activating enzyme in 1993, and our journey for answers has brought us to many surprising discoveries, from a direct coordination of SAM to a unique iron in a [4Fe-4S] cluster, to our recent discovery of an organometallic intermediate and our ability to quantitatively produce and characterize the long-sought 5’-deoxyadenosyl radical intermediate. These adventures, and what we have learned along the way about this fundamentally novel chemistry is described in this review.

Introduction

The group of radical S-adenosyl-L-methionine (radical SAM or RS) enzymes is among the largest enzyme superfamilies in nature, with hundreds of thousands of unique sequences identified spanning all kingdoms of life.1, 2 We published a JBIC mini-review on these enzymes in early 2001,3 essentially concurrent with a paper by Heidi Sofia identifying this superfamily for the first time.4 Our JBIC review described seven different enzyme systems that appeared to have mechanistic similarities in using iron-sulfur clusters and SAM to initiate radical reactions: pyruvate formate-lyase activating enzyme (PFL-AE), the anaerobic ribonucleotide reductase activating enzyme (RNR-AE), the benzylsuccinate synthase activating enzyme, lysine 2,3-aminomutase (LAM), biotin synthase (BioB), lipoate synthase (LipA), and spore photoproduct lyase (SPL).3 With the bioinformatics studies by Sofia and coworkers,4 and more recently by Babbitt, Holiday, and their coworkers,5, 6 this small group of enzymes has expanded to encompass a very large and diverse superfamily of enzymes catalyzing some of the most challenging reactions known in biology.1 RS enzymes have received growing attention in large part because they appear in so many important biological pathways, catalyzing a diverse array of reactions important for life processes.

Our interests have long centered on the fundamental chemistry of radical initiation: how does the ancient and ubiquitous [4Fe-4S] cofactor work in conjunction with SAM to generate a central 5’-dAdo• radical intermediate that initiates the chemistry on substrate in all these enzymes? Our thinking has been inspired by the early recognition by Perry Frey and coworkers,7–9 and Joachim Knappe and coworkers,10 that SAM-dependent radical enzymes bear key similarities to the adenosylcobalamin (coenzyme B12)-dependent radical enzymes. This review will focus on the structure and properties of the RS [4Fe-4S] cluster, how it interacts with SAM, and the current understanding of the mechanism of radical initiation.

A Site-Differentiated [4Fe-4S] Cluster and its Interaction with SAM

The presence of a conserved CX3CX2C motif was one of the early indicators of similarity among radical SAM enzymes, and remains the most characteristic sequence feature, although variations in this motif are now known in the superfamily. This three-cysteine motif, together with the early evidence for a [4Fe-4S] cluster in these enzymes,11–16 pointed to the presence of a site-differentiated cluster, where one iron site is distinguished by having non-cysteine ligation. The first experimental evidence for site differentiation was provided through specific isotopic labeling of the [4Fe-4S] cluster of PFL-AE, coupled with Mössbauer spectroscopic analysis (Fig. 1).17 Specific isotopic labeling was accomplished by exposing purified PFL-AE containing a [4Fe-4S]2+ cluster to O2, which caused cluster oxidation and loss of the unique iron (the one not coordinated by cysteine) to provide a [3Fe-4S]1+ cluster bound to the protein. This protein was then made anaerobic again, and treated with mild reductant and 57Fe2+; the resulting protein had a [4Fe-4S] cluster with the unique site labeled with 57Fe. Addition of SAM to this protein resulted in a change in the isomer shift of the unique site from 0.42 to 0.72 mm/s, which we interpreted as resulting from SAM coordination to the unique Fe.17

Figure 1.

Figure 1.

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Mössbauer spectroscopic studies of PFL-AE provide evidence of SAM coordination. Left, the [4Fe-4S] cluster was specifically labeled with 57Fe in the unique site. Right, Mössbauer spectra in the absence (A) and presence (B) of SAM, and (C) difference of A-B. Adapted with permission from Krebs et al17

Coordination of SAM to the unique iron of the [4Fe-4S] cluster was subsequently demonstrated using isotopic labeling of SAM together with electron-nuclear double resonance (ENDOR) spectroscopy (Fig. 2).18 SAM was labeled at the amino group with 15N, and at the carboxylate with 17O and with 13C, and when the labeled SAMs were added to PFL-AE in the [4Fe-4S]1+ state, ENDOR signals were observed that were attributed to direct coordination of the amino and carboxylate groups of SAM to the unique iron of the [4Fe-4S]1+ cluster.18, 19 This was the first demonstration for any radical SAM enzyme that SAM directly coordinated an iron of the [4Fe-4S] cluster; subsequent ENDOR studies on LAM showed the same coordination mode,20 and now numerous X-ray structures of RS enzymes with SAM bound have reiterated the same bidentate SAM coordination to the unique iron.21–26 Clearly, coordination of SAM to the unique iron of the activesite [4Fe-4S] cluster is a unifying structural feature of RS enzymes, but why? One answer seems to be that this places the sulfonium sulfur of SAM within orbital overlap of the [4Fe-4S]+ cluster, a close association also revealed through ENDOR spectroscopic studies.27 This close association is thought to provide a pathway for inner-sphere electron transfer from the reduced [4Fe-4S]+ cluster to the sulfonium of SAM to initiate reductive cleavage, as discussed further in the next section.

Figure 2.

Figure 2.

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ENDOR spectroscopic studies of PFL-AE demonstrated that SAM interacts directly with the [4Fe-4S] cluster via coordination of the amino and carboxylate of SAM to the unique iron of the cluster.

The X-ray crystal structure of PFL-AE provides a representative view of the active site of a RS enzyme.25 When the enzyme was crystallized in the presence of SAM and a 7-mer peptide corresponding to the site of H-atom abstraction (G734 of pyruvate formate-lyase) flanked by the three amino acids on each side, the SAM and peptide are found well-ordered in the active site, with SAM coordinated to the unique iron of the [4Fe-4S] cluster as previously determined by ENDOR studies (Fig. 3).18, 25 In this configuration, the sulfonium sulfur of SAM is 3.2 Å from the unique iron, and 3.9 Å from the nearest cluster sulfide, consistent with the orbital overlap observed in ENDOR studies.25, 27 The S-5’C bond is trans to the S(SAM)---Fe vector, a geometric arrangement common to all known canonical RS structures. The Cα of G734 is positioned 4.1Å from the C5’ of SAM, seemingly perfectly positioned for H atom abstraction immediately upon formation of the 5’-dAdo• upon reductive cleavage of SAM.25 An interesting feature of the PFL-AE active site that is not universal to RS enzymes is the presence of a catalytically important monovalent cation that is bridged to the unique iron of the [4Fe-4S] cluster by the carboxylate of SAM (Fig. 4).28

Figure 3.

Figure 3.

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Active site of PFL-AE, with SAM (bright green) bound to the unique iron of the [4Fe-4S] cluster. The 7-mer peptide substrate analog of the natural substrate PFL is shown in teal. PDB 3CB8. Adapted from Vey et al.25

Figure 4.

Figure 4.

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Alternate view of the active site of PFL-AE, focusing on the monovalent cation site. The [4Fe-4S] cluster (rust and gold) is coordinated by SAM (teal with blue – nitrogen, red – oxygen, and yellow – sulfur) through the amino and carboxylate moieties. The carboxylate moiety also coordinates the monovalent cation K+, as do several amino acid residues.

Roles of the [4Fe-4S] Cluster and SAM in Initiating Radical Catalysis

Radical SAM enzymes require reducing conditions for activity. One of the earliest reports of this requirement was by Knappe et al. in 1969, where they showed that activation of pyruvate formate-lyase required a flavoprotein and other undefined components, which could be replaced by a chemical system of “high reducing power.”29 We now know that flavodoxin can serve the function of providing the reducing power for a number of different RS enzymes, including PFL-AE.30–33 In the absence of the biological reductant, chemical reductants such as dithionite or photoreduction using deazariboflavin can be used for many of these enzymes. Perry Frey and coworkers showed that these strong reductants reduced lysine 2,3-aminomutase (LAM) to a state containing a [4Fe-4S]+ cluster, and that this cluster provides catalytic activity in the presence of SAM and substrate.34 We later showed that in PFL-AE, the reduced [4Fe-4S]+ provides the electron required for the reductive cleavage of SAM and the subsequent formation of the glycyl radical on PFL: there was a 1:1 ratio between the quantity of [4Fe-4S]+ in the PFL-AE prior to reaction, and the quantity of Gly• in PFL after reaction.35 These results provided important new insights into the role for the [4Fe-4S] cluster in RS enzymes: the [4Fe-4S]+ is the catalytically active state of RS enzymes, and it provides an electron to reductively cleave the S-C5’ bond of SAM to generate the central 5’-dAdo• radical intermediate (Fig. 5). Given the orbital overlap between the sulfonium of SAM and the [4Fe-4S] cluster demonstrated through ENDOR studies,27 the electron is thought to be provided by inner-sphere electron transfer, with efficient configurational interaction between the [4Fe-4S]+ donor and the S-C5’ σ* orbitals facilitating S-C5’ bond cleavage.36

Figure 5.

Figure 5.

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In RS enzymes, a reduced [4Fe-4S]+ cluster provides the electron required for the reductive cleavage of SAM (step 1). The resulting 5’-dAdo• radical abstracts an H-atom from substrate R-H (step 2).

Probing the Mechanism of Radical Initiation in the Radical SAM Enzymes

Although 5’-dAdo• has been implicated as the central intermediate in RS enzymes, it is a species that has never been directly observed; rather, its involvement in catalysis has been inferred by the transfer of isotopic labels from substrate into product 5’-deoxyadenosine or into SAM itself. The elusive nature of 5’-dAdo• was attributed to its high reactivity and expected very short lifetime. In order to provide a probe of this key intermediate, Perry Frey and coworkers developed an analog of SAM, 3’,4’-anhydro-S-adenosyl-L-methionine (anSAM), which has a C3’-C4’ double bond (Fig. 6).37, 38 They showed that anSAM is catalytically competent to function in place of SAM with LAM, however turnover with anSAM leads to buildup of the allylically-stabilized anAdo• radical, which they characterized by EPR. We subsequently used this anSAM/LAM system to generate samples with anAdo• in the active site with specifically isotopically labeled substrates; we observed that the 5’-C of anAdo• is in close proximity to the C3-H target of H-atom abstraction on lysine, and that C5’ had moved only a total of 1.5 Å upon S-C bond cleavage.39 The active site appears to impose stringent control on this intermediate radical, allowing it to move only slightly upon S-C bond cleavage: in other words, the anAdo• radical and its close cousin the 5’-dAdo• radical are ‘never free’ during turnover.39

Figure 6.

Figure 6.

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Reductive cleavage of SAM (top) and the analog anSAM (bottom). SAM reductive cleavage leads to the elusive 5’-dAdo• radical, while anSAM reductive cleavage gives the allylically stabilized anAdo• radical.

In our continuing efforts to provide evidence for the mechanism of radical initiation, we pursued rapid freeze-quench experiments of the PFL-AE reaction: PFL-AE in its catalytically active [4Fe-4S]+ state was mixed with PFL and SAM and quenched at 77 K on a millisecond timescale. EPR spectroscopy revealed a new radical species that was due neither to the starting [4Fe-4S]+ state of PFL-AE, nor to the product glycyl radical of PFL (Figure 7).40 Using electron-nuclear double resonance (ENDOR) spectroscopy in conjunction with SAM that was isotopically labeled with 13C, 2H, or 15N in specific atom positions, as well as PFL-AE labeled with 57Fe in the [4Fe-4S] cluster, we were able to show that this new radical species is an organometallic intermediate (named Ω) in which the S-C5’ bond of SAM is cleaved, and the adenosyl moiety of SAM is directly bound to the unique iron of the [4Fe-4S]3+ cluster of PFL-AE through an Fe-C bond (Figure 8).40 The observation of Ω was a surprise, and led us to question the mechanism by which it forms, its relationship to the presumably universal intermediate 5’-dAdo•, and whether Ω is unique to PFL-AE or involved more broadly in the radical SAM superfamily.41 The formation of Ω could go via initial reductive cleavage of SAM to generate the 5’-dAdo• intermediate, followed by oxidative addition of the 5’-dAdo• to the [4Fe-4S]2+ cluster; alternatively, one could posit a nucleophilic attack of the unique iron of the [4Fe-4S]+ on the 5’C of SAM to directly yield Ω. While neither of these possibilities has been disproven, the typical geometry of SAM binding to the [4Fe-4S] cluster in RS enzymes places the 5’C pointing away from the unique iron, making the nucleophilic mechanism implausible in the absence of significant structural rearrangement.

Figure 7.

Figure 7.

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Comparison of the EPR spectra of the starting [4Fe-4S]+ cluster of the active state of PFL-AE, with SAM bound (left), the product glycyl radical of PFL (right), and the intermediate Ω.

Figure 8.

Figure 8.

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Structure of Ω as determined by ENDOR spectroscopy.

The question of the ubiquity of Ω in the RS superfamily was answered when we carried out RFQ experiments on a series of RS enzymes spanning a range of subclasses in the superfamily: in every case tested, Ω was detected via EPR spectroscopy.42 The observation of Ω in divergent enzymes across the RS superfamily suggests that it is a central intermediate, fundamental to the mechanism of radical initiation.41, 42 The mechanistically central nature of Ω during radical initiation in RS enzymes requires a shift in the accepted paradigm for RS mechanisms: rather than reductive cleavage of SAM directly liberating 5’-dAdo• for hydrogen atom abstraction from substrate, we now propose that reductive cleavage of SAM is mechanistically coupled to Ω formation in these enzymes.41, 42 Further, Ω may serve as a way to store the nascent 5’-dAdo•, which is liberated upon homolytic Fe-C5’ bond cleavage, in direct analogy to the Co-C5’ bond homolysis to liberate 5’-dAdo• in radical-B12 enzymes.41, 42 Interestingly, an Ω-like organometallic intermediate has also been identified in the non-canonical RS enzyme Dph2,43 providing yet further evidence that organometallic chemistry may be an essential aspect of iron-sulfur cluster and SAM-based radical initiation.

Our ability to trap Ω led us to consider whether we might be able to generate the 5’-dAdo• by photolysis of Ω. The Co-C5’ bond of adenosylcobalamin has been shown to be photochemically active,44, 45 however the putative 5’-dAdo• generated by photolysis was trapped near the paramagnetic Co(II) and thus not amenable to detailed characterization due to spin-spin coupling of the 5’-dAdo• and paramagnetic Co(II) center.46 In contrast, we reasoned that if we could photolyze the Fe-C5’ bond of Ω, we would generate the 5’-dAdo• trapped near the diamagnetic [4Fe-4S]2+ cluster, enabling the characterization of the 5’-dAdo• radical via EPR spectroscopy. In the course of doing these experiments, we also carried out photolysis of the PFL-AE [4Fe-4S]1+/SAM complex in the absence of substrate, and we found that at 12 K it undergoes essentially quantitative photoinduced electron transfer to generate a 5’-dAdo• and [4Fe-4S]2+.47 This efficient photoinduced electron transfer in PFL-AE was quite surprising, as such a process has not been previously reported for any RS enzyme, or for iron-sulfur proteins more generally. That the process quantitively generated the elusive 5’-dAdo• radical, a central radical intermediate to both B12 and RS enzymes that had been sought by researchers for over half a century, was equal parts stunning and gratifying. While much remains to be learned about the photophysics and biological relevance of photoinduced ET in RS enzymes, this discovery has provided us with a powerful new tool to examine mechanism. Through use of isotopically labeled SAMs in this experiment, we unequivocally demonstrated that the radical species formed is 5’-dAdo•, and through spectroscopy and computation we provided a precise picture of the nature of this long-sought radical species.47

Concluding Remarks

Helmut Beinert published a JBIC minireview in 2000 in which he described iron-sulfur clusters as “ancient structures, still full of surprises.”48 That descriptor is even more true now: the discovery of the RS superfamily, and its vast reach in nature, has brought and continues to bring surprises in terms of the reactions catalyzed and the pathways in which these enzymes play key roles. Further, the fundamental chemistry of radical initiation in RS enzymes keeps reminding us to expect the unexpected. RS enzymes have brought us direct coordination of SAM to an iron of a [4Fe-4S] cluster as a unifying structural feature of the catalytically poised state of these enzymes.18 They have revealed the exquisite control possible in enzyme active sites, even when dealing with extraordinarily reactive primary carbon radical intermediates.39, 49 RS enzymes have revealed novel chemistry, such as the organometallic Ω intermediate: never before seen, but central to the mechanisms of these ubiquitous enzymes.40, 42 Most recently, RS enzymes have provided a novel photochemical route to generating radicals, allowing us for the first time to trap and characterize the 5’-dAdo• so broadly relevant in biological radical catalysis.47 So we agree with Beinert: iron-sulfur clusters really are “still full of surprises,” and we are looking forward to the next surprises these ancient structures choose to reveal!

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