Reversible Formation of Alkyl Radicals at [Fe4S4] Clusters and Its Implications for Selectivity in Radical SAM Enzymes

Article abstract of DOI:10.1021/jacs.0c05590

All kingdoms of life use the transient 5′-deoxyadenosyl radical (5′-dAdoa ) to initiate a wide range of difficult chemical reactions. Because of its high reactivity, the 5′-dAdo?must be generated in a controlled manner to abstract a specific H atom and avoid unproductive reactions. In radical S-Adenosylmethionine (SAM) enzymes, the 5′-dAdo?is formed upon reduction of SAM by an [Fe4S4] cluster. An organometallic precursor featuring an Fe-C bond between the [Fe4S4] cluster and the 5′-dAdo group was recently characterized and shown to be competent for substrate radical generation, presumably via Fe-C bond homolysis. Such reactivity is without precedent for Fe-S clusters. Here, we show that synthetic [Fe4S4]-Alkyl clusters undergo Fe-C bond homolysis when the alkylated Fe site has a suitable coordination number, thereby providing support for the intermediacy of organometallic species in radical SAM enzymes. Addition of pyridine donors to [(IMes)3Fe4S4-R]+ clusters (R = alkyl or benzyl; IMes = 1,3-dimesitylimidazol-2-ylidene) generates Ra , ultimately forming R-R coupled hydrocarbons. This process is facile at room temperature and allows for the generation of highly reactive radicals including primary carbon radicals. Mechanistic studies, including use of the 5-hexenyl radical clock, demonstrate that Fe-C bond homolysis occurs reversibly. Using these experimental insights and kinetic simulations, we evaluate the circumstances in which an organometallic intermediate can direct the 5′-dAdo?toward productive H-Atom abstraction. Our findings demonstrate that reversible homolysis of even weak M-C bonds is a feasible protective mechanism for the 5′-dAdo?that can allow selective X-H bond activation in both radical SAM and adenosylcobalamin enzymes.

Full text of DOI:10.1021/jacs.0c05590

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Article
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Reversible Formation of Alkyl Radicals at [FeS] Clusters
and Its Implications for Selectivity in Radical SAM Enzymes
Alexandra C Brown, and Daniel L. M. Suess
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c05590 • Publication Date (Web): 22 Jul 2020
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Reversible Formation of Alkyl Radicals at [Fe4S4]
Clusters and Its Implications for Selectivity in Radical
SAM Enzymes
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Alexandra C. Brown and Daniel L. M. Suess*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
*suess@mit.edu
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Abstract:
All kingdoms of life use the transient 5′-deoxyadenosyl radical (5′-dAdo•) to initiate a wide range
of difficult chemical reactions. Because of its high reactivity, the 5′-dAdo• must be generated in a
controlled manner to abstract a specific H atom and avoid unproductive reactions. In radical S–
adenosylmethionine (SAM) enzymes, the 5′-dAdo• is formed upon reduction of SAM by an [Fe4S4]
cluster. An organometallic precursor featuring an Fe–C bond between the [Fe4S4] cluster and the 5′-dAdo
group was recently characterized and shown to be competent for substrate radical generation, presumably
via Fe–C bond homolysis. Such reactivity is without precedent for Fe–S clusters. Here we show that
synthetic [Fe4S4]–alkyl clusters undergo Fe–C bond homolysis when the alkylated Fe site has a suitable
coordination number, providing support for the intermediacy of organometallic species in radical SAM
enzymes. Addition of pyridine donors to [(IMes)3Fe4S4–R]+ clusters (R = alkyl or benzyl; IMes = 1,3-
dimesitylimidazol-2-ylidene) generates R•, ultimately forming R–R coupled hydrocarbons. This process
is facile at room temperature and allows for the generation of highly reactive radicals, including primary
carbon radicals. Mechanistic studies, including use of the 5-hexenyl radical clock, demonstrate that Fe–C
bond homolysis occurs reversibly. Using these experimental insights and kinetic simulations, we evaluate
the circumstances in which an organometallic intermediate can direct the 5′-dAdo• toward productive H-
atom abstraction. Our findings demonstrate that reversible homolysis of even weak M–C bonds is a
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feasible protective mechanism for the 5′-dAdo• that can allow selective X–H bond activation in both
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radical SAM and adenosylcobalamin enzymes.
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Introduction:
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Nature utilizes radical chemistry for a wide range of challenging reactions including cofactor
maturation, antibiotic synthesis, and secondary metabolism.1–4 The most common radical initiator is the
5′-deoxyadenosyl radical (5′-dAdo•), which is generated in adenosylcobalamin and radical S-
adenosylmethionine (SAM) enzymes.1,3 Because the 5′-dAdo• is a primary carbon radical, it can abstract
H atoms from strong X–H bonds and initiate reactions that proceed through high-energy intermediates.5
Although this enables thermodynamically and kinetically challenging reactions to proceed, it introduces
an additional difficulty: how to generate and utilize the 5′-dAdo• in a controlled fashion so as to avoid
unproductive reactivity.
The 5′-dAdo• is derived from relatively unreactive precursors, either by homolysis of the Co–C
bond in adenosylcobalamin1 or by reductive cleavage of an S–C bond in SAM (Figure 1).3,6,7 In both
adenosylcobalamin and radical SAM enzymes, a dramatic rate enhancement for production of the 5′-
dAdo• is observed only when all reaction components are present (i.e., substrate and adenosylcobalamin
for adenosylcobalamin enzymes8–11 or substrate, SAM, and a reduced [Fe4S4] cluster for radical SAM
enzymes12). But even with this safety mechanism operative, it is possible for the 5′-dAdo• to react
unproductively, for example by abstracting the wrong H atom from the substrate or the protein. How
selective H-atom abstraction occurs upon generation of the 5′-dAdo• is therefore a critical subject of
investigation. Structural modeling and spectroscopic studies of adenosylcobalamin enzymes suggest that
the 5′ carbon must traverse several ångstroms (ca. 6 Å) between the Co center and the target substrate H
atom.13–16 In contrast, structural and spectroscopic studies of radical SAM enzymes with SAM and
substrate bound reveal that the substrate X–H bond is positioned in close proximity to the 5′-dAdo carbon
(ca. 4 Å, corresponding to only ca. 3 Å to the target H atom; Figure 1, inset).17,18 Based on these findings,
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S
O
O
A
O
S
O
O
X–H
(substrate)
H2N
S
H2N
A
+ e–
O
OH OH
5′-dAdo•
OH
Fe
OH
Fe
X• + 5′-dAdoH
+
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S
S
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Fe
Fe
Fe
(Cys)S
S
(Cys)S
S
~ 5 Å
Fe
Fe
S(Cys)
S(Cys)
(Cys)S
(Cys)S
product
S
S
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3.8 Å
4.9 Å
+ e–
S
O
~ 2 Å
A
O
O
H2N
Fe
S
S
OH
OH
Fe
Fe
(Cys)S
S
Fe
S(Cys)
(Cys)S
S
organometallic
intermediate
Figure 1. Proposed mechanism for 5′-dAdo• formation in radical SAM enzymes showing
the bond-distance changes incurred upon Fe–C bond formation and homolysis. (Inset)
Structure of the lysine 2,3-aminomutase active site (PDB ID: 2A5H). From the available
structural and spectroscopic data, the 5′ carbon appears to be primed for selective H-atom
abstraction, but formation and homolysis of an Fe–C bond would require significant
rearrangement in the active site with the 5′ carbon moving away from the substrate X–H
bond to form an Fe–C bond and back toward the substrate to undergo H-atom abstraction.
it was proposed17 that the closer proximity of the substrate’s target H atom and the 5′-carbon in radical
SAM enzymes compared to adenosylcobalamin enzymes may explain why radical SAM enzymes
outnumber19,20 adenosylcobalamin enzymes by a factor of ~104: because radical SAM enzymes position
the substrate X–H bond adjacent to the nascent 5′-dAdo•, their active sites are predisposed to perform
selective H-atom abstraction upon generation of the 5′-dAdo•.
This narrative has been somewhat complicated by the discovery of an unexpected intermediate in
5′-dAdo• formation: an organometallic species—observed in a wide range of radical SAM enzymes—that
features a bond between an Fe of the [Fe4S4] cluster and the 5′-dAdo carbon, akin to the Co–C bond in
adenosylcobalamin (Figure 1).21,22 Efforts to understand this species’ precise geometric and electronic
structure are ongoing and its role as an intermediate in 5′-dAdo• generation raises several questions. First,
because Fe–C bond homolysis is an unprecedented reaction for Fe–S clusters, it is unclear if and how the
Fe–C bond in an alkylated [Fe4S4] cluster can be sufficiently weakened to generate primary carbon
radicals. Second, the careful positioning of the 5′ carbon near the target H atom—certainly an important
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factor for selectivity—must also be considered in light of the geometric changes required to form and
break an Fe–C bond (Figure 1);23 if such dramatic rearrangements occur during catalysis, then rigid
positioning of the substrate and SAM cannot fully account for the selectivity of radical SAM enzymes.
Therefore, other mechanisms for achieving selectivity must be considered, particularly those that take into
account the dynamic nature of the active site.
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We herein address these questions with a combination of model chemistry and kinetic simulations.
In contrast to synthetic models of adenosylcobalamin enzymes, which have provided insights into the
structure and reactivity of adenosylcobalamin,24–33 there are no functional models of the organometallic
intermediate in radical SAM enzymes (i.e., alkylated [Fe4S4] clusters that generate alkyl radicals). Using
synthetic [Fe4S4]–alkyl clusters, we show that even highly reactive, primary carbon radicals can be
generated from [Fe4S4]–alkyl clusters and in doing so delineate the coordination-chemistry requirements
for Fe–C bond homolysis. Notably, we find that Fe–C bond homolysis is both facile and reversible. Using
kinetic simulations, we then evaluate the circumstances in which reversible Fe–C bond homolysis can
affect the selectivity of H-atom abstraction. Our findings suggest that, by slowing the rates of all reactions
of the 5′-dAdo•, an organometallic intermediate may be key to achieving selective reactivity in radical
SAM enzymes.
Results and Discussion
Synthesis of alkylated [Fe4S4] clusters
We recently reported the first [Fe4S4]–alkyl cluster, which is stable with respect to Fe–C bond
homolysis at room temperature.34 Our current study centers on the chemistry of alkylated [Fe4S4] clusters
supported by the N-heterocyclic carbene IMes (IMes = 1,3-dimesitylimidazol-2-ylidene); the complement
of three strongly binding IMes ligands increases the stability of these clusters and makes them suitable for
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reactivity and mechanistic studies. Synthetic access to IMes-supported [Fe4S4]–alkyl clusters is afforded
by treatment of (IMes)3Fe4S4Cl (1)35 with the appropriate Grignard reagent to provide
(IMes)3Fe4S4(benzyl) (2) and (IMes)3Fe4S4(octyl) (3) in 64% and 66% yield, respectively (Figure 2).
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Cl
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IMes
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IMes
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IMes
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[Cp2Co]+
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RMgCl
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2: R = benzyl
3: R = octyl
[2]+: R = benzyl
[3]+: R = octyl
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NaBArF
1 equiv. DMAP
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[4]+
Figure 2. Synthesis of alkylated [Fe4S4]+/2+ clusters and an [Fe4S4]+–DMAP adduct. (Inset)
Thermal ellipsoid (50%) plot of [2][OTf] (left) and [4][OTf] (right). Color scheme: carbon
(gray), iron (orange), sulfur (yellow), nitrogen (blue), oxygen (red), fluorine (green).
The 1H NMR resonances from the IMes ligands in 2 and 3 are similar to those observed for 1. The α
protons of the benzyl and octyl groups are observed at 223 and 258 ppm for 2 and 3, respectively; the β
protons of the octyl group in 3 are observed at 22 ppm. EPR spectroscopic analysis establishes that both
clusters have an S = ½ ground spin state (Figures S53 and S54). The one-electron oxidized clusters
[(IMes)3Fe4S4(benzyl)][BArF ] ([2]+, [BArF ] = [tetrakis(3,5-trifluoromethyl)phenyl]borate)]) and
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[(IMes)3Fe4S4(octyl)][BArF ] ([3]+) were synthesized by reaction of 2 or 3 with [Cp2Co][BArF ]. Upon
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oxidation, the resonances in the 1H NMR spectrum corresponding to the α protons of the benzyl and octyl
groups shift upfield to 68 ppm for [2]+ and 75 ppm for [3]+; the β protons in [3]+ also shift upfield to –3.5
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ppm. These shifts are similar to the 1H NMR shifts observed in the previously reported [Fe4S4]2+–Et
cluster34 and are indicative of a diamagnetic ground state with thermally populated, paramagnetic excited
states. Clusters 2 and [2]+ have been crystallographically characterized (Figure 2 (inset) and S80); their
metrical parameters are similar to those of 1 and of the previously reported alkyl cluster.34
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Observation of Fe–C bond homolysis
In comparison to the organometallic intermediate characterized in radical SAM enzymes—which
is consumed in minutes at 170 K21—the alkylated clusters described above are strikingly stable. We
attribute this to the coordination number of the alkylated Fe site: homolysis of the Fe–C bond would lead
to a high-energy intermediate with a three-coordinate Fe site. In contrast, homolysis of the Fe–C bond in
the organometallic intermediate in radical SAM enzymes, which is thought to have a six-coordinate Fe
site, would generate an energetically feasible, five-coordinate Fe site. We therefore hypothesized that
addition of an exogenous ligand to the synthetic clusters could lead to Fe–C bond homolysis through an
intermediate with a five-coordinate Fe site. Indeed, reaction of [2]+ with an excess of a pyridine, such as
4-N,N-dimethylaminopyridine (DMAP), results in a rapid reaction at room temperature to generate the
reduced [Fe4S4]+ cluster [(IMes)3Fe4S4(DMAP)][BArF ] ([4]+, 95 ± 3%) and bibenzyl (92 ± 3 % yield)
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(Scheme 1A). The identity of [4]+ was confirmed by its independent synthesis and characterization (Figure
2). Fe–C bond homolysis extends to other alkyl groups including those whose corresponding radicals are
much more reactive than the benzyl radical. For example, when DMAP is added to [3]+, [4]+ (92 ± 3 %)
and hexadecane (75 ± 8 % yield) are rapidly formed (Scheme 1B). The rate of C–C bond formation scales
with the substituent Hammett parameters;36 when DMAP (σp = –0.83) is added to [2]+, the reaction is
complete in less than 10 min, whereas the reaction of [2]+ with pyridine (σp = 0.0) results in ca. 90%
conversion in 15 min and that of [2]+ with 4-trifluoromethylpyridine (CF3-py, σp = 0.54) takes over 24 h
to reach 90% completion (Scheme 1C). The rates increase with the donicity of the substituted pyridine,
consistent with pyridine coordination preceding Fe–C bond homolysis. Fe–C bond homolysis is not
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Scheme 1. Formation of alkyl radicals from [Fe4S4] clusters. (A) Generation and trapping of benzyl
radicals upon treatment of [2]+ with DMAP. (B) Generation and trapping of octyl radicals upon
treatment of [3]+ with DMAP. (C) Reaction of [2]+ with substituted pyridines.
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limited to pyridinyl donors; addition of quinuclidine generates the quinuclidine adduct (98 ± 3 %) and
bibenzyl (90 ± 4 %).
The reactions of [2]+ and [3]+ with exogenous ligands are consistent with donor-induced Fe–C
bond homolysis followed by combination of the radical with another benzyl or alkyl group to form
bibenzyl or hexadecane (the C–C bond-forming step could occur by one or more pathways involving a
radical; see SI p. S39). Such mechanisms involving M–C bond homolysis would align our findings with
those observed for adenosylcobalamin model complexes24,29,33,37 and those proposed for organometallic
intermediates in radical SAM enzymes.21,22,38 However, these observations do not rule out other
conceivable mechanisms that proceed without formation of organic radicals (i.e., those in which bibenzyl
or hexadecane is formed through concomitant Fe–C bond cleavage and C–C bond formation). No
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intermediates were observable by NMR or EPR spectroscopies, so to distinguish between these
mechanistic possibilities we performed a series of experiments to assay for the generation of organic
radicals.
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As an initial test, we carried out the reaction of [2]+ and [3]+ with DMAP in the presence of a
radical trap. We chose Bu3SnH because it does not react with either cluster in the absence of DMAP and
the expected organic products (toluene for [2]+ and octane for [3]+) are readily detected by 1H NMR
spectroscopy and GC–MS, respectively. When DMAP is added to [2]+ in the presence of 20 equiv. of
Bu3SnH, the reduced cluster [4]+ is formed in less than 10 minutes. Alkyl groups are detected as a mixture
of toluene (34 ± 3%), bibenzyl (33 ± 3%), and Bu3SnBn (17 ± 3%) (Scheme 1A). That both bibenzyl and
toluene were observed in this reaction indicates incomplete trapping of the benzyl radicals by Bu3SnH and
suggests that the pathways that lead to bibenzyl and toluene occur with competitive rates. Addition of
DMAP to [3]+ in the presence of Bu3SnH induces similar reactivity with production of octane (56 ± 6%;
Scheme 1B). The reaction of [2]+ with DMAP was also carried out in the presence of Bu3SnD (90% D),
which resulted in the formation of bibenzyl (54 ± 3%), toluene (7 ± 3%), d1-toluene (13 ± 3%) and
Bu3SnBn (11 ± 3%) (Scheme 1A). Formation of d1-toluene indicates that Bu3SnD is the deuterium-atom
source and the lower deuterium incorporation into toluene (1:1.9 H:D) compared to Bu3SnH/D (1:9 H:D)
is indicative of a kinetic isotope effect for H/D-atom abstraction from Bu3SnH/D.
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Because radical traps can alter the major mechanistic pathway of the reaction—and therefore not
provide definitive evidence of free-radical intermediates—we pursued the use of radical clocks as a
complementary test for the generation of free radicals. For these purposes, we wanted a radical clock that
rearranges on a timescale similar to or faster than the rate of other reactions involving organic radicals,
which we estimated based on the following observations. In the reaction of [2]+ with DMAP, the benzyl
radical is only partially trapped by Bu3SnH, suggesting that other reactions leading to bibenzyl formation
are competitive with trapping. Because H–SnBu3 abstraction by an organic radical occurs at ~106 s–1,39
we predicted that a radical clock with a similar rate constant would exhibit at least partial rearrangement
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under the Fe–C bond homolysis conditions. We therefore selected the 5-hexenyl radical clock, which
rearranges to the cyclopentylmethyl radical with a rate constant of 1.8 x 105 s–1 (20 °C),40,41,42 and assayed
for organic products derived from the 5-hexenyl and/or cyclopentylmethyl radicals.
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To test these predictions, we prepared (IMes)3Fe4S4(5-hexenyl) (5) and [(IMes)3Fe4S4(5-
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hexenyl)][BArF ] ([5]+) using the methodology described above. The 1H NMR spectra of 5 and [5]+ are
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similar to those of 3 and [3]+; the α and β protons of the 5-hexenyl group shift from 256 ppm and 24.5
ppm, respectively, to 74 ppm and -4.0 ppm, respectively, upon oxidation of 5 to [5]+. Addition of excess
DMAP to [5]+ leads to immediate formation of [4]+ and coupled organic fragments. Integration of the 1H
NMR spectrum suggests that approximately 72 ± 2 % of the alkyl groups are cyclized, while 28 ± 2% of
the alkyl groups still contain alkene resonances (Figures S50 and S51). The observation of both cyclized
and uncyclized organic products strongly supports that the Fe–C bond undergoes homolysis upon addition
of DMAP to [5]+ and suggests that the rates of subsequent steps involving the organic radical are indeed
competitive with cyclization.
Evidence for reversible Fe–C bond homolysis
We also carried out the reaction of [5]+ with an excess of CF3-py. As expected, addition of CF3-
py induces consumption of [5]+ and formation of the CF3-py adduct, [6]+. This reaction takes more than
24 h to reach completion (much slower than when DMAP is added) because of the diminished equilibrium
for CF3-py binding. Intriguingly, a second species, characterized by peaks in the 1H NMR spectrum at 75
ppm and –3 ppm, increases in intensity over the course of this reaction (Figure 3). Given the similarity of
the 1H NMR resonances to those of the other [Fe4S4]2+–alkyl clusters described herein, we hypothesized
that this new species was [(IMes)3Fe4S4(cyclopentylmethyl)][BArF ] ([7]+), containing a
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cyclopentylmethyl group derived from the 5-hexenyl radical; its identity was confirmed by independent
synthesis of [7]+. We note that [5]+ does not convert to [7]+ in the absence of added pyridine. The
conversion of [5]+ to [7]+ via organic radicals demonstrates that Fe–C bond homolysis is reversible,
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Figure 3. Partial 1H NMR spectrum of the
reaction between [5]+ and CF3-py showing the
decay of [5]+ and the growth of [7]+ during the
course of the reaction. The top trace is an
authentic sample of [7]+.
whereby pyridine binding to [5]+ induces formation of the transient 5-hexenyl radical, which rearranges
to the cyclopentylmethyl radical and recombines with [6]+ to give [7]+ (Scheme 2A).
Based on these findings, we predicted that Fe–C bond homolysis in the reaction of [2]+ with
pyridines should also be reversible, and that if this was the case, [(IMes)3Fe4S4(pyridine)][BArF ] ([8]+)
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would inhibit the radical termination steps that lead to bibenzyl. To test this, we added pyridine to [2]+ in
the presence and absence of additional [8]+ (Scheme 2B); we chose to perform this test with pyridine and
[8]+ rather than DMAP and [4]+ to slow the reaction so that the effect of the additional pyridine adduct
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would be easier to observe by H NMR spectroscopy. In the absence of added [8]+, the reaction of [2]+
and pyridine to yield [8]+ and bibenzyl is ca. 90% complete in 15 minutes. Strikingly, in the presence of
10 equiv. of added [8]+, the reaction is only ca. 50% complete after 2 h. Taken together, these observations
demonstrate rapid and reversible alkyl radical formation from [Fe4S4] clusters. That [Fe4S4] clusters
exhibit this reactivity, which has also been observed in other open-shell metal complexes37,43–46—most
notably adenosylcobalamin models—further deepens the connections between the chemistry of
adenosylcobalamin and radical SAM enzymes.
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Scheme 2. Evidence for reversible Fe–C bond homolysis. (A) Proposed mechanism for the conversion
of [5]+ to [7]+. (B) Reaction of [2]+ with pyridine in the presence of an excess of [8]+ decreases the rate
of bibenzyl formation.
Kinetic simulations of reversible Fe–C bond homolysis in radical SAM enzymes
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Our results demonstrate that even primary alkyl radicals can be produced from [Fe4S4]–alkyl
clusters, providing support for the intermediacy of organometallic species in radical SAM enzymes.21,22,38
We now consider how an organometallic intermediate that generates an alkyl radical can impact catalysis
in radical SAM enzymes. First, an organometallic precursor to the 5′-dAdo• would clearly decrease the
population of the 5′-dAdo•, with a stronger Fe–C bond shifting the homolysis equilibrium towards the
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organometallic species. An Fe–C bond that is too strong could shut down 5′-dAdo• production entirely,
but our results suggest that radical SAM enzymes may avoid this scenario by virtue of the coordination
geometry at the unique Fe site: coordination of the amine (and likely the carboxylate) of methionine
renders the apical Fe site five- or six-coordinate. As we have shown here, coordination numbers greater
than four for the alkylated Fe results in a dramatically weakened Fe–C bond.
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However, even a weak Fe–C bond would significantly deplete the population of the 5′-dAdo•. This
would decrease the rates of all subsequent steps, but how would it impact selectivity for productive versus
unproductive reactions? Of course, if the 5′-dAdo• and substrate are perfectly positioned for abstraction
of only the desired H atom, the selectivity for the productive pathway would be perfect, and an
organometallic intermediate would have no impact on selectivity. Even if the 5′-dAdo• can undergo
unproductive reactions, such as abstracting the wrong H atom from the substrate or protein, the selectivity
would depend only on the relative rates of the productive and unproductive pathways (Figure 4A and 4B).
An organometallic intermediate can only affect the selectivity of the H-atom abstraction step if an
additional, kinetically coupled process is operative. Multiple selectivity-influencing processes can be
envisioned including bond rotations, rearrangements in the active site, or larger conformational changes.
Regardless of their precise nature, such processes will affect the selectivity of the reaction only if they can
outcompete X–H bond activation. Under these conditions, an organometallic intermediate can affect
selectivity by slowing the rates of X–H bond activation to allow these processes to occur.
We evaluated this hypothesis using a simplified kinetic model consisting of a radical SAM enzyme
for which the selectivity of X–H bond activation is dictated by whether the protein is in a productive state
(denoted P and representing states leading to the substrate radical) or an unproductive state (denoted U
and representing states leading to any other reaction of the 5′-dAdo• ) (Figure 4C). In choosing rate
constants for these simulations, we assumed that recombination of the 5′-dAdo• with the cluster is rapid
(k–1 = 1011 s–1), that interconversion between protein states is slow (k2 = 102 s–1 and k–2 = 1 s–1, favoring
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Figure 4. (A) Kinetic model and (B) energy diagram for a system in which the 5′-dAdo• reacts
through a branching path with fixed rates of X–H abstraction. The presence of the organometallic
intermediate has no effect on the selectivity. (C) Kinetic model that invokes interconversion between
productive (P) and unproductive (U) states in the absence (black) and presence (black and red) of an
organometallic intermediate. (D) Quantitative energy diagram for the system depicted in C showing
the effect of the organometallic intermediate using barriers calculated from the rate constants listed
below. As the Fe–C bond strength increases, the barrier to state interconversion becomes lower than
the barrier to homolysis and X–H bond activation. (E) Simulations showing the selectivity of the
reaction as a function of the rate of state interconversion and Fe–C bond strength. (F) Simulations
showing the selectivity of the reaction as a function of the rate of X–H bond activation and Fe–C bond
strength. k–1 (1011 s–1), k2 (102 s–1), and k3 (105 s–1) were held constant and k1 was varied to give the
indicated Fe–C bond strengths.
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the productive state by ~ 2 kcal/mol), and that Fe–C bond homolysis occurs slower than X–H bond
activation (k1 = 103 s–1, k3 = 105 s–1). Further discussion of these rate constants is in the SI. Under these
conditions, the slow rate of state interconversion favors X–H bond activation from the initial state; if the
radical is formed in the unproductive state, state interconversion must outcompete unproductive X–H bond
activation for productive reactivity to occur. We therefore initialized our simulations with the 5′-dAdo• in
the unproductive state as a worst-case scenario to determine if an organometallic intermediate can recover
selectivity for the productive reaction.
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With these rate constants and in the absence of the organometallic intermediate, 99% of the radical
is funneled to undesired pathways because, as noted above, H-atom abstraction is much faster than state
interconversion. However, if an organometallic intermediate is formed, the H-atom abstraction step is
sufficiently slowed such that the reaction occurs with 99% selectivity for the productive pathway (note
that given the difference in energy of the productive and unproductive states, the maximum selectivity
under thermodynamic control is 99%). Thus, if SAM is cleaved when the protein is in an unproductive
state (e.g., if the initial substrate positioning is somehow perturbed), formation of the Fe–C bond could
buy time for the protein to convert to a state that yields productive reactivity. Figure 4D illustrates the
energetics of this process: introducing an Fe–C bond raises the barrier to X–H bond activation relative to
state interconversion. This shifts the reaction from being under kinetic control, in which X–H bond
activation outcompetes state interconversion, to being under Curtin-Hammett control, in which the relative
X–H bond activation barriers control the selectivity.
We also probed how selectivity is affected by the individual kinetic parameters. Figure 4E shows
the reaction outcome as a function of the rate of state interconversion with a range of Fe–C bond strengths;
as the Fe–C bond strength is increased, higher selectivity for the productive reaction can be obtained at
slower rates of state interconversion. This demonstrates how even a weak Fe–C bond in an organometallic
intermediate can allow for relatively slow processes to impact the selectivity of the reaction. An analogous
effect is observed when the rate of (both productive and unproductive) X–H bond activation is varied
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(Figure 4F); increasing the Fe–C bond strength allows for high selectivity even with very fast rates of X–
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Overall, these results support a mechanism by which reversible formation of an organometallic
intermediate can affect the selectivity in radical SAM reactions: it increases the range of kinetic parameters
(X–H abstraction rates and state interconversion rates) for which interconversion between productive and
unproductive states can occur, allowing these processes to outcompete what may be intrinsically very fast
unproductive reactions.
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Connection between reversible M–C bond homolysis in radical SAM and adenosylcobalamin enzymes
The protective mechanism described above is agnostic with respect to the identity of the metal
fragment, and therefore may apply equally well to adenosylcobalamin enzymes for which reversible Co–
C bond cleavage has been observed.45-47 Evidence for reversible Co–C bond homolysis was provided by
rate measurements using protiated and deuterated substrate, which showed an H/D kinetic isotope effect
on the apparent rate of Co–C bond homolysis that results from kinetic coupling of Co–C bond homolysis
and H-atom abstraction.47–50 Similar kinetic experiments carried out with radical SAM enzymes could
determine if reversible Fe–C bond cleavage occurs during turnover of radical SAM enzymes. The
observation of a kinetic isotope effect for consumption of the organometallic intermediate when the
substrate is deuterated would simultaneously provide evidence that the organometallic intermediate is on-
path for productive chemistry and that Fe–C bond homolysis is reversible in enzymatic systems.
Conclusion:
The observation of facile Fe–C bond homolysis from [Fe4S4]–alkyl clusters demonstrates the
feasibility of 5′-dAdo• formation from organometallic intermediates in radical SAM enzymes and links
the chemistry of radical SAM and adenosylcobalamin model systems. Similarly to Co–C bond homolysis
in adenosylcobalamin model systems, Fe–C bond homolysis from [Fe4S4]–alkyl clusters occurs reversibly
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to generate primary carbon radicals, but unlike in adenosylcobalamin models, homolysis of Fe–C bonds
in [Fe4S4]-alkyl clusters is facile at room temperature. Overall, these findings suggest that in both
adenosylcobalamin enzymes and radical SAM enzymes, reversible homolysis of M–C bonds can allow
for selective X–H bond activation by kinetically coupling reactions of the 5′-dAdo• to dynamic processes
within or beyond the protein active site. Studies of the dynamics of these proteins will help illuminate how
radical SAM and adenosylcobalamin enzymes utilize reversible M–C bond homolysis as a strategy to
control the 5′-dAdo• and will contribute to our understanding of the differences between these two enzyme
families.
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Acknowledgements:
We thank Dr. Peter Müller for assistance with XRD experiments, Dr. Fang Wang for assistance with GC-
MS measurements, and Profs. Alexander Radosevich and Alison Wendlandt for critical feedback on the
manuscript. Research reported in this publication was supported by the National Institute of General
Medical Sciences of the National Institutes of Health under award number R01GM136882 and by
fellowships for ACB from the National Science Foundation (Graduate Research Fellowship #1122374)
and the Fannie and John Hertz Foundation.
Supporting Information:
Experimental procedures, spectra, additional discussion, and script for kinetic simulations
This material is available free of charge via the Internet at http://pubs.acs.org.
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