S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-Adenosyl-l-methionine (SAM) in the Radical SAM Enzyme HydG

Article abstract of DOI:10.1002/anie.202014337

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5′-deoxyadenosyl radical (5′-dAdo^.). A [4Fe-4S]⁺ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5′-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5′-dAdo^.. We present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE-bound HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77 K, the ethyl radical adds to the [4Fe-4S]²⁺ cluster, generating an ethyl-[4Fe-4S]³⁺ organometallic species termed Ω_E.

Full text of DOI:10.1002/anie.202014337

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-
Adenosyl-l-methione (SAM) in the Radical SAM Enzyme HydG
Authors: Stella Impano, Hao Yang, Eric M. Shepard, Ryan Swimley,
Adrien Pagnier, William E. Broderick, Brian M. Hoffman, and
Joan B. Broderick
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202014337
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10.1002/anie.202014337
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RESEARCH ARTICLE
S-Adenosyl-L-ethionine is a Catalytically Competent Analog of S-
Adenosyl-L-methione (SAM) in the Radical SAM Enzyme HydG
Stella Impano,[a] Hao Yang,[b] Eric M. Shepard,[a] Ryan Swimley,[a] Adrien Pagnier,[a] William E.
Broderick,[a] Brian M. Hoffman,*[a] Joan B. Broderick*[a]
[a]
Dr. S. Impano, Dr. E. M. Shepard, Mr. R. Swimley, Dr. A. Pagnier, Prof. W. E. Broderick, Prof. J. B. Broderick
Department of Chemistry & Biochemistry
Montana State University
Bozeman, MT. USA. 59717
E-mail: jbroderick@montana.edu
Dr. H. Yang, Prof. B. M. Hoffman
Department of Chemistry
[b]
Northwestern University
Evanston, IL. USA 60208
Supporting information for this article is given via a link at the end of the document.
Abstract: Radical S-adenosyl-L-methionine (SAM) enzymes initiate
biological radical reactions with the 5’-deoxyadenosyl radical (5’-
dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω
organometallic intermediate in which the 5’-deoxyadenosyl moiety is
directly bound to the unique iron of the [4Fe-4S] cluster, with
subsequent liberation of 5’-dAdo•. Here we present synthesis of the
SAM analog S-adenosyl-L-ethionine (SAE) and show SAE is a
mechanistically-equivalent SAM-alternative for HydG, both supporting
enzymatic turnover of substrate tyrosine and forming the
organometallic intermediate Ω. Photolysis of SAE bound to HydG
forms an ethyl radical trapped in the active site. The ethyl radical
withstands prolonged storage at 77 K and its EPR signal is only
partially lost upon annealing at 100 K, making it significantly less
reactive than the methyl radical formed by SAM photolysis. Upon
annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster,
generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.
AE) [4Fe-4S]+:SAM complex cleaves the S-C5’ bond of SAM to
generate 5’-dAdo• trapped in the PFL-AE active site, enabling the
first direct characterization of this important radical intermediate.[9]
HydG is a RS enzyme essential for the maturation of the [FeFe]-
hydrogenase (Fig 1), catalyzing the radical decomposition of
tyrosine to produce CO and CN–, both of which end up as ligands
in the H-cluster at the active site of [FeFe]-hydrogenase.[10-13]
Radical formation occurs at a site-differentiated [4Fe-4S] cluster
bound near the N-terminus of HydG, which coordinates SAM and
catalyzes reversible reductive cleavage of SAM to generate the
5’-dAdo• species. This ultimately abstracts an H atom from the
amino of tyrosine,[13-15] through a mechanism that proceeds via
the
Ω organometallic intermediate central to RS enzyme
reactions.[2] The initial products of tyrosine cleavage are p-cresol
and dehydroglycine;[16] the latter migrates towards an auxiliary
cluster that is involved in the subsequent chemistry, ultimately
forming CN– and CO.[17] These diatomic products, perhaps
together with iron in the form of a small-molecule synthon,[18-20]
are delivered to the GTPase scaffold protein HydF.[21-24] The RS
enzyme HydE is proposed to provide the dithiomethylamine
(DTMA) ligand that forms a 2Fe H-cluster precursor on HydF
([2Fe]F), which is then delivered to HydA to produce the active
hydrogenase.[25-27] A recent report provides intriguing evidence
that the substrate of HydE is a HydG-generated cysteine-
coordinated organometallic synthon.[28]
Introduction
Catalysis by radical S-adenosyl-L-methionine (radical SAM or RS)
enzymes is accomplished via the reductive cleavage of SAM to
initially generate an organometallic intermediate Ω comprising a
5’-deoxyadenosyl ligand covalently bound to the unique iron of
the active-site [4Fe-4S]3+ cluster.[1-2] The intermediate Ω then
liberates a 5’-deoxyadenosyl radical (5’-dAdo•) via homolytic Fe-
C bond cleavage, and this radical abstracts an H-atom from
substrate to initiate the wide variety of reactions performed by the
RS superfamily.[3-7] Suess and coworkers have recently
synthesized a model for alkyl-[4Fe-4S]3+ clusters that shows
spectroscopic properties similar to Ω.[8] The discovery of Ω
reinforced the remarkable catalytic parallels between RS and
adenosylcobalamin (B12) radical enzymes, with both using
homolytic metal-carbon bond cleavage of an organometallic
species to generate the central 5’-dAdo• intermediate responsible
for H-atom abstraction.[1-2] Despite its central role in radical
initiation in both enzyme families, the 5’-dAdo• radical remained
an elusive species for nearly 60 years, escaping direct
observation and characterization. Our recent discovery of novel
photochemistry of SAM-bound [4Fe-4S]+ clusters in RS enzyme
active sites changed this: we showed that photoinduced electron
transfer in the pyruvate formate-lyase activating enzyme (PFL-
Reduced HydG with SAM bound to the [4Fe-4S]+ cluster
furthermore undergoes cryogenic photoinduced electron transfer
from the cluster to SAM, resulting in regiospecific cleavage of the
S-CH3 bond, rather than the S-C5’ bond (as in the case of PFL-
AE).[29-30] This generates a •CH3 radical in the active site, as
characterized by electron paramagnetic resonance (EPR)
spectroscopy.[29] We found that this •CH3 undergoes rapid
rotational diffusion even at 40 K, and converts to a slower-
tumbling state at lower temperatures.[29] The •CH3 radical decays
rapidly at 77 K with a half-life of minutes,[29] in contrast to the 5’-
dAdo• radical formed by photolysis of [4Fe-4S]+:SAM in PFL-AE,
which is indefinitely stable at 77 K.[9] We concluded that the •CH3
is intrinsically more reactive than 5’-dAdo•, primarily because of
its small size and ability to undergo rapid translational diffusion in
search of a reaction partner.[29]
1
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NH2
NH2
N
Ω
NH2
N
NH2
N
N
N
N
N
N
N
N
N
RH
RH
R
RH
N
N
O
N
N
N
O
O
H
O
H3C
H3C
H3C
H3C
OH
H
H3C
S
OH
S
OH
S
S+
OH
H
H
OH
OH
OH
OH
1+
O
O
O
3+
O
2+
O
2+
O
O
O
N
H2
N
H2
N
H2
N
H2
O
O
C
5’-dAdo•
5’-dAdoH
C
CO
CN-
24 Å
NH3
HydG
SAM
HO
HO
Figure 1. Top: Radical initiation mechanism in RS Enzymes. Lower Left: HydG structure showing the RS [4Fe-4S] cluster and the auxiliary cluster. Lower Right:
HydG catalyzed tyrosine cleavage into diatomic CO and CN- ligands.
This report focuses on the reactivity of the ethyl analog of SAM,
S-adenosyl-L-ethionine (SAE) with HydG. SAE can form in vivo in
yeast and in mammals fed a diet containing ethionine,[31-32] and
has been shown to inhibit some methyltransferase reactions,[33-36]
but we are not aware of any prior studies of SAE reactivity with
RS enzymes. Ji et al. have reported on the use of the SAM analog
allyl-SAM in mechanistic investigations of the radical SAM
enzyme NosN.[37] We report here the enzymatic synthesis and
characterization of SAE, and show that HydG can utilize SAE as
an effective alternative cosubstrate to SAM under normal
enzymatic turnover conditions, producing the Ω intermediate and
allowing efficient catalytic turnover of substrate L-tyrosine. We
also report that cryogenic photolysis of the HydG [4Fe-4S]+:SAE
complex results in S-CH2CH3 homolytic bond cleavage to form a
•CH2CH3 radical trapped in the HydG active site, which we have
characterized by EPR spectroscopy. This ethyl radical has
significantly greater stability than the methyl radical studied
previously, persisting under prolonged storage at 77 K and
through annealing at 100 K.[29] Upon annealing to higher
temperatures this radical adds to the [4Fe-4S]2+ cluster to
generate an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.
Our results provide a window into the remarkable versatility of RS
enzymes in generating alkyl radicals to initiate diverse biological
radical reactions.
approximately 25% that of a typical SAM preparation, most likely
reflecting a lower specific activity for SAM synthetase with
ethionine vs. methionine. The synthesis of SAE was verified by
NMR (Fig S1) and HPLC-MS (Fig S2). The NMR showed
characteristic resonances for the ethyl group vs. methyl group of
SAM. Close examination of the characteristic triplet for the methyl
group of the -CH2CH3 at 1.45 ppm reveals a smaller triplet slightly
upfield (1.41 ppm), which is due to the R,S-diastereomer of SAE.
Relative integration gives approximately 10% of the R,S product
in the synthesized SAE, indicating some racemization occurred
during the workup and purification procedures, similar to what is
observed for SAM.[39]
EPR Evidence for SAE Coordination to the HydG [4Fe-4S] Cluster.
HydG is a RS enzyme with two [4Fe-4S] clusters: the RS [4Fe-
4S] cluster that binds near the N-terminal of the protein ([4Fe-
4S]RS), and an auxiliary cluster that binds near the C-terminus
([4Fe-4S]aux). Under anoxic and reducing conditions, both
clusters are in the EPR-active S=1/2 [4Fe-4S]+ cluster state and
have distinct EPR g-values: for the [4Fe-4S]+RS, g = [2.03, 1.935,
1.893], and for the [4Fe-4S]+aux, g = [2.027, 1.919, 1.888] (Fig 2,
Fig S3). Upon addition of SAM, the EPR signal of the [4Fe-4S]+
RS
is perturbed, with g = [1.998 1.88 1.834], while that of the [4Fe-
4S]+
is unchanged, Fig 2. The g-values were obtained by
aux
simulations of the observed spectra in Fig 2 with a two cluster
model (Figs S3, S4); the obtained g-values agree well with
previously reported values.[13]
Results and Discussion
The changes in the EPR spectra of [4Fe-4S]+RS upon addition of
Synthesis of SAE. SAE was synthesized in vitro from ATP and
ethionine using E. coli SAM synthetase. SAE was purified on a
cation exchange column, eluting at the same concentration of HCl
eluent as required for elution of SAM.[38] The yield of SAE from the
enzymatic synthesis and subsequent purification was
SAM arise from coordination of the SAM amino and carboxylate
[3, 25]
moieties to the unique iron of the [4Fe-4S]+
,
RS
as has been
observed in a wide range of RS enzymes, although the details
vary from enzyme to enzyme and can depend on buffer conditions
2
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10.1002/anie.202014337
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deoxymyoglobin serving as a detector of the product CO.[11] The
results (Fig 3) show that SAE is able to replace SAM during HydG
catalysis, with assays containing SAE showing CO production
rates (apparent first-order rate, k = 0.035 ± 0.003 min-1) that were
80% of those observed with SAM (k = 0.044 ± 0.004 min-1). LC-
MS analysis confirms that this SAE-supported turnover is
accompanied by the production of 5’-deoxyadenosine, a normal
product of SAM turnover; thus SAE, like SAM, supports normal
catalytic function of HydG, wherein the interaction of SAE (or
HydG
HydG + SAM
HydG + SAE
SAM) with the [4Fe-4S]+ results in reductive S-C5’ bond
RS
cleavage to produce a 5’-dAdo• radical which abstracts a H atom
from substrate L-tyrosine to initiate the conversion of tyrosine to
p-cresol, CO, and CN-.
3000
3250
3500
3750
4000
Magnetic Field (Gauss)
Figure 2. EPR spectral evidence for SAE binding to the [4Fe-4S]+ cluster of
HydG. CW-EPR spectra of reduced HydG (top) is due to the overlay of signals
arising from [4Fe-4S]+ and [4Fe-4S]+AUX. Addition of SAM to reduced HydG
RS
(middle) perturbs the [4Fe-4S]+ EPR signal while the [4Fe-4S]+
signal
AUX
RS
remains unchanged. Addition of SAE to reduced HydG (lower) also perturbs the
[4Fe-4S]+ EPR signal while the [4Fe-4S]+
signal remains unchanged.
RS
AUX
Conditions: [HydG] = 1 mM, [dithionite] = 3 mM, [SAM] or [SAE] = 5 mM. EPR
parameters: microwave frequency, 9.37 GHz; T = 12 K; modulation amplitude,
10 G.
as well as the monovalent cation present in the assay buffer.[40]
Also impacting the EPR spectra of HydG is the potential for an
additional FeII bridged to the [4Fe-4S]aux cluster via a cysteine
ligand; the presence of this 5th iron leads to an EPR-silent state
for the auxiliary cluster,[41] and therefore the contribution of the
auxiliary cluster to the EPR signal can vary depending on iron
loading.
Because of the sensitivity of the EPR signature for the [4Fe-4S]+
RS
cluster to SAM coordination, we used EPR spectroscopy to probe
whether the SAM analog SAE would bind to the unique iron of the
HydG [4Fe-4S]1+RS as does SAM. When SAE is added to HydG,
the EPR spectrum of [4Fe-4S]+RS cluster changes to one with g =
[2.012 1.876 1.854], different from the spectrum of unliganded
[4Fe-4S]1+RS and similar to the spectrum when SAM binds (Fig 2,
S5). This indicates that SAE binds to the [4Fe-4S]1+RS cluster in a
manner similar to that of SAM, presumably with chelation of the
unique iron through the amino nitrogen and carboxylate oxygen
Figure 3. HydG activity assays with SAE and tyrosine. Top, UV-visible spectral
changes as CO produced by HydG catalysis binds to H64L deoxy-Mb. Bottom,
kinetic traces for the HydG-catalyzed formation of CO with SAM (squares) and
SAE (circles). The red lines represent exponential fits to the data used to
determine kcat values provided in the text.
of SAE. The SAE-bound [4Fe-4S]+ has somewhat different g-
RS
values from the SAM-bound [4Fe-4S]+RS, as revealed through
simulations (Figs S4, S5); these differences likely reflect
perturbations in the unique iron coordination environment due to
the greater steric bulk of ethyl vs. methyl. Specifically, replacing
the methyl of SAM with an ethyl in SAE would likely change the
positioning of the positively-charged sulfonium relative to the
[4Fe-4S]+RS, which change would propagate to the Fe chelate and
would be expected to alter the EPR signal arising from this cluster.
Rapid freeze-quench (RFQ) experiments were carried out to
define the catalytic pathway for the reaction of HydG with SAE
and tyrosine. The RFQ experiments were performed by rapidly
mixing reduced HydG with a solution containing (tyrosine+SAM)
or (tyrosine+SAE) and freeze-quenching at 500 ms. The EPR
spectrum of HydG+(tyrosine+SAM) quenched 500 ms after
mixing shows the disappearance of the cluster signal of [4Fe-
4S]1+RS+SAM at g^=1.864, and the appearance of the
organometallic intermediate Ω with g|| = 2.035, g⊥ = 2.004, Fig 4,
as previously reported.[2] The EPR spectrum of freeze quenched
HydG+(tyrosine+SAE) is virtually identical to the Ω captured
SAE is a Catalytically Functional Cofactor. To determine whether
SAE can serve as a functional cofactor in RS enzyme turnover,
enzymatic activity assays were carried out under anoxic
conditions with reduced HydG, SAE, and tyrosine, with
3
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during RFQ with SAM and HydG (Fig 4), as well as the
organometallic Ω intermediates previously shown to be central to
catalysis in a wide range of radical SAM enzymes.[1-2]
observed upon photolysis of HydG complexed to SAM,[29] and
also different from the signal observed for the 5’-dAdo• generated
on photolysis of PFL-AE/SAM.[9] This multiline signal is associated
with the •CH2CH3 radical liberated by photolytic cleavage of SAE
in HydG. This is established by simulation of the EPR spectrum
on the basis of a carbon spin center coupled to the two •CH2- a-
protons, each with anisotropic hyperfine tensors, A(1H) = [80, 40,
60] MHz, and to three b-protons from a non-rotating methyl group,
the three having isotropic couplings, aiso(1Ha, 1Hb, 1Hc) = (110 MHz,
20 MHz and 20 MHz), Fig 5, upper. The photoinduced S-CH2CH3
cleavage of SAE occurs much more rapidly under steady-state
photolysis than does the photoinduced S-CH3 cleavage of SAM,
which suggests more efficient photoinduced electron transfer
within [4Fe-4S]1+RS:SAE than [4Fe-4S]1+RS:SAM.
Together, the enzymatic and spectroscopic results indicate that in
the presence of substrate and at optimal temperatures for
catalysis the ethyl-for-methyl substitution does not perturb the
local active site environment sufficiently to disrupt the normal
catalytic function of HydG. SAE in the HydG active site is bound
in a catalytically competent state and oriented in a manner that
favors reductive cleavage of the SAE S-5C’ bond, followed by
formation of Ω, liberation of 5’-dAdo•, and efficient catalytic
reaction with substrate to initiate the conversion of substrate
tyrosine to p-cresol, CO, and CN-.
On extending the time of radiation up to 1hr, the EPR signal of
•CH2CH3 gradually evolves into a radical signal with sharper and
more resolved 1H-hyperfine-split peaks, while maintaining the
same EPR breadth and total spin integration, Fig 5, lower. The
spectrum of the 1hr-radiation generated radical is well simulated
Figure 4. X-band CW-EPR of the catalytically central Ω trapped in HydG (250
µM) on reaction with tyrosine and SAM (red) or tyrosine and SAE (black)
quenched at 500 ms. Conditions: T = 12 K; microwave power, 2 mW; modulation
amplitude, 5 G; microwave frequency, 9.37 GHz; both have been annealed to
150K for 1 min to remove a very weak impurity signal.
Photolysis of SAE bound to HydG. We recently demonstrated that
blue-light photolysis of SAM bound to the active site [4Fe-4S]+
cluster of PFL-AE in the absence of PFL results in photoinduced
electron transfer from the reduced iron-sulfur cluster to SAM,
reductively cleaving the SAM to produce a 5’-deoxyadenosyl
radical (5’-dAdo•) trapped in the active site.[9] Subsequent studies
showed that photolysis of HydG [4Fe-4S]+-SAM complex in the
absence of substrate tyrosine instead led to a methyl radical
(•CH3) trapped in the active site; that is, in HydG the photoinduced
electron transfer led to homolytic S-CH3 bond cleavage, rather
than S-C5’ bond cleavage as in PFL-AE.[29]
Figure 5. X-band CW-EPR spectra of •CH2CH3 (top) and •CH2CH3* (bottom)
after 450 nm photolysis of [4Fe-4S]1+RS:SAE complex of HydG for 5 mins and 1
hr. The simulation (red) parameters for •CH2CH3 and •CH2CH3* are supported
in Table 1. EPR parameters: temperature, 40 K; microwave frequency, 9.37
GHz; modulation amplitude, 3 G.
Table 1. Simulation[a] parameters for •CH2CH3 and •CH2CH3*
Spin Hamiltonian
•CH2CH3
•CH2CH3*
g
2.003,2.002,2.001
80,40,60
80,40,60
110,110,110
20,20,20
2.005,2.004,2.003
80,40,60
80,40,60
100,100,115
25,25,30
To test the sensitivity of photoinduced S-C cleavage to variations
in SAM, we carried out cryogenic (12 K) 450 nm photolysis of the
[4Fe-4S]+-SAE complex of HydG in the absence of tyrosine. The
EPR spectrum subsequent to 5 mins of photolysis reveals the
appearance of a new radical species concomitant with the loss of
a majority of the [4Fe-4S]1+ cluster signal (Fig 5). This contrasts
with photolysis of the [4Fe-4S]+-SAM complex of HydG, which
requires about 1 hour to go to completion.[29]
A(a-1Ha) MHz[b]
A(a-1Hb) MHz
A(b-1Ha) MHz
A(b-1Hb) MHz
A(b-1Hc) MHz
20,20,20
25,25,30
[a]The homogenous EPR linewidth for the simulation of •CH2CH3 is 30 MHz, and
for the simulation of •CH2CH3* is 6 MHz; this simulation further includes
inhomogeneous line-broadening with HStrain = (40, 40, 10) MHz. [b]Euler angle
(a,b,g) = (60,0,0) for 1Ha of •CH2-, and (a,b,g) = (-60,0,0) for 1Hb of •CH2-,
corresponding to ∠1Ha-C-1Hb = 120°.
The new radical species exhibits a multiline EPR signal at 40 K,
clearly different than the distinct 1:3:3:1 methyl radical signal
4
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with only slight adjustment of the spin-Hamiltonian parameters for
•CH2CH3 (Fig 5, lower, Table 1), showing that the properties of
the radical itself are not altered. Instead, the enhanced resolution
results from a decreased linewidth (decreased homogeneous
linewidth, but with the introduction of a heterogeneous linewidth
component in simulations; Table 1). This change suggests that
the prolonged illumination has relaxed the environment of the
•CH2CH3 radical, and we denote the resulting radical center,
•CH2CH3*. We attribute this phenomenon to local photothermal
annealing in the vicinity of the cluster caused by the release as
heat of the energy of multiple photons absorbed during the
extended illumination period by the [4Fe-4S]2+ cluster proximate
to the ethyl radical. It is well known that absorption of a single
photon of this energy can cause extremely large transient
increases in temperature of the absorbing cofactor, with smaller
but substantial temperature increases in the protein vicinity as the
energy flows through the protein.[42-43] Such a temperature
increase would be greatly enhanced at the cryogenic
temperatures employed here, as the protein heat capacity is
strongly decreased at such temperatures.
radicals affords a major increase in stability at cryogenic
temperatures, likely a result of decreased translational freedom of
the radical in the active site leading to a diminished susceptibility
to quenching reactions. Indeed, the ethyl radical behaves similarly
to the much larger 5’-dAdo• radical generated by photolysis of
SAM bound to reduced PFL-AE, which is stable indefinitely at 77
K.[9]
When a sample that exhibits the •CH2CH3 signal formed by
photolysis at 12 K (Fig 5 upper) was annealed for 1 minute at
150K and then at 200 K for 1 min, its intensity decreases and the
signal becomes distorted, Fig 6, in a manner suggesting the
conversion of the •CH2CH3 radical into a second paramagnetic
species that shows no 1H splittings. Upon further annealing at
220K for 1 min, •CH2CH3 is completely gone, and the spectrum
exhibits the signal from the new paramagnetic species. The new
signal, with a substantial g-anisotropy (g = [2.02 2.002 1.984]);
Fig 6) and an absence of hyperfine splittings is analogous to, but
more rhombic than, the signal of the
Ω organometallic
intermediate (g = [2.035, 2.004, 2.004]) that is observed widely
across the RS superfamily, in which the SAM-derived 5’-dAdo
moiety is covalently bound to the unique iron of the [4Fe-4S]
cluster. This new signal is also similar to the methyl-[4Fe-4S]3+ ΩM
observed upon annealing of •CH3 in HydG (g = [2.02 2.005
1.987]),[29] as well as the methyl-[4Fe-4S]3+ model complex
recently synthesized by Suess and coworkers (g = [2.101, 2.050,
2.042]).[8] We therefore assign the new signal (Fig 6) to an
organometallic species formed by reaction of •CH2CH3 with the
Thermal-annealing of •CH2CH3 and •CH2CH3*: The •CH2CH3
radical formed directly by photolysis of the HydG [4Fe-4S]+-SAE
complex at 12 K (Fig 5 upper) is indefinitely stable at 77 K, as is
the photothermally annealed •CH2CH3* radical. These results are
in sharp contrast to our observation that the methyl radical
generated by photolysis of the HydG/SAM complex decays at 77
K with a half-life of minutes.[29] Thus, the modest difference in
steric bulk between the 2-carbon ethyl and the 1-carbon methyl
Figure 7. X-band EPR spectra of •CH2CH3* radical formed under prolonged
photolysis of HydG/SAE and its behavior under step-annealing at 100 K for 25
min, 210 K for 5 min, and 220 K for 1min. The EPR intensity is normalized to
the gain level. Simulation of WE*: g = [2.035, 2.001, 1.984], LW = 30 MHz,
HStrain = [65, 25, 70] MHz. Conditions: microwave frequency, 9.37 GHz;
modulation amplitude 2 G; T = 40 K.
Figure 6. X-band EPR spectra of •CH2CH3 radical formed after photolysis of
HydG/SAE for 5 mins (top, same as the top spectrum in Figure 5), and its
behavior under step-annealing at 150 K for 1 min, 200 K for 1 min, and 220 K
for 1min. Simulation of WE: g = [2.02, 2.002, 1.983], LW = 36 MHz, HStrain =
[115, 20, 45] MHz. Conditions: microwave frequency, 9.37 GHz; modulation
amplitude, 5 G; T = 40 K.
5
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RESEARCH ARTICLE
[4Fe-4S]2+ cluster to create a CH3CH2-[4Fe-4S]3+ adduct, denoted
ΩE. Equivalent annealing of the •CH2CH3* sample (Fig 7) likewise
causes the loss of the •CH2CH3* radical signal and the
appearance a signal whose substantial g anisotropy again
indicates the formation of an CH3CH2-[4Fe-4S]3+ organometallic,
but with g-values (g = [2.035, 2.001, 1.984]) and linewidths slightly
different from those of ΩE; it is thus denoted ΩE* (Fig 7). The
difference presumably reflects a conformational difference arising
from the photothermal relaxation of the protein environment
induced by the prolonged irradiation.
Thermal annealing of the active-site trapped ethyl radical yields
an organometallic species ΩE in which the ethyl group is
covalently bound to the unique iron of the active site [4Fe-4S]
cluster. This center is slightly different when produced by photo-
annealing the •CH2CH3 and •CH2CH3* environment, hence the
two are termed ΩE and ΩE*. Thus, SAE is capable of forming
organometallic alkyl-[4Fe-4S]3+ species in HydG with two different
alkyl groups, Ω and ΩE, depending on the method of S-C bond
cleavage (photolytic or enzymatic turnover). Given that a third
alkyl-[4Fe-4S]3+ species, with a methyl-group bound to Fe, was
formed in the RS enzyme HydG when SAM was utilized,[29] these
results demonstrate a remarkable versatility of RS enzymes in
generating alkyl radicals and organometallic alkyl-[4Fe-4S]3+
species.
The signals from both these organometallic ΩE and ΩE* species
are more intense than that of ΩM formed in HydG upon
photolysis/annealing of the HydG/SAM complex. This suggests
that the larger ethyl radical is more constrained within the active
site than •CH3, and so more of the ethyl radical is converted into
an organometallic intermediate instead of diffusing away to
undergo nonspecific reactions within the protein matrix.
Acknowledgements
The involvement of Ω as an intermediate in the RS reductive
cleavage mechanism was surprising when first reported,[1] but it
Preparation and characterization of HydG was funded by the U.S.
Department of Energy, Office of Basic Energy Sciences grant DE-
SC0005404 (to JBB and EMS). All other work was funded by the
NIH (GM 111097 to BMH and GM 131889 to JBB). The authors
thank Dr. Maike Blakeley for assistance with NMR spectroscopy
and analysis.
was subsequently shown to be central in catalysis across the RS
4, 7]
superfamily.[2,
However, the present observation of an ΩE
species (CH3CH2-[4Fe-4S]3+) in HydG provides now the third
organometallic, alkyl-[4Fe-4S]3+ complex, along with Ω and ΩM,[29]
observed in RS enzymes. The photolysis/annealing method
utilized to generate ΩM[29] and ΩE appears to be versatile, and may
allow for the synthesis of other alkyl-[4Fe-4S]3+ species. While ΩM
and ΩE are not involved in catalysis, they highlight the ability of
site-differentiated [4Fe-4S] clusters to form stable alkyl-
complexes. As expanded by the recent synthesis and
characterization of a CH3-[4Fe-4S]3+ complex by Suess and
coworkers,[8] these results reveal an unexpectedly rich
organometallic chemistry of [4Fe-4S] clusters.
Keywords: S-adenosylethionine • ethyl radical • radical SAM •
EPR • organometallic
[1]
[2]
M. Horitani, K. A. Shisler, W. E. Broderick, R. U.
Hutcheson, K. S. Duschene, A. R. Marts, B. M. Hoffman,
J. B. Broderick, Science 2016, 352, 822-825.
A. S. Byer, H. Yang, E. C. McDaniel, V. Kathiresan, S.
impano, A. Pagnier, H. Watts, C. Denler, A. L. Vagstad, J.
Piel, K. S. Duschene, E. M. Shepard, T. P. Shields, L. G.
Scott, E. A. Lilla, K. Yokoyama, W. E. Broderick, B. M.
Hoffman, J. B. Broderick, J. Am. Chem. Soc. 2018, 140,
8634-8638.
Conclusion
[3]
[4]
J. B. Broderick, B. R. Duffus, K. S. Duschene, E. M.
Shepard, Chem. Rev. 2014, 114, 4229-4317.
W. E. Broderick, B. M. Hoffman, J. B. Broderick, Acc.
Chem. Res. 2018, 51, 2611-2619.
SAM is widely distributed in all organisms, and plays essential
roles not only in the chemistry of the RS superfamily, but also in
methyltransferases and other reactions.[44] SAM is synthesized in
vivo from ATP and methionine by the enzyme SAM synthetase.
While SAE has been detected in living systems under conditions
where ethionine is available, few studies have focused on the
impact of SAE on enzymatic systems that use SAM as a cofactor,
such as the RS enzymes. Here we provide a study of SAE as an
analog of SAM in a RS enzyme, demonstrating that SAE is a
catalytically functional cofactor that supports the same catalytic
chemistry, via the same mechanism, as the natural cofactor SAM.
[5]
[6]
P. A. Frey, Annu. Rev. Biochem. 2001, 70, 121-148.
P. A. Frey, O. T. Magnusson, Chem. Rev. 2003, 103,
2129-2148.
[7]
[8]
W. E. Broderick, J. B. Broderick, J. Biol. Inorg. Chem.
2019, 24, 769-776.
A. McSkimming, A. Sridharan, N. B. Thompson, P. Müller,
D. L. M. Suess, J. Am. Chem. Soc. 2020, 142, 14314-
14323.
[9]
H. Yang, E. C. McDaniel, S. Impano, A. S. Byer, R. J.
Jodts, K. Yokoyama, W. E. Broderick, J. B. Broderick, B.
M. Hoffman, J. Am. Chem. Soc. 2019, 141, 12139-12146.
R. C. Driesener, M. R. Challand, S. E. McGlynn, E. M.
Shepard, E. S. Boyd, J. B. Broderick, J. W. Peters, P. L.
Roach, Angew. Chem. Int. Ed. Engl. 2010, 49, 1687-1690.
E. M. Shepard, B. R. Duffus, S. E. McGlynn, M. R.
Challand, K. D. Swanson, P. L. Roach, J. W. Peters, J. B.
Broderick, J. Am. Chem. Soc. 2010, 132, 9247-9249.
J. M. Kuchenreuther, S. J. George, C. S. Grady-Smith, S.
P. Cramer, J. R. Swartz, PLoS ONE 2011, 6, e20346.
R. C. Driesener, B. R. Duffus, E. M. Shepard, I. R. Bruzas,
K. S. Duschene, N. J.-R. Coleman, A. P. G. Marrison, E.
Salvadori, C. W. M. Kay, J. W. Peters, J. B. Broderick, P.
L. Roach, Biochemistry 2013, 52, 8696-8707.
B. R. Duffus, S. Ghose, J. W. Peters, J. B. Broderick, J.
Am. Chem. Soc. 2014, 136, 13086-13089.
[10]
[11]
We also show that photoinduced electron transfer of the SAE-
HydG complex results in homolytic cleavage of a different
sulfonium S-C bond (S-CH2CH3) than is cleaved catalytically (S-
C5’), leading to formation of an ethyl radical. Interestingly,
prolonged irradiation of [4Fe-4S]2+ after SAE cleavage causes
[12]
[13]
RS
a photothermal local annealing of the protein environment near
•CH2CH3, which results in better-resolved and sharper 1H-
hyperfine split peaks for the •CH2CH3* radical (Fig 5). Contrary to
the rotationally and translationally mobile methyl radical, which
decays in hours at 77K,[29] the larger ethyl radical is less mobile
and is indefinitely stable at liquid nitrogen temperatures.
[14]
6
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10.1002/anie.202014337
Angewandte Chemie International Edition
RESEARCH ARTICLE
[15]
Y. Nicolet, L. Zeppieri, P. Amara, J. C. Fontecilla-Camps,
Angew. Chem. 2014, 126, 12034-12038.
E. Pilet, Y. Nicolet, C. Mathevon, T. Douki, J. C.
Fontecilla-Camps, M. Fontecave, FEBS Lett. 2009, 583,
506-511.
Hodgson, B. Hedman, U. Bergmann, K. J. Gaffney, E. I.
Solomon, Science 2017, 356, 1276-1280.
G. D. Markham, in Nature Encyclopedia of Life Sciences,
Nature Publishing Group, London, 2002.
[16]
[44]
[17]
[18]
A. Pagnier, L. Martin, L. Zeppieri, Y. Nicolet, J. C.
Fontecilla-Camps, Proc. Natl. Acad. Sci. U. S. A. 2016,
113, 104-109.
J. M. Kuchenreuther, W. K. Myers, D. L. M. Suess, T. A.
Stich, V. Pelmenschikov, S. A. Shiigi, S. P. Cramer, J. R.
Swartz, R. D. Britt, S. J. George, Science 2014, 343, 424-
427.
[19]
D. L. M. Suess, C. C. Pham, I. Bürstel, J. R. Swartz, S. P.
Cramer, R. D. Britt, J. Am. Chem. Soc. 2016, 138, 1146-
1149.
[20]
[21]
G. D. Rao, L. Z. Tao, D. L. M. Suess, R. D. Britt, Nat.
Chem. 2018, 10, 555-560.
E. M. Shepard, S. E. McGlynn, A. L. Bueling, C. Grady-
Smith, S. J. George, M. A. Winslow, S. P. Cramer, J. W.
Peters, J. B. Broderick, Proc. Natl. Acad. Sci. U.S.A. 2010,
107, 10448-10453.
[22]
[23]
I. Czech, A. Silakov, W. Lubitz, T. Happe, FEBS Lett.
2010, 584, 638-642.
A. G. Scott, R. K. Szilagyi, D. W. Mulder, M. W. Ratzloff,
A. S. Byer, P. W. King, W. E. Broderick, E. M. Shepard, J.
B. Broderick, Dalton Trans. 2018, 47, 9521-9535.
A. S. Byer, E. M. Shepard, M. W. Ratzloff, J. N. Betz, P.
W. King, W. E. Broderick, J. B. Broderick, J. Biol. Inorg.
Chem. 2019, 24, 783-792.
J. N. Betz, N. W. Boswell, C. J. Fugate, G. L. Holliday, E.
Akiva, A. G. Scott, P. C. Babbitt, J. W. Peters, E. M.
Shepard, J. B. Broderick, Biochemistry 2015, 54, 1807-
1818.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
E. M. Shepard, F. Mus, J. Betz, A. Byer, B. R. Duffus, J.
W. Peters, J. B. Broderick, Biochemistry 2014, 53, 4090-
4104.
J. B. Broderick, A. S. Byer, K. S. Duschene, B. R. Duffus,
J. N. Betz, E. M. Shepard, J. W. Peters, J. Biol. Inorg.
Chem. 2014, 19, 747-757.
L. Tao, S. A. Pattenaude, S. Joshi, T. P. Begley, T. B.
Rauchfuss, R. D. Britt, J. Am. Chem. Soc. 2020, 142,
10841-10848.
H. Yang, S. Impano, E. M. Shepard, C. D. James, W. E.
Broderick, J. B. Broderick, B. M. Hoffman, J. Am. Chem.
Soc. 2019, 141, 16117-16124.
S. Impano, H. Yang, R. J. Jodts, A. Pagnier, R. Swimley,
E. C. McDaniel, E. M. Shepard, W. E. Broderick, J. B.
Broderick, B. M. Hoffman, in review 2020.
F. Schlenk, J. L. Dainko, S. M. Stanford, Arch. Biochem.
Biophys. 1959, 83, 28-34.
R. C. Smith, W. D. Salmon, Arch. Biochem. Biophys.
1965, 111, 191-196.
J. D. Finkelstein, J. J. Martin, Biochem. Biophys. Res.
Comm. 1984, 118, 14-19.
[31]
[32]
[33]
[34]
B. G. Moore, R. C. Smith, Can. J. Biochem. 1969, 47, 561-
565.
[35]
[36]
[37]
R. Cox, C. C. Irving, Cancer Res. 1977, 37, 222-225.
B. G. Moore, Can. J. Biochem. 1970, 48, 702-705.
X. Ji, D. Mandalapu, J. Cheng, W. Ding, Q. Zhang,
Angew. Chem. Int. Ed. 2018, 57, 6601-6604.
A. S. Byer, E. C. McDaniel, S. impano, W. E. Broderick, J.
B. Broderick, Methods Enzymol. 2018, 606, 269-318.
J. Zhang, J. P. Klinman, Anal. Biochem. 2015, 476, 81-83.
K. A. Shisler, R. U. Hutcheson, M. Horitani, K. S.
Duschene, A. V. Crain, A. S. Byer, E. M. Shepard, A.
Rasmussen, J. Yang, W. E. Broderick, J. L. Vey, C. L.
Drennan, B. M. Hoffman, J. B. Broderick, J. Am. Chem.
Soc. 2017, 139, 11803-11813.
[38]
[39]
[40]
[41]
P. Dinis, D. L. M. Suess, S. J. Fox, J. E. Harmer, R. C.
Driesener, L. De La Paz, J. R. Swartz, J. W. Essex, R. D.
Britt, P. L. Roach, Proc. Natl. Acad. Sci. U. S. A. 2015,
112, 1362-1367.
[42]
[43]
P. Li, P. M. Champion, Biophys. J. 1994, 66, 430-436.
M. W. Mara, R. G. Hadt, M. E. Reinhard, T. Kroll, H. Lim,
R. W. Hartsock, R. Alonso-Mori, M. Chollet, K. O.
7
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RESEARCH ARTICLE
Entry for the Table of Contents
A
OH
O
OH
H
H2C
H
C
H3C
S
hn
NH2
O
O
+ Tyrosine
1+
•CH2CH3
Ω
ΩE
The radical SAM enzyme HydG can use S-adenosyl-L-ethionine (SAE) in place of SAM during catalysis. HydG reacts with SAE and
tyrosine to form the organometallic intermediate Ω, similar to reaction with SAM. The SAE-bound [4Fe-4S]+ cluster of HydG
undergoes cryogenic blue light photolysis to generate an ethyl radical, which upon annealing forms an organometallic species in
which an ethyl is bound to the unique iron of a [4Fe-4S]3+ cluster.
Institute and/or researcher Twitter usernames: BroderickLabFeS
8
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