Organometallic Complex Formed by an Unconventional Radical S-Adenosylmethionine Enzyme

Article abstract of DOI:10.1021/jacs.6b04155

Pyrococcus horikoshii Dph2 (PhDph2) is an unusual radical S-adenosylmethionine (SAM) enzyme involved in the first step of diphthamide biosynthesis. It catalyzes the reaction by cleaving SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. To probe the reaction mechanism, we synthesized a SAM analogue (SAM_CA), in which the ACP group of SAM is replaced with a 3-carboxyallyl group. SAM_CA is cleaved by PhDph2, yielding a paramagnetic (S = 1/2) species, which is assigned to a complex formed between the reaction product, α-sulfinyl-3-butenoic acid, and the [4Fe-4S] cluster. Electron-nuclear double resonance (ENDOR) measurements with ¹³C and ²H isotopically labeled SAM_CA support a π-complex between the C=C double bond of α-sulfinyl-3-butenoic acid and the unique iron of the [4Fe-4S] cluster. This is the first example of a radical SAM-related [4Fe-4S]⁺ cluster forming an organometallic complex with an alkene, shedding additional light on the mechanism of PhDph2 and expanding our current notions for the reactivity of [4Fe-4S] clusters in radical SAM enzymes.

Full text of DOI:10.1021/jacs.6b04155

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Communication
An organometallic complex formed by an unconventional radical SAM enzyme
Min Dong, Masaki Horitani, Boris Dzikovski, Maria-Eirini Pandelia,
Carsten Krebs, Jack H. Freed, Brian M. Hoffman, and Hening Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04155 • Publication Date (Web): 28 Jul 2016
Downloaded from http://pubs.acs.org on July 31, 2016
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An organometallic complex formed by an unconventional radical
SAM enzyme
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Min Dong, 1,# Masaki Horitani, 2,# Boris Dzikovski,1 Maria-Eirini Pandelia3, Carsten Krebs4, Jack H.
Freed,1 Brian M. Hoffman2,* and Hening Lin1,5*
1Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Cornell University, Ithaca,
New York, 14853. 2Department of Chemistry, Northwestern University, Evanston, Illinois 60208. 3Department of
Biochemistry, Brandeis University, MA 02453. 4Department of Chemistry and Department of Biochemistry and Mo-
lecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802. 5Howard Hughes Medical
Institute, Cornell University, Ithaca, New York, 14853.
Supporting Information Placeholder
ABSTRACT: Pyrococcus horikoshii Dph2 (PhDph2) is an
unusual radical SAM enzyme involved in the first step of
diphthamide biosynthesis. It catalyzes the reaction by cleav-
ing S-adenosylmethionine (SAM) to generate a 3-amino-3-
carboxy propyl (ACP) radical. To probe the reaction mecha-
nism, we synthesized a SAM analog (SAMCA), in which the
ACP group of SAM is replaced with a 3-carboxyallyl group.
SAMCA is cleaved by PhDph2, yielding a paramagnetic (S =
1/2) species, which is assigned to a complex formed between
the reaction product, α-sulfinyl-3-butenoic acid, and the
The formation of the ACP radical is supported by the
PhDph2-catalyzed generation of 2-aminobutyric acid and
homocysteine sulfinic acid in the absence of the substrate
protein, PhEF2.3 However, the ACP radical has not been di-
rectly observed even when we carried out freeze quench-
electron paramagnetic resonance (EPR) experiments (Figure
S1). This is not surprising because the ACP radical is expected
to be short-lived. Following the strategy developed by Frey
[4Fe-4S] cluster. Electron-nuclear double resonance
2
(ENDOR) measurements with 13C and H isotopically labeled
SAMCA support a π-complex between the C=C double bond
of α-sulfinyl-3-butenoic acid, and the unique iron of the
and coworkers to generate a more stable allylic analog of the
[4Fe-4S] cluster. This is the first example of a radical SAM
5′
-dA•6.7, we synthesized a SAM analog, SAMCA, in which the
related [4Fe-4S]+ cluster forming an organometallic complex
with an alkene, shedding additional light into the mecha-
nism of PhDph2 and expanding our current notions for the
reactivity of [4Fe-4S] clusters in radical SAM enzymes.
ACP group of SAM was replaced with a 3-carboxyallyl group
(Scheme 2). If PhDph2 could accept SAMCA as a substrate, a
3-carboxyallyl radical would be generated, which should be
more stable and allow direct observation by EPR. As shown
below, even with SAMCA, we could not detect the radical in-
termediate. However, surprisingly, we found that a dithionite
quenched radical product formed an organometallic complex
with the Fe-S cluster, revealing an interesting reactivity of
the [4Fe-4S] cluster in RS enzymes.
Diphthamide is a post-translationally modified histidine res-
idue on archaeal and eukaryotic translation elongation factor
2 (EF2), a protein essential for ribosomal protein synthesis.
Its biosynthesis involves at least seven proteins.1,2 The first
step involves the transfer of 3-amino-3-carboxypropyl (ACP)
group derived from S-adenosyl-L-methionine (SAM) to the
histidine residue of EF2. In vitro, the [4Fe-4S]-containing
radical SAM (RS) enzyme PhDph23 or the eukaryotic Dph1-
Dph2 homodimer4 is sufficient for this step, with dithionite
as the reducing agent. In contrast to classical RS enzymes
that cleave the C5 ,Ade–S bond of SAM to form 5 -
deoxyadenosyl radical (5 -dA•) (Scheme 1A)5, PhDph2
cleaves the Cγ,Met–S bond to form an ACP radical (Scheme
1B).3
Scheme 2. Structures of SAMCA and aza-SAMCA
.
We initially examined whether SAMCA could serve as a sub-
strate of PhDph2. High-performance liquid chromatography
(HPLC) showed that all the SAMCA was converted to 5
′ -
Scheme 1. PhDph2 is different from classical RS enzymes.
deoxy-5 -methylthioadenosine (MTA) within 20 min, where-
′
as in the absence of dithionite or PhDph2, no SAMCA was
cleaved (Figure 1A). These results demonstrate that PhDph2
is able to catalyze the cleavage of the ‘correct’ C-S bond of
SAMCA. In the presence of the substrate protein PhEF2, the
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same reaction product was detected (Figure S2). However,
signal as that of CA was obtained, supporting that CA is a
product complex (Figure S5).
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3
PhEF2 was not modified by SAMCA. This was likely because
SAMCA generated a more stable radical (see more experi-
mental evidence for the stability below), which was not ac-
tive enough to react with PhEF2.
To understand the structure of CA, we analyzed the reaction
1
products using H-NMR. Several new peaks were observed
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from the reaction containing PhDph2, SAMCA, and dithionite
(Figure S6), which were absent in control reactions without
dithionite. Some of the new peaks were assigned to MTA.
Crotonic acid and its isomer, 2-butenoic acid, which are the
hydrogen abstraction products of the carboxyallyl radical,
were not formed by comparison to the spectra of their stand-
ards. Three new peaks (a-c) were assigned to γ-
sulfinylcrotonic acid (Figure S6) and the remaining three
peaks (d, e and f) around 5.1 and 5.8 ppm were assigned to α-
sulfinyl-3-butenoic acid (Figure S6); the remaining proton on
α-sulfinyl-3-butenoic acid is localized at 3.4 ppm based on
1H-1H COSY (correlation spectroscopy) (Figure S14, S15). Us-
ing 1,1-2H2-SAMCA as the substrate, peaks c, e and f (Figure
S7) disappeared, which supported the assignments. The as-
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signments were confirmed by H/13C HMBC (Heteronuclear
Multiple Bond Correlation), HSQC (Heteronuclear Single
Quantum Correlation) and 1H-1H COSY (Figure S8 - S15). The
reaction products were also chemically modified to allow
detection by LC-MS (Figure S16). Our data thus demonstrat-
ed the formation of γ-sulfinylcrotonic and α-sulfinyl-3-
butenoic acid, which likely resulted from the recombination
Figure 1. A). HPLC traces of the SAMCA cleavage reaction
(reaction time 20 min) catalyzed by PhDph2 at room tem-
perature. B). X-band CW EPR spectra of PhDph2 in the ab-
sence and presence of SAMCA. A spectral simulation for the S
= 1/2 species is shown as green dotted line. T = 12 K.
-
•
of the carboxyallyl radical with the dithionite-derived SO2
radical, similar to previously reported reactions of radicals
with dithionite 3,12,13. The NMR signal intensities suggested
roughly equal formation of the two products.
We then prepared freeze quench samples and used EPR to
detect radical signals. The dithionite-reduced PhDph2 in the
absence of SAMCA showed a typical [4Fe-4S]1+ EPR signal at 12
K (Figure 1B),3 which was temperature-dependent and
broadened beyond detection at 30 K (Figure S1). When
PhDph2 in the presence of dithionite was allowed to react
with SAMCA for 30 s, the [4Fe-4S]1+ signal was replaced by a
nearly axial signal, with a split feature at g1 (Figure 1B). The
ratio of this g1 splitting at X- and Q-band scaled with the
ratio of microwave frequencies (Figure S3), showing that the
splitting represents two different conformations and not a
hyperfine interaction with a paramagnetic nucleus. Simula-
tion of the spectra gave for the one conformer principal g-
values g = [2.092, 2.010, 1.990], and for the second one g =
[2.074, 2.010, 1.990]. Both signals exhibited similar relaxation
properties and were hardly detectable above 70 K (Figure S1).
The large g-anisotropy and the absence of proton hyperfine
splittings suggest that it is not a free allyl radical.6,8 In con-
trast, the signals of the new species formed in the reaction of
the reduced PhDph2 with SAMCA exhibited an “isotropic” g-
value, giso = (g11+g22+g33)/3 > ge = 2.0023,9 which is reminis-
cent of that observed in high potential iron-sulfur proteins
(HiPIP) and [4Fe-4S]3+ intermediates trapped with the [4Fe-
4S] proteins IspG10 and IspH11. With 57Fe-enriched PhDph2,
the EPR signal became broadened (Figure 1B), further sup-
porting that the new species (termed CA) is associated with
the [4Fe-4S] cluster.
We then performed 35 GHz CW/pulse 13C, 1H and 2H ENDOR
with 1-13C, 2-13C, 3-13C, 1,1-2H2 and 1,1,2,3-2H4 labeled SAMCA
(see Scheme 2). Field-modulated 35 GHz CW ENDOR spec-
tra obtained from reactions with 1-13C, 2-13C labeled SAMCA
both exhibit 13C doublets with broad component lines, split
by the 13C hyperfine coupling, A ~ 5-8 MHz (Figure 2). An
estimate of the magnitude of the hyperfine coupling tensors
for these two 13C sites of SAMCA (Table S1) was obtained
through analysis of 2D field-frequency patterns of ENDOR
spectra collected across the EPR envelopes of the SAMCA
isotopologs (Figures S17, S18). The analysis showed compara-
ble isotropic hyperfine tensors, Aiso ~ 7 MHz for both 1-13C
and 2-13C. For 3-13C labeled SAMCA, a considerably smaller
isotropic coupling, Aiso ~ 0.6 MHz, was estimated using Mims
pulsed ENDOR (Figure 2A, Inset).
When we monitored the reaction at different time points,
the EPR signal of CA persisted for >30 min, even after all
SAMCA was consumed (Figure S4). This suggested that CA
was a complex of the reaction product with the [4Fe-4S]1+
cluster, the latter regenerated by dithionite reduction of the
[4Fe-4S]2+ produced in the reaction. To test this hypothesis,
protein-free aliquots of the reaction products were added to
‘fresh’ dithionite-reduced PhDph2. The same anisotropic EPR
Figure 2. (A) 35 GHz CW 13C ENDOR spectra for CA with 1-
13C and 2-13C-labeled SAMCA collected at g2; the doublets are
split by the hyperfine coupling, and centered at the Larmor
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frequency as described in SI. Inset: Mims ENDOR spectra of
with aza-SAMCA showed only a [4Fe-4S]+ signal (Figure S23)
that was similar to that of the reduced cluster in PhDph2 in
the absence of SAMCA. Therefore, CA is not formed by bind-
ing of uncleaved SAMCA to the [4Fe-4S] cluster. Addition of
crotonic acid to PhDph2 also failed to generate the CA spe-
cies. Thus, the sulfinic group in α-sulfinyl-3-butenoic acid is
likely involved in the formation of CA. Similarly, adding
allylsulfinic acid to PhDph2 did not generate the CA signal
either. Thus, the carboxyl group in α-sulfinyl-3-butenoic acid
is also necessary. The results suggest that both the sulfinic
group and the carboxylate of α-sulfinyl-3-butenoic acid bind
to the Fe-S cluster, facilitating the formation of a π-complex
between that Fe and the C=C double bond of the product
(Figure 3).
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3-13C SAMCA. (B) H Davies ENDOR difference spectra of CA
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generated by subtracting from the spectrum of H-SAMCA
(natural abundance) the spectra for 1,1,2,3-2H-labeled SAMCA
(upper) and 1,1-2H-labeled SAMCA (lower). Spectra were col-
lected at g2 and normalized by EPR echo height.
To further probe the structure of CA, 1H and 2H pulse
ENDOR measurements were performed with 1,1-2H2, and
1,1,2,3-2H4 labeled SAMCA. Only the pulsed ENDOR tech-
niques resolved individual features, but the lower sensitivity
of pulsed vs CW ENDOR allowed spectra to be collected only
at the intensity maximum of the EPR envelope, near g2. The
1H signals from the labeled sites were obtained by
subtracting the spectra obtained using the natural-
abundance SAMCA (Figure 2B). After subtraction of the 1,1-2H2
spectrum, a doublet of 1H peaks assignable to the 1,1-1H nuclei
was obtained with coupling constant of A ~ 7 MHz;
subtraction with the 1,1,2,3-2H4 labeled SAMCA showed an
enhanced intensity for this doublet (Figure 2B), indicating
that the 1,1-1H and 2-1H nuclei had comparable couplings
(assigned based on 2-13C data). In addition, weak broad
features corresponding to a doublet with A ~ 2 MHz were
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assigned to 3-1H. Mims H ENDOR spectra (Figure S19 and
S20) provided support for this assignment. The substantial
isotropic couplings to 1-13C and 2-13C (Aiso ~ 7 MHz), the devi-
ations of the g-values from g~2, and the 57Fe broadening im-
ply the spin is primarily localized on the cluster, but with
close C-Fe interaction.
Figure 3. Plausible structures of CA and formation mecha-
nism.
CA can have two formal resonance structures (Figure 3,
structures a and b). To further probe the oxidation state of
CA, we attempted to reduce CA by low-temperature γ-
irradiation (cryoreduction)16 (Figure S24). The EPR signal did
not decrease upon γ-irradiation, as would be expected for a
[4Fe-4S]3+ cluster. Similarly, extra dithionite could not reduce
CA, which is inconsistent with the expectation for a [4Fe-
4S]3+ cluster (Figure S25)17. In contrast, when we tried to oxi-
dize CA with K3[Fe(CN)6], CA was oxidatively degraded to a
[3Fe-4S]+ cluster (Figure S25), an outcome commonly ob-
served in similar treatments of [4Fe-4S]+ clusters of RS en-
zymes with chemical oxidants. Thus, the Fe-S core in CA
behaves like a [4Fe-4S]+ cluster. We also sought to use 57Fe
Mössbauer spectroscopy. However, owing to the rather mod-
est yield of CA (50% of total Fe) and the presence of at least
one additional EPR-silent Fe-containing product, the analysis
of the spectra does not permit definitive conclusions about
the electronic structure of CA (Figures S26, 27 and 28).
The similarity of 1-13C and 2-13C hyperfine couplings, as well
as the similarity of the 1,1-2H and the 2-2H couplings, sug-
gests that both 1-C and 2-C have similar direct interactions
with the unique Fe of the [4Fe-4S] cluster. In contrast, the
weaker 3-13C and 3-2H hyperfine couplings show that 3-C
does not experience a significant direct interaction with the
Fe. These results suggest that CA only consists of one of the
two products, α-sulfinyl-3-butenoic acid, coordinated to the
unique Fe site of the [4Fe-4S] cluster. If CA also involved a
complex with γ-sulfinylcrotonic acid, 2-C and 3-C should
have similar strong hyperfine couplings, and 1-C should have
weaker coupling, contrary to our observations (Figure 2A).
We further confirmed this by utilizing the instability of α-
sulfinyl-3-butenoic acid. After incubating the protein-free
aliquots of the reaction products at room temperature
overnight, the peaks corresponding to α-sulfinyl-3-butenoic
acid disappeared in the 1H-NMR spectrum (Figure S21), while
the signals from γ-sulfinylcrotonic acid remained. The insta-
bility of α-sulfinyl-3-butenoic acid is likely due to the acidity
of the α-proton, which is adjacent to two strong electron-
withdrawing groups.14 When the mixture containing only the
γ-sulfinylcrotonic acid product was added to ‘fresh’
dithionite-reduced PhDph2, the signal associated with CA
was not be detected by EPR (Figure S22). This result supports
the notion that γ-sulfinylcrotonic acid does not form an EPR-
active complex with the [4Fe-4S] cluster and that CA is best
With the characterization of reaction products and the struc-
ture of CA, we proposed a plausible mechanism for the for-
mation of CA (Figure 3). The [4Fe-4S]+ cluster in PhDph2
provides one electron to SAMCA, cleaving the CCA-S bond and
generating a 3-carboxyallyl radical, MTA, and a [4Fe-4S]2+
cluster. The 3-carboxyallyl radical reacts with a dithionite-
-
•
derived SO2 radical, forming γ-sulfinylcrotonic acid and α-
sulfinyl-3-butenoic acid. The sulfinic group, the carboxyl
group, and the C=C double bond of α-sulfinyl-3-butenoic
acid, coordinate the [4Fe-4S]+ cluster (reduced by extra di-
thionite) to generate the π-complex. The coordination of the
sulfinic and carboxyl group helps to place the double bond in
a favorable position for π interactions with the unique Fe.
The two conformers revealed by EPR (Figure 1B) could be
attributed to different binding orientations of the α-sulfinyl-
described as
a complex formed between α-sulfinyl-3-
butenoic acid and the [4Fe-4S] cluster.
We then probed whether the sulfinyl and carboxyl of α-
sulfinyl-3-butenoic acid groups are required for CA for-
mation. We synthesized a non-cleavable analog of SAMCA
,
aza-SAMCA (Scheme 2)15. The freeze quench-EPR experiment
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3-butenoic acid to [4Fe-4S]+ cluster (the three chelating func-
tional groups may exchange positions).
ACKNOWLEDGMENT
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8
This work is supported by NIH (GM088276, P41-GM103521,
and GM111097). We thank Dr. Ivan Keresztes and Mr. Antho-
ny Condo for assistance with NMR; Prof. H. Halpern at Uni-
versity of Chicago for access to the 60Co Gammacell irradia-
tor.
In summary, using SAMCA, we have detected an EPR-active
species, CA, formed in the enzymatic reaction of PhDph2.
Various experiments demonstrate that CA is a complex
formed between the [4Fe-4S]1+ cluster and one of the reac-
tion products, α-sulfinyl-3-butenoic acid, coordinated to the
unique cluster Fe through the sulfinyl and carboxylate ox-
ygens, with the C=C double bond forming an organometallic
π-complex with the Fe. Although we could not accumulate
the radical intermediate that we set out to detect with
SAMCA, formation of both α-sulfinyl-3-butenoic acid and γ-
sulfinylcrotonic acids provides additional support for the
radical mechanism of PhDph2 reaction, as a nucleophilic
mechanism cannot account for their formation. Interestingly,
although the EPR g tensors of CA shows similarity to those of
[4Fe-4S]3+-like clusters observed in two enzymes involved in
the non-mevalonate pathway, IspG and IspH, the cluster in
CA behaves like a [4Fe-4S]+ cluster.
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The present case can be described as the reaction of the
unique Fe of the [4Fe-4S]+ cluster with the alkene product to
form a stable organometallic complex. Such an alkene com-
plex has reported for the nitrogenase FeMo-cofactor and the
ethylenic product of alkyne reduction18, and for mutants of
IspH and its substrate HMBPP.11,19 However, similar organo-
metallic complex had not been demonstrated for RS enzymes
until very recently, when the [4Fe-4S]2+ cluster of the RS en-
zyme, pyruvate formate-lyase activating enzyme is shown to
react with the 5 -dA• to form a highly reactive organometal-
lic intermediate.20 This study and our current result suggest
that the unique iron of [4Fe-4S] clusters in RS enzymes can
readily form a C-Fe complex. Such a property not only may
help to explain the unusual chemistries catalyzed by the
[4Fe-4S]-containing RS enzymes, but also may open up new
avenues to tune the activity or develop new chemistry for
this enzyme class or for other [4Fe-4S]-containing enzymes.
ASSOCIATED CONTENT
Supporting Information
Experimental materials and methods, supporting figures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Wang, W.; Wang, K.; Liu, Y. L.; No, J. H.; Li, J.; Nilges, M. J.;
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AUTHOR INFORMATION
Corresponding Authors
Brian M. Hoffman (bmh@northwestern.edu) and Hening Lin
(hl379@cornell.edu).
Author Contributions
#MD and MH contributed equally to this work.
Notes
The authors declare no competing financial interests.
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