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Deprotonation of Enoxy Radicals Theoretical Validation of a 50-Year-Old Mechanistic Proposal.

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Mechanisms of Enzyme Catalysis
Deprotonation of Enoxy Radicals: Theoretical
Validation of a 50-Year-Old Mechanistic
- H2O
David M. Smith, Wolfgang Buckel, and Hendrik Zipse*
The dehydration or a,b elimination of water from biomolecules is a very common enzymatic reaction. Almost all
dehydratases catalyze the removal of an hydroxy group in the
b position of an electron-withdrawing carboxylate, thioester,
or carbonyl group [Eq. (1)].
– OH
R1 = OH, SR, R
Scheme 1. Proposed mechanism for the reversible syn, a,b elimination
of water from (R)-2-hydroxyglutaryl-CoA (3).
The CHa bond of compounds such as 1 is activated and
can be deprotonated relatively easily by a basic residue of an
enzyme. In a typical substrate, such as 3-hydroxybutyryl-CoA
(1 with R1 = SCoA, R2 = CH3, R3 = H), the relevant CHa
bond has an associated pKa of approximately 21 in aqueous
solution. Upon binding to enoyl-CoA hydratase (crotonase),
this pKa is reduced to approximately 8 by specific interactions
with the enzyme. An essential glutamate residue appears to
act as the base in the abstraction of the activated aliphatic
Several glutamate-fermenting anaerobic bacteria contain
a dehydratase, which catalyses the reversible syn, a,b elimination of water from (R)-2-hydroxyglutaryl-CoA (3) to (E)glutaconyl-CoA (4) (Scheme 1). In this case, however, the
CHb bond to be cleaved is not activated (pKa 40) and in
the reverse direction the OH group adds to the more
electron-rich a-C atom of the polarized double bond. To
explain these observations, a mechanism has been proposed
in which the carbonyl group undergoes an “Umpolung” by
one-electron reduction.[4] The resulting substrate-derived
ketyl radical anion 5 (Scheme 1) may eliminate the a hydroxy
[*] Prof. Dr. H. Zipse, Dr. D. M. Smith
Department Chemie, Ludwig-Maximilians-Universit*t
Butenandtstrasse 13, 82131 M/nchen (Germany)
Fax: (+ 49) 89-2180-77738
Prof. Dr. W. Buckel
Laboratorium f/r Mikrobiologie, Philipps-Universit*t
Karl-von-Frisch Strasse, 35032 Marburg (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(SPP “Radicals in Enzymatic Catalysis”). H.Z. and W.B. thank the
Fonds der Chemischen Industrie for continuous support and
D.M.S. thanks the Alexander von Humboldt Foundation for a
research fellowship. We thank Prof. V. Barone for helpful discussions and technical assistance.
Supporting information for this article is available on the WWW
under or from the author.
Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870
group, leading to the enoxy radical 6. Deprotonation (Hb) of
this radical would yield the product-related ketyl radical
anion 7, which could be oxidized to the product 4.[5–7]
The biochemical analyses of the two-component 2hydroxyglutaryl-CoA dehydratase systems from the intestinal
bacteria Acidaminococcus fermentans and Clostridium symbiosum indeed reveal the possibility of an electron transfer
such as the one shown in Scheme 1.[8–10] Component A from
A. fermentans, a homodimeric protein with a [4Fe-4S]1+/2+
cluster between the two subunits, transfers an electron to
component D under hydrolysis of ATP. Component D is a
heterodimeric protein containing molybdenum(vi), reduced
riboflavin-5’-phosphate (FMNH2), and a [4Fe-4S]2+ cluster.
This ATP-driven electron transfer causes the formation of
Mov, which through oxidation to Movi quite probably
generates the ketyl radical anion 5. Oxidation of the second
ketyl radical anion 7 is likely to reform Mov, so that a single
electron transfer between the two components is sufficient to
induce many catalytic cycles.
2-Hydroxyglutaryl-CoA dehydratase is not the only
enzyme involved in the cleavage of an unactivated CHb
bond. For example, the dehydration of 4-hydroxybutyrylCoA to crotonyl-CoA, catalyzed by an oxygen-sensitive
[4Fe-4S] cluster and FAD-containing enzyme from Clostridium aminobutyricum, also involves the loss of a proton from
the b position of a thioester. In this case, it has been proposed
that an enoxy radical (similar to 6 in Scheme 1) is generated
by radical abstraction of the a hydrogen atom. Deprotonation
of the b C atom of this radical leads to the ketyl radical anion
of 4-hydroxycrotonyl-CoA, which can expel the hydroxy
group to yield the dienoxy radical. Re-addition of the initially
abstracted hydrogen atom at C4 yields the final product,
The key feature of the proposed mechanisms for both
these enzymes is that the b H atom is lost from the enoxy
radical (e.g. 6) and not from the substrate itself (e.g. 3).
DOI: 10.1002/anie.200250502
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Almost 50 years ago, Sir John W. Cornforth suggested that an
enoxy radical (derived by a-H-atom abstraction in a mechanism similar to that proposed for the dehydration of 4hydroxybutyryl-CoA) might undergo facile deprotonation to
a ketyl radical anion.[11] However, this suggestion has not
since been proven by any experimental or theoretical data.
Herein, we aim to quantify the acidity of the b position of the
enoxy radical. We wish to determine whether the acidity of
this radical is sufficiently enhanced over the closed-shell
thioester to explain the apparent deprotonation at the
unactivated position, or whether additional factors must be
invoked to arrive at a satisfactory mechanism.
The acidities of simple thioesters have been studied
experimentally by Richard and Amyes in D2O at 25 8C, and a
pKa value of 21 0.5 was determined for methyl thioacetate.[15] Much lower values were estimated for substituted
phenylacetyl-CoA derivatives based on the measured values
of analogously substituted phenlyacetone compounds by
Ghisla and co-workers.[16] The estimated pKa value for 4nitrophenylacetyl-CoA amounts to 13.6. An additional observation made in this latter study is the short lifetime of the
anions of phenylacetyl-CoA derivatives in aqueous basic
solution, presumably as a result of rapid hydrolysis.
The theoretical prediction of absolute pKa values has
become possible in recent years owing to the advent of
accurate continuum-solvation methods.[12–14] Even though the
accuracy of this approach for compounds with protic hydrogen atoms is quite impressive, its application to acidic CH
open-shell systems has not yet been tested. Therefore, in this
study, we apply an indirect approach in which the acidities of
the enoyl radicals are compared with the known values of the
closely related thioesters. Specifically, we compare herein the
a deprotonation of methyl thiopropionate 8 (to give the
corresponding anion 9) with the b deprotonation of the
propionate radical 10 to give the radical anion of methyl
thioacrylate 11 (Scheme 2).
Figure 1 shows the optimum B3LY/Paug-cc-pVDZ structures of 8–11. Although rather short CS bond lengths are
found for the neutral thioesters 8 and 10, in line with
expectations for a typical CS single bond, much longer
distances are found for the deprotonated forms 9 and 11. This
trend is particularly notable in 9 with a CS bond length of
1.97 F. The bond lengthening is partly a result of charge
transfer from the acyl moiety to the sulfur atom, as can be
seen by comparing the sulfur atomic charges in the neutral
Scheme 2. Model systems used to calculate pKa values.
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Structures of thioesters 8–11 optimized at the Becke3LYP/
aug-cc-pVDZ level of theory. Data in brackets were obtained with the
CPCM model for aqueous solvation.
systems (+ 0.18 e in 8 and + 0.20 e in 10) with those in the
anionic systems (0.07 e in 9 and 0.02 e in 11). The increase
in negative charge at sulfur, as well as the observed CS bond
stretching, suggests that the short lifetime of methyl thioacyl
anions in aqueous solution may indeed be due to the rapid
release of methylthiolate anions under these conditions.
Deprotonation also leads to a shortening of the bond
between the carbonyl carbon atom and the a-C atom. This is
most pronounced in the closed-shell system with CC bond
lengths of 1.52 F in 8 and only 1.37 F in 9. The bond
shortening is, in comparison, much smaller in the open-shell
systems 10 and 11. These structural changes are accompanied
by an increase in the atomic charge of the a-C atom, which is
almost neutral in 8 and 10 with charges of 0.03 e and
+ 0.09 e, respectively, but clearly negative in 9 and 11 with
respective atomic charges of 0.35 e and 0.22 e. This
deprotonation-induced bond shortening is to be expected as
a consequence of resonance stabilization of the developing
negative charge by the adjacent carbonyl group. The effect is
larger in the more localized case of 8 than in the corresponding open-shell system 10, in which the CC bond length is
already shortened as a result of resonance stabilization of the
initial radical.
Most of the unpaired spin density of 10 is localized at the
a-C atom (NPA coefficient 0.75) with additional contributions from the carbonyl oxygen atom (0.18). This is certainly
in accordance with expectations for a heteroallylic radical
such as 10. The spin density is slightly more delocalized in 11
with coefficients of 0.55 at the terminal b-C atom, 0.25 at the
carbonyl carbon atom, and 0.17 at the carbonyl oxygen atom.
This is very much in line with expectation for the LUMO
structure of a,b-unsaturated carbonyl compounds.[17] The
Lewis structures shown in Scheme 2 are most suitable to
reflect the situation in both 10 and 11.
Deprotonation of thioester 8 to yield a free proton and
anion 9 is strongly endothermic in the gas phase (Table 1).
Zero-point-corrected relative energies at 0 K, values includ-
Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870
Table 1: Free energies of deprotonation and the difference in acidity
between 8 and 10.[a]
Gas phase
DG (B3LY/Paug-cc-pVDZ)
DG (G3(MP2)(þ)-RAD(p))
+ 1472.2
+ 1488.7
+ 1424.4
+ 1434.8
Aqueous solution (implicit
DG (G3(MP2)(þ)-RAD(p))
∆∆Gsolv = ∆G2 – ∆G1
+ 149.6
Aqueous solution (explicit
DG (G3(MP2)(þ)-RAD(p))
+ 106.9
[a] DDG in kJ mol , pKa values are dimensionless, values for T = s98 K.
[b] Obtained by using the CPCM continuum-solvation model, see text.
[c] Obtained by using a periodic box of 566 TIP3P water molecules, see
ing enthalpic corrections to 298 K, and values including both
enthalpic and entropic corrections are all in agreement with
the fact that the radical 10 is approximately 50 kJ mol1 more
acidic than the closed-shell ester 8. Because of this invariance,
we have chosen to include only the free-energy differences in
Table 1. Combination of the more reliable G3(MP2)(þ)RAD(p) energies with thermochemical corrections calculated
at the B3LY/Paug-cc-pVDZ level yields free-energy values
that are approximately 6 kJ mol1 larger than those calculated
at the lower level. Our best estimate for the difference in free
energy of deprotonation between 8 and 10 in the gas phase
therefore amounts to 53.9 kJ mol1 at 298 K. According to
DG = RT lnK, this free-energy difference will effectively
lower the pKa value of 10 by 9.4 units relative to that of 8.
For use in aqueous solution, the gas-phase values must be
corrected for differences in free energies of solvation of all
four species involved. The structures of thioesters 8–11 could,
however, conceivably undergo considerable changes upon
aqueous solvation, and therefore the structures were reoptimized using the CPCM continuum model. The structural
parameter most strongly affected by the presence of the
solvent field is the CS ester bond in anions 9 and 11
(Figure 1). Most other structural parameters show only a
small solvent effect.
Examination of the free energies of solvation from the
implicit (CPCM) approach shows that aqueous solvation has
a more pronounced effect on the dissociation of 8 than on that
of 10. This appears to be primarily a result of better solvation
of the more localized enolate anion 9 relative to the
delocalized radical anion 11. The acidity difference predicted
by the implicit solvation model is therefore lower than that
calculated in the gas phase. After the inclusion of the CPCM
solvation effects, the acidity enhancement is calculated to be
42.7 kJ mol1 or 7.5 pKa units at 298 K.
In addition to the implicit solvent calculations outlined
above, we also carried out explicit evaluation of the effect of
solvation on the acidity difference, in a periodic box of 566
TIP3P water molecules. As the direct calculation of charge
separation (such as that involved in deprotonation) is
difficult, we made use of the thermodynamic cycle shown in
Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870
= ∆G4 – ∆G3
Scheme 3. Free-energy cycle used in conjunction with the explicit solvation model.
Scheme 3.[18] Instead of directly calculating the difference in
free-energy of solvation (DG2DG1), we combined the
difference in solvation energies between the two neutral
systems 8 and 10 (DG3) with the difference in solvation
energies between the two anionic systems 9 and 11 (DG4).
Because free energy is a state function, the equation shown at
the bottom of Scheme 3 holds and we obtain a valid result for
the relative free energy of solvation.
The explicit solvation calculations provide support for our
previous conclusion regarding the more favorable solvation of
9 with respect to 11. In addition, the agreement between the
two approaches (implicit and explicit) is quite impressive,
with the explicit approach predicting an acidity difference
between 8 and 10 of 38.8 kJ mol1 or 6.7 pKa units. Given the
uncertainties involved in the calculations, our best estimate
for the pKa enhancement of 10 over 8 is then 7.1 0.4.
Assuming a pKa value of 21 0.5 for the a-CH group of
thioester 8 in aqueous solution, we would therefore predict a
pKa value of 14 for the b-CH group in radical 10. Although a
pKa value of 14 for the b position might not be considered
particularly acidic, it should be noted that the pKa value of the
proton at C2 of thiaminodiphosphate (12.7 0.1)[19] is in the
same range. In many enzymatic reactions, the carbanion of
thiaminodiphosphate is known to act as a nucleophile in CC
bond cleavage adjacent to carbonyl groups. For example, the
decarboxylation of pyruvate to acetaldehyde is one reaction
to make use of such a mechanism. Furthermore, the b position
in 10 is calculated to be more activated (acidic) than the
a position in typical hydrolyase substrates such as 3-hydroxybutyryl-CoA. However, as in the case of the latter substrate, it
is likely that the enzyme is still required to provide additional
stabilizing interactions to deprotonate the enoxy radical
efficiently under normal physiological conditions.
Given that the pKa value of the b position in closed-shell
substrates such as 3 is approximately 40, our prediction
corresponds to a radical-induced pKa enhancement of more
than 25 orders of magnitude! This far exceeds the established
radical-induced pKa enhancement for the hydroxy groups in
aliphatic alcohols.[20] For example, the pKa values of the
hydroxy groups of ethylene glycol are shifted from values of
around 17 to 9.8 upon radical formation. Although significant,
this corresponds to a radical-induced pKa shift of “only” 7 pKa
As mentioned previously, 2-hydroxyglutaryl-CoA dehydratase is not the only enzyme that employs a radical
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
mechanism to activate the b position of carbonyl compounds.
This fact, combined with the magnitude of the calculated pKa
enhancement in the current work, makes it appear likely that
in the anaerobic world there are more enzymes to be
discovered that make use of the facile deprotonation of an
enoxy radical.[19]
Experimental Section
Geometry optimizations were performed for all four species (8, 9, 10,
and 11) in the gas phase at the Becke3 LYP/aug-cc-pVDZ level of
theory.[21, 22] Zero-point vibrational energies and thermochemical
corrections were calculated at this same level without any scaling.
The charge- and spin-density distributions were analyzed by the
Natural Population Analysis (NPA) method.[23]
Improved energetics were evaluated with a slightly modified
version of the G3(MP2)(þ)-RAD(p) procedure.[24] This technique is a
modification of the G3(MP2) method[25] in which a restricted-openshell coupled-cluster calculation (RCCSD(T)/6-31 + G(d)) replaces
the UQCISD(T)/6-31G(d) calculation and the basis-set extension is
evaluated with restricted-open-shell perturbation theory (ROMP2)
rather than with the unrestricted formalism (UMP2). The only
difference between the method outlined in reference [24] and that
employed in this case is the use of geometries and frequencies
obtained with the Becke3LYP/aug-cc-pVDZ level of theory.
We chose to include the effects of aqueous solvation with two
different approaches. The first (implicit) approach employed the
CPCM continuum-solvation method in combination with the UAHF
cavity model at the Becke3LYP/aug-cc-pVDZ level of theory.[26] To
account for structural relaxation in the aqueous environment, we
reoptimized all structures with the CPCM model prior to calculating
solvation energies. In the second (explicit) approach, we solvated
each molecule in a periodic box of 566 TIP3P[27] waters and used
thermodynamic integration[28] to calculate relative free energies of
solvation. Further information on these calculations can be found in
the Supporting Information. The CCSD(T) calculations were performed with MOLPRO,[29] other gas-phase and implicit solvation
calculations employed Gaussian 98,[30] while all explicit solvation
calculations were carried out with the AMBER[31] program suite.
Received: November 7, 2002 [Z50502]
Keywords: ab initio calculations · acidity · CH activation ·
enzyme catalysis · radicals
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