Mechanisms of Enzyme Catalysis

Deprotonation of Enoxy Radicals: TheoreticalValidation of a 50-Year-Old MechanisticProposal**

David M. Smith, Wolfgang Buckel, and Hendrik Zipse*

The dehydration or a,b elimination of water from biomole-cules is a very common enzymatic reaction. Almost alldehydratases catalyze the removal of an hydroxy group in theb position of an electron-withdrawing carboxylate, thioester,or carbonyl group [Eq. (1)].

The C�Ha bond of compounds such as 1 is activated andcan be deprotonated relatively easily by a basic residue of anenzyme. In a typical substrate, such as 3-hydroxybutyryl-CoA(1 with R1= SCoA, R2=CH3, R3=H), the relevant C�Ha

bond has an associated pKa of approximately 21 in aqueoussolution. Upon binding to enoyl-CoA hydratase (crotonase),this pKa is reduced to approximately 8 by specific interactionswith the enzyme. An essential glutamate residue appears toact as the base in the abstraction of the activated aliphaticproton.[1–3]

Several glutamate-fermenting anaerobic bacteria containa dehydratase, which catalyses the reversible syn, a,b elimi-nation of water from (R)-2-hydroxyglutaryl-CoA (3) to (E)-glutaconyl-CoA (4) (Scheme 1). In this case, however, theC�Hb bond to be cleaved is not activated (pKa� 40) and inthe reverse direction the OH� group adds to the moreelectron-rich a-C atom of the polarized double bond. Toexplain these observations, a mechanism has been proposedin which the carbonyl group undergoes an “Umpolung” byone-electron reduction.[4] The resulting substrate-derivedketyl radical anion 5 (Scheme 1) may eliminate the a hydroxy

group, leading to the enoxy radical 6. Deprotonation (Hb) ofthis radical would yield the product-related ketyl radicalanion 7, which could be oxidized to the product 4.[5–7]

The biochemical analyses of the two-component 2-hydroxyglutaryl-CoA dehydratase systems from the intestinalbacteria Acidaminococcus fermentans and Clostridium sym-biosum indeed reveal the possibility of an electron transfersuch as the one shown in Scheme 1.[8–10] Component A fromA. fermentans, a homodimeric protein with a [4Fe-4S]1+/2+

cluster between the two subunits, transfers an electron tocomponent D under hydrolysis of ATP. Component D is aheterodimeric protein containing molybdenum(vi), reducedriboflavin-5’-phosphate (FMNH2), and a [4Fe-4S]2+ cluster.This ATP-driven electron transfer causes the formation ofMov, which through oxidation to Movi quite probablygenerates the ketyl radical anion 5. Oxidation of the secondketyl radical anion 7 is likely to reform Mov, so that a singleelectron transfer between the two components is sufficient toinduce many catalytic cycles.

2-Hydroxyglutaryl-CoA dehydratase is not the onlyenzyme involved in the cleavage of an unactivated C�Hb

bond. For example, the dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, catalyzed by an oxygen-sensitive[4Fe-4S] cluster and FAD-containing enzyme from Clostri-dium aminobutyricum, also involves the loss of a proton fromthe b position of a thioester. In this case, it has been proposedthat an enoxy radical (similar to 6 in Scheme 1) is generatedby radical abstraction of the a hydrogen atom. Deprotonationof the b C atom of this radical leads to the ketyl radical anionof 4-hydroxycrotonyl-CoA, which can expel the hydroxygroup to yield the dienoxy radical. Re-addition of the initiallyabstracted hydrogen atom at C4 yields the final product,crotonyl-CoA.[7]

The key feature of the proposed mechanisms for boththese enzymes is that the b H atom is lost from the enoxyradical (e.g. 6) and not from the substrate itself (e.g. 3).

O

R1

OH

Hα

R3R2

O

R1

R3R2

–H2O

1 2

R1 = OH, SR, R

(1)O

SCoA

Hβ

– OH

O

SCoA

Hβ

HO

– H

O

SCoA

O

SCoA

Hβ

HO

O

SCoA- H2O

3 4

_

•

_

• _

+ e

5

6

7

+

_

_– e

•

–OOC

–OOC

–OOC

–OOC

–OOC

Scheme 1. Proposed mechanism for the reversible syn, a,b eliminationof water from (R)-2-hydroxyglutaryl-CoA (3).

[*] Prof. Dr. H. Zipse, Dr. D. M. SmithDepartment Chemie, Ludwig-Maximilians-Universit*tButenandtstrasse 13, 82131 M/nchen (Germany)Fax: (+49)89-2180-77738E-mail: zipse@cup.uni-muenchen.de

Prof. Dr. W. BuckelLaboratorium f/r Mikrobiologie, Philipps-Universit*tKarl-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 theFonds der Chemischen Industrie for continuous support andD.M.S. thanks the Alexander von Humboldt Foundation for aresearch fellowship. We thank Prof. V. Barone for helpful discus-sions and technical assistance.

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

AngewandteChemie

1867Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870 DOI: 10.1002/anie.200250502 � 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Almost 50 years ago, Sir John W. Cornforth suggested that anenoxy radical (derived by a-H-atom abstraction in a mech-anism similar to that proposed for the dehydration of 4-hydroxybutyryl-CoA) might undergo facile deprotonation toa ketyl radical anion.[11] However, this suggestion has notsince been proven by any experimental or theoretical data.Herein, we aim to quantify the acidity of the b position of theenoxy radical. We wish to determine whether the acidity ofthis radical is sufficiently enhanced over the closed-shellthioester to explain the apparent deprotonation at theunactivated position, or whether additional factors must beinvoked to arrive at a satisfactory mechanism.

The acidities of simple thioesters have been studiedexperimentally by Richard and Amyes in D2O at 25 8C, and apKa value of 21� 0.5 was determined for methyl thioace-tate.[15] Much lower values were estimated for substitutedphenylacetyl-CoA derivatives based on the measured valuesof analogously substituted phenlyacetone compounds byGhisla and co-workers.[16] The estimated pKa value for 4-nitrophenylacetyl-CoA amounts to 13.6. An additional obser-vation made in this latter study is the short lifetime of theanions of phenylacetyl-CoA derivatives in aqueous basicsolution, presumably as a result of rapid hydrolysis.

The theoretical prediction of absolute pKa values hasbecome possible in recent years owing to the advent ofaccurate continuum-solvation methods.[12–14] Even though theaccuracy of this approach for compounds with protic hydro-gen atoms is quite impressive, its application to acidic C�Hopen-shell systems has not yet been tested. Therefore, in thisstudy, we apply an indirect approach in which the acidities ofthe enoyl radicals are compared with the known values of theclosely related thioesters. Specifically, we compare herein thea deprotonation of methyl thiopropionate 8 (to give thecorresponding anion 9) with the b deprotonation of thepropionate radical 10 to give the radical anion of methylthioacrylate 11 (Scheme 2).

Figure 1 shows the optimum B3LY/Paug-cc-pVDZ struc-tures of 8–11. Although rather short C�S bond lengths arefound for the neutral thioesters 8 and 10, in line withexpectations for a typical C�S single bond, much longerdistances are found for the deprotonated forms 9 and 11. Thistrend is particularly notable in 9 with a C�S bond length of1.97 F. The bond lengthening is partly a result of chargetransfer from the acyl moiety to the sulfur atom, as can beseen by comparing the sulfur atomic charges in the neutral

systems (+ 0.18 e in 8 and + 0.20 e in 10) with those in theanionic systems (�0.07 e in 9 and �0.02 e in 11). The increasein negative charge at sulfur, as well as the observed C�S bondstretching, suggests that the short lifetime of methyl thioacylanions in aqueous solution may indeed be due to the rapidrelease of methylthiolate anions under these conditions.

Deprotonation also leads to a shortening of the bondbetween the carbonyl carbon atom and the a-C atom. This ismost pronounced in the closed-shell system with C�C bondlengths of 1.52 F in 8 and only 1.37 F in 9. The bondshortening is, in comparison, much smaller in the open-shellsystems 10 and 11. These structural changes are accompaniedby an increase in the atomic charge of the a-C atom, which isalmost neutral in 8 and 10 with charges of �0.03 e and+ 0.09 e, respectively, but clearly negative in 9 and 11 withrespective atomic charges of �0.35 e and �0.22 e. Thisdeprotonation-induced bond shortening is to be expected asa consequence of resonance stabilization of the developingnegative charge by the adjacent carbonyl group. The effect islarger in the more localized case of 8 than in the correspond-ing open-shell system 10, in which the C�C bond length isalready shortened as a result of resonance stabilization of theinitial radical.

Most of the unpaired spin density of 10 is localized at thea-C atom (NPA coefficient 0.75) with additional contribu-tions from the carbonyl oxygen atom (0.18). This is certainlyin accordance with expectations for a heteroallylic radicalsuch as 10. The spin density is slightly more delocalized in 11with coefficients of 0.55 at the terminal b-C atom, 0.25 at thecarbonyl carbon atom, and 0.17 at the carbonyl oxygen atom.This is very much in line with expectation for the LUMOstructure of a,b-unsaturated carbonyl compounds.[17] TheLewis structures shown in Scheme 2 are most suitable toreflect the situation in both 10 and 11.

Deprotonation of thioester 8 to yield a free proton andanion 9 is strongly endothermic in the gas phase (Table 1).Zero-point-corrected relative energies at 0 K, values includ-

O

SCH3

H3C

HHα

O

SCH3

C

H

Hβ

HH

–H+

O

SCH3H

HH

O

SCH3

H3C

H–H+

8 9

_

10 11

_• •

Scheme 2. Model systems used to calculate pKa values.

Figure 1. Structures of thioesters 8–11 optimized at the Becke3LYP/aug-cc-pVDZ level of theory. Data in brackets were obtained with theCPCM model for aqueous solvation.

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1868 � 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870

ing enthalpic corrections to 298 K, and values including bothenthalpic and entropic corrections are all in agreement withthe fact that the radical 10 is approximately 50 kJ mol�1 moreacidic than the closed-shell ester 8. Because of this invariance,we have chosen to include only the free-energy differences inTable 1. Combination of the more reliable G3(MP2)(þ)-RAD(p) energies with thermochemical corrections calculatedat the B3LY/Paug-cc-pVDZ level yields free-energy valuesthat are approximately 6 kJmol�1 larger than those calculatedat the lower level. Our best estimate for the difference in freeenergy of deprotonation between 8 and 10 in the gas phasetherefore amounts to 53.9 kJmol�1 at 298 K. According toDG=�RT lnK, this free-energy difference will effectivelylower the pKa value of 10 by 9.4 units relative to that of 8.

For use in aqueous solution, the gas-phase values must becorrected for differences in free energies of solvation of allfour species involved. The structures of thioesters 8–11 could,however, conceivably undergo considerable changes uponaqueous solvation, and therefore the structures were reopti-mized using the CPCM continuum model. The structuralparameter most strongly affected by the presence of thesolvent field is the C�S ester bond in anions 9 and 11(Figure 1). Most other structural parameters show only asmall solvent effect.

Examination of the free energies of solvation from theimplicit (CPCM) approach shows that aqueous solvation hasa more pronounced effect on the dissociation of 8 than on thatof 10. This appears to be primarily a result of better solvationof the more localized enolate anion 9 relative to thedelocalized radical anion 11. The acidity difference predictedby the implicit solvation model is therefore lower than thatcalculated in the gas phase. After the inclusion of the CPCMsolvation effects, the acidity enhancement is calculated to be42.7 kJmol�1 or 7.5 pKa units at 298 K.

In addition to the implicit solvent calculations outlinedabove, we also carried out explicit evaluation of the effect ofsolvation on the acidity difference, in a periodic box of 566TIP3P water molecules. As the direct calculation of chargeseparation (such as that involved in deprotonation) isdifficult, we made use of the thermodynamic cycle shown in

Scheme 3.[18] Instead of directly calculating the difference infree-energy of solvation (DG2�DG1), we combined thedifference in solvation energies between the two neutralsystems 8 and 10 (DG3) with the difference in solvationenergies between the two anionic systems 9 and 11 (DG4).Because free energy is a state function, the equation shown atthe bottom of Scheme 3 holds and we obtain a valid result forthe relative free energy of solvation.

The explicit solvation calculations provide support for ourprevious conclusion regarding the more favorable solvation of9 with respect to 11. In addition, the agreement between thetwo approaches (implicit and explicit) is quite impressive,with the explicit approach predicting an acidity differencebetween 8 and 10 of 38.8 kJmol�1 or 6.7 pKa units. Given theuncertainties involved in the calculations, our best estimatefor 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 ofthioester 8 in aqueous solution, we would therefore predict apKa value of 14 for the b-CH group in radical 10. Although apKa value of 14 for the b position might not be consideredparticularly acidic, it should be noted that the pKa value of theproton at C2 of thiaminodiphosphate (12.7� 0.1)[19] is in thesame range. In many enzymatic reactions, the carbanion ofthiaminodiphosphate is known to act as a nucleophile in C�Cbond cleavage adjacent to carbonyl groups. For example, thedecarboxylation of pyruvate to acetaldehyde is one reactionto make use of such a mechanism. Furthermore, the b positionin 10 is calculated to be more activated (acidic) than thea position in typical hydrolyase substrates such as 3-hydroxy-butyryl-CoA. However, as in the case of the latter substrate, itis likely that the enzyme is still required to provide additionalstabilizing interactions to deprotonate the enoxy radicalefficiently under normal physiological conditions.

Given that the pKa value of the b position in closed-shellsubstrates such as 3 is approximately 40, our predictioncorresponds to a radical-induced pKa enhancement of morethan 25 orders of magnitude! This far exceeds the establishedradical-induced pKa enhancement for the hydroxy groups inaliphatic alcohols.[20] For example, the pKa values of thehydroxy groups of ethylene glycol are shifted from values ofaround 17 to 9.8 upon radical formation. Although significant,this corresponds to a radical-induced pKa shift of “only” 7 pKa

units.As mentioned previously, 2-hydroxyglutaryl-CoA dehy-

dratase is not the only enzyme that employs a radical

Table 1: Free energies of deprotonation and the difference in aciditybetween 8 and 10.[a]

Level 9�8 11�10 DDG DpKa[a]

Gas phaseDG (B3LY/Paug-cc-pVDZ) +1472.2 +1424.4 47.8 8.4DG (G3(MP2)(þ)-RAD(p)) +1488.7 +1434.8 53.9 9.4

Aqueous solution (implicitsolvation)[b]

DG (G3(MP2)(þ)-RAD(p)) +149.6 +106.9 42.7 7.5

Aqueous solution (explicitsolvation)[c]

DG (G3(MP2)(þ)-RAD(p)) 38.3 6.7

[a] DDG in kJmol� , 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, seetext.

8aq 9aq

10aq 11aq

∆G1

∆G2

∆G3 ∆G4

∆∆Gsolv = ∆G2 – ∆G1

= ∆G4 – ∆G3

Scheme 3. Free-energy cycle used in conjunction with the explicit solva-tion model.

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1869Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870 www.angewandte.org � 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 thatin the anaerobic world there are more enzymes to bediscovered that make use of the facile deprotonation of anenoxy radical.[19]

Experimental SectionGeometry optimizations were performed for all four species (8, 9, 10,and 11) in the gas phase at the Becke3LYP/aug-cc-pVDZ level oftheory.[21,22] Zero-point vibrational energies and thermochemicalcorrections were calculated at this same level without any scaling.The charge- and spin-density distributions were analyzed by theNatural Population Analysis (NPA) method.[23]

Improved energetics were evaluated with a slightly modifiedversion of the G3(MP2)(þ)-RAD(p) procedure.[24] This technique is amodification of the G3(MP2) method[25] in which a restricted-open-shell coupled-cluster calculation (RCCSD(T)/6-31+G(d)) replacesthe UQCISD(T)/6-31G(d) calculation and the basis-set extension isevaluated with restricted-open-shell perturbation theory (ROMP2)rather than with the unrestricted formalism (UMP2). The onlydifference between the method outlined in reference [24] and thatemployed in this case is the use of geometries and frequenciesobtained with the Becke3LYP/aug-cc-pVDZ level of theory.

We chose to include the effects of aqueous solvation with twodifferent approaches. The first (implicit) approach employed theCPCM continuum-solvation method in combination with the UAHFcavity model at the Becke3LYP/aug-cc-pVDZ level of theory.[26] Toaccount for structural relaxation in the aqueous environment, wereoptimized all structures with the CPCM model prior to calculatingsolvation energies. In the second (explicit) approach, we solvatedeach molecule in a periodic box of 566 TIP3P[27] waters and usedthermodynamic integration[28] to calculate relative free energies ofsolvation. Further information on these calculations can be found inthe Supporting Information. The CCSD(T) calculations were per-formed with MOLPRO,[29] other gas-phase and implicit solvationcalculations employed Gaussian 98,[30] while all explicit solvationcalculations were carried out with the AMBER[31] program suite.

Received: November 7, 2002 [Z50502]

.Keywords: ab initio calculations · acidity · C�H activation ·enzyme catalysis · radicals

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1870 � 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2003, 42, 1867 – 1870