Download Radical species in the catalytic pathways of enzymes from anaerobes

FEMS Microbiology Reviews 22 (1999) 523^541
Radical species in the catalytic pathways of enzymes from
anaerobes
Wolfgang Buckel a; *, Bernard T. Golding
b
b
a
Laboratorium fuër Mikrobiologie, Philipps-Universitaët, Marburg, Germany
Department of Chemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, UK
Received 17 June 1998; received in revised form 27 October 1998 ; accepted 29 October 1998
Abstract
Radicals are reactive intermediates of growing importance in enzymatic catalysis. There are reactions in which neutral
radicals participate and those where radical anions are involved. The former class is illustrated by lysine 2,3-aminomutase and
also by enzymes dependent on coenzyme B12 , that catalyse carbon skeleton rearrangements (e.g. glutamate mutase). A
substrate-based radical for both lysine 2,3-aminomutase and glutamate mutase has been characterised by EPR spectroscopy.
Representatives of the second class are 2-hydroxyglutaryl-CoA dehydratase, benzoyl-CoA reductase, DNA photolyase and
chorismate synthase, all of which may generate the radical anion by one-electron reduction. 4-Hydroxybutyryl-CoA
dehydratase, pyruvate formate lyase, and the coenzyme B12 -dependent eliminases (ribonucleotide reductase, ethanolamine
ammonia lyase and diol dehydratase) could be examples of radical anion formation by one-electron oxidation. The electronrich ketyl-like radical anions cause the elimination of an adjacent group. The advantages of using radicals as intermediates in
enzymatic transformations are their high reactivity and special properties. However, this reactivity includes rapid bimolecular
combination with dioxygen and radicals are therefore primarily utilised as intermediates by anaerobic organisms. z 1999
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Substrate-based radical; Coenzyme B12 -dependent carbon skeleton mutase and eliminase ; Radical anion; Umpolung ; Lysine 2,3aminomutase ; 2-Hydroxyglutaryl-CoA dehydratase ; Benzoyl-CoA reductase ; DNA photolyase ; Chorismate synthase; 4-HydroxybutyrylCoA dehydratase; Pyruvate formate lyase
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Detection of substrate-based radicals . . . . . . . . . . . . . . . . . . . . . . . .
3. Mechanisms of enzymatic reactions initiated by the 5P-deoxyadenosyl
4. Umpolung via radical anions, a new concept in enzymatic catalysis .
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +49 (6421) 28 1527; Fax: +49 (6421) 28 8979; E-mail: [email protected]
0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
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W. Buckel, B.T. Golding / FEMS Microbiology Reviews 22 (1999) 523^541
1. Introduction
A century ago in Munich, Edward Buchner prepared the ¢rst active cell-free extract from brewer's
yeast. This opened the door for the puri¢cation of
enzymes (OXjSWg = in yeast, [1]) and led to the discovery of the main metabolic routes by Otto Warburg, Sir Hans Krebs, Feodor Lynen and other biochemists. In 1959, the organic chemist Sir John W.
Cornforth summarised these achievements in an important review [2] and proposed plausible mechanisms for the enzymatic reactions of fatty acid and
cholesterol biosynthesis in which, with one exception, no radicals were involved. The exception was
the FAD-containing acyl-CoA dehydrogenase in
which £avin semiquinone [3] was thought to participate. At that time all known enzymatic catalyses, in
which C^H and C^C bonds were formed or broken,
were written as two-electron steps, although Leonor
Michaelis had proposed that `all oxidations of organic molecules proceed in successive univalent
steps' [4]. Horace Albert Barker, the discoverer of
the coenzyme form of vitamin B12 in glutamate mutase from Clostridium tetanomorphum [5], hardly considered radicals as important intermediates in enzymatic catalysis. Even Robert H. Abeles, who has
uncovered so many unusual enzymatic reactions, is
quoted as saying: ``if you can formulate on paper a
mechanism in two-electron steps, then there is no
radical involved'' (Perry Frey, University of Wisconsin, Madison, WI, USA, personal communication).
Today it is generally accepted that under aerobic, as
well as anaerobic conditions, cleavage of all unactivated C^H bonds occurs via radical intermediates,
although the true pathway with mono-oxygenases
such as cytochrome P450 may have cationic character
[6]. In addition there is an increasing number of reactions which can be formulated in two-electron
steps but proceed via radicals, e.g. the reactions catalysed by galactose oxidase [7,8] and pyruvate ferredoxin oxidoreductase (Fig. 1) [9,10]. There is even
evidence for the participation of radicals in NAD‡ and thiamine diphosphate-dependent pyruvate dehydrogenase. Presumably, the hydroxyethylthiamine di-
Fig. 1. Proposed mechanism of pyruvate ferredoxin oxidoreductase.
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525
Fig. 2. Proposed mechanism of lysine-2,3-aminomutase. PxCHO = pyridoxal-5P-phosphate ; Ado-CH3 = 5P-deoxyadenosine.
phosphate intermediate can only be oxidised in oneelectron steps, as in pyruvate ferredoxin oxidoreductase (Perry Frey, personal communication).
Despite the slow acceptance of radicals in enzymatic catalysis, they are apparently much more common than hitherto anticipated. The use of radicals as
intermediates by enzymes opens new reaction paths
and enables the metabolism of otherwise refractory
compounds. Since they are highly reactive towards
dioxygen, radicals are found especially in catalysis by
enzymes from anaerobic micro-organisms, which are
highlighted in this review. The main topic is the formation and the properties of substrate-based radicals. In two sections the detection of neutral radicals
and their possible rearrangements are discussed. A
third section focuses on the intermediacy of ketyllike radical anions, which form an emerging concept
in biological catalysis.
2. Detection of substrate-based radicals
Helmut Beinert pioneered EPR spectroscopy in
enzyme research and was the ¢rst to detect £avin
semiquinones and iron-sulfur clusters in proteins
[3]. Though several enzymes are known to stabilise
£avin semiquinones [11], ironically the only radical
mechanism postulated by Cornforth in his review of
1959 cannot be accepted any longer [12]. Later
Orme-Johnson, Beinert and Blakley discovered the
catalytically competent EPR signal of the coenzyme
B12 -dependent ribonucleotide triphosphate reductase
from Lactobacillus leichmannii [13], which was ¢nally
identi¢ed by the group of JoAnne Stubbe, in collaboration with Gary Gerfen, as a protein-based thiyl
radical interacting with cob(II)alamin at a distance
î [14]. Actually, the groups of Frey and
of about 6 A
Reed were the ¢rst to characterise thoroughly a substrate-derived radical in an enzymatic reaction, the
pyridoxal-5P-phosphate-dependent equilibration of Llysine [(2S)-2,6-diaminohexanoate] with L-lysine
[(3S)-3,6-diaminohexanoate] catalysed by lysine-2,3aminomutase from Clostridium subterminale [15]
(Fig. 2). This reaction is the ¢rst step in the L-lysine
fermentation pathway leading to ammonia, acetate
and butyrate. The migration of the amino group
from the K- to the L-position enables the subsequent
cleavage of the carbon skeleton into a C2 and a C4
fragment. As in coenzyme B12 -dependent reactions
(see below), a hydrogen atom is initially abstracted
from the L-carbon by the 5P-deoxyadenosyl radical,
which in this case is generated by one-electron reduction of S-adenosylmethionine to methionine mediated by the [4Fe-4S] cluster of the enzyme. The for-
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mation of the 5P-deoxyadenosyl radical from S-adenosylmethionine occurs irreversibly and only in the
complete absence of oxygen. It contrasts with the
reversible formation of the same radical by homolysis of the cobalt-carbon bond of coenzyme B12 ,
which also occurs in aerobic organisms. Hence,
H.A. Barker called S-adenosylmethionine `poor
man's coenzyme B12 ' (cited in [16]).
After abstraction of the L-Si-hydrogen from L-lysine, the amino group, which was previously combined with pyridoxal-5P-phosphate by imine formation, migrates from the K- to the L-position (1C2).
Finally, a hydrogen is donated back from the methyl
group of 5P-deoxyadenosine to the K-position. The
most persistent radical in this process, (3S)-3-(pyridoximino-5P-phosphate)-6-aminohexanoate-2-yl (Kradical of L-lysine attached to pyridoxal-5P-phosphate, 2, Fig. 2), was extensively characterised by
EPR spectroscopy [15].
In the early seventies, EPR spectra of the coenzyme B12 -dependent enzymes diol dehydratase and
ethanolamine ammonia lyase were published, but
the nature of the radicals could not be resolved
(see discussion in [15]). Recently, a substrate-derived
radical was characterised with the coenzyme B12 -dependent glutamate mutase from Clostridium cochlea-
rium. Upon addition of the substrate L-glutamate to
the holoenzyme, an EPR spectrum with a spin concentration of 50% of that of the enzyme was observed, which was related to that of cob(II)alamin,
but di¡ered in g-value and coupling constant [17].
The spectrum was simulated by assuming a dipolar
interaction and exchange coupling of the substratederived 4-glutamyl radical (3, Fig. 3) with cob(II)î , similar to the
alamin at a distance of 6.5 þ 0.9 A
thiyl radical of ribonucleotide reductase [18].
3. Mechanisms of enzymatic reactions initiated by the
5P-deoxyadenosyl radical
Glutamate mutase from clostridia initiates a fermentation pathway via pyruvate to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The biological signi¢cance of the rearrangement
of the linear carbon skeleton of (S)-glutamate to the
branched one of (2S,3S)-3-methylaspartate lies in the
subsequent facile elimination of ammonia to yield
mesaconate (methylfumarate). Hydration to (S)-citramalate (2-methylmalate), and the subsequent
retro-Claisen condensation to acetate and pyruvate,
conclude the special part of the fermentation path
Fig. 3. Proposed fragmentation/recombination mechanism of glutamate mutase.
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527
Fig. 4. Proposed addition/elimination mechanism of 2-methyleneglutarate mutase. The coenzyme (cf. Fig. 3) is omitted. Only the Z-isomer
of 2-[methylene-2 H]methyleneglutarate and the E-isomer of (R)-3-methyl[methylene-2 H]itaconate are shown.
[19]. A related rearrangement is found in the pathway of nicotinate fermentation to ammonia, acetate,
carbon dioxide and propionate by Clostridium barkeri. In a series of redox, hydrolytic and elimination
reactions, nicotinate is converted to 2-methyleneglutarate whereby the original carbon skeleton is still
retained. In the subsequent coenzyme B12 -dependent
rearrangement of 2-methyleneglutarate to (R)-3methylitaconate, a derivative of a C5 -dicarboxylate
is again converted to a more branched C4 -dicarboxylate, which can be readily degraded via pyruvate to
the ¢nal products [20,21]. Methylmalonyl-CoA mutase is the best known coenzyme B12 -dependent enzyme of this group of carbon skeleton mutases, since
it occurs in many Bacteria and Eucarya including
human mitochondria [22]. These three carbon skeleton mutases have several features in common, including a related cobalamin-binding domain with a
conserved histidine that is coordinated to cobalt
([23^25]; G. Broëker, F. Kroll and W. Buckel, unpublished results) and similar substrate- and inhibitorinduced EPR spectra [17,26^29]. Whereas the formation of the substrate-derived radicals in these three
enzymes has been established, their subsequent rearrangement to the product related radicals is still a
matter of controversy and will be discussed in this
section.
For glutamate mutase [21,26,30], it has been pro-
posed that the 4-glutamyl radical (3) fragments into
acrylate (5) and the 2-glycinyl radical (4), which recombine to the product-related 3-methyleneaspartate
radical (6, Fig. 3). This proposal is supported by the
observation that only the combination of glycine and
acrylate, but not the individual compounds, inhibits
glutamate mutase at the same concentration as that
of the substrate. Furthermore, glycine+acrylate together induce an EPR spectrum related to that derived from the substrate glutamate or the product 3methylaspartate [26]. In a similar way, the 2-methylene-4-glutarate radical (7, Fig. 4) could fragment
into 2-acrylate radical and acrylate during catalysis
by 2-methyleneglutarate mutase, and the 3-succinylCoA radical could yield formyl-CoA radical and
acrylate with methylmalonyl-CoA mutase. 2-Methyleneglutarate mutase is indeed inhibited by two acrylates, as shown by a square dependence on the acrylate concentration. Furthermore acrylate also induces
an EPR spectrum [26,31]. In contrast, formyl-CoA
and acrylate inhibit methylmalonyl-CoA mutase very
weakly and induce no EPR spectrum [27].
For methylmalonyl-CoA mutase and 2-methyleneglutarate mutase, an alternative `addition-elimination' pathway has to be considered for these enzymes
(see Fig. 4 for 2-methyleneglutarate mutase). Methylmalonyl-CoA and 2-methyleneglutarate each contain an sp2 -centre to which the free electron in the
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substrate-derived radical could add with the formation of the corresponding cyclopropyloxy or cyclopropylmethylene radical (8). Homolytic cleavage of
the newly formed C^C bond in the three-membered
ring regenerates the starting radical, whereas homolysis of an adjacent C^C bond gives the product-related radical (9). Evidence in support of this mechanism was initially thought to have been obtained for
2-methyleneglutarate mutase. The enzyme was found
to catalyse the rotation of the exo-methylene group
of 2-methyleneglutarate at a rate comparable to that
of substrate turnover1 , indicating the transient conversion of the exo-methylene group into a methylene
radical, which can rotate with a relatively low energy
barrier [32]. However, the steric course described can
also be explained by a fragmentation-recombination
mechanism (cf. glutamate mutase, Fig. 3). Following
abstraction of 4-HRe by the 5P-deoxyadenosyl radical
and fragmentation of the substrate-derived radical to
acrylate and the 2-acrylate radical, which is most
likely linear [33], a rotation of 180³ before its recombination with acrylate could explain the observed
steric course. The rotation could either involve the
whole acrylate radical molecule along its longitudinal
axis or an internal rotation at the bond between the
carboxyl group and the radical carbon.
In another experiment the possible intermediacy of
the 1-methylenecyclopropane-1,2-dicarboxylate radical in the 2-methyleneglutarate mutase reaction was
checked by testing all four stereoisomers of the stable analogue 1-methylcyclopropyl-1,2-dicarboxylate
and none was inhibitory [26]. An inhibition should
be only observable, however, if the analogue matches
a transition state, which is stabilised by the enzyme.
If the rate-limiting steps with the highest transition
states in the mutase reaction are formation of the
substrate-derived radical from the substrate and decomposition of the product-related radical to the
1
In the paper by Edwards et al. [32] the rate of the rotation was
estimated by the rate of the formation of the E-isomer of 2-(methylene-2 H1 )methyleneglutarate from the Z-isomer, which occurred
about 5 times slower than the rate of the formation of the product
(R)-3-methylitaconate. Since the equilibrium between 2-methyleneglutarate and (R)-3-methylitaconate was already reached after 6%
conversion of the less branched substrate, up to 94% of the Zisomer had not participated in catalysis under these conditions.
Hence, the conclusion drawn in this paper that rotation was slower
than carbon-skeleton rearrangement was incorrect.
product, the enzyme may not speci¢cally interact
with the cyclic radical and hence is not inhibited
by the analogue. Therefore, this negative result cannot disprove the addition-elimination mechanism
(Esteban Pombo-Villar, Novartis, Basel, Switzerland,
personal communication). With methylmalonyl-CoA
mutase, the 3 H-transfer rates were measured from 5Pdeoxy[5P-3 H]adenosylcobalamin to the substrate-derived radical and to the product-related radical.
Since there was no di¡erence whether the reaction
was initiated with succinyl-CoA or methylmalonylCoA, it has been concluded that the energy barrier
between the two radicals is indeed very low compared to those of the H-transfers [34].
L-Ketoalkyl radicals, which can be considered as
models for the radicals involved in the catalysis of
methylmalonyl-CoA mutase, were reported not to
rearrange, however, via cyclic intermediates, but to
undergo fragmentation-recombination via an acyl
radical and ethene ([35], see also [36]). In contrast,
a related rearrangement of 2-formylalkyl radicals
was formulated with an intermediate cyclopropyloxy
radical [37].
Furthermore, it has been argued that the steric
course of the conversion of the 3-methylene group
of succinyl-CoA to the methine group of (R)-methylmalonyl-CoA catalysed by methylmalonyl-CoA
mutase can be explained by a fragmentation-recombination mechanism, in which the intermediate acrylate performs a `£ip-over' in order to provide the
observed retention of con¢guration [26]. Stereospeci¢c substitution of the 3-Re-methylene hydrogen,
which is abstracted by the 5P-deoxadenosyl radical,
by deuterium, sometimes may cause the enzyme to
take the 3-Si-hydrogen because of a large isotope
e¡ect, D (kcat /Km )W7. Despite this `error in cryptic
stereospeci¢city' [38], by omitting the £ip-over of
acrylate, the product methylmalonyl-CoA will have
the correct R-con¢guration. An alternative explanation for the steric course of methylmalonyl-CoA mutase has been presented in terms of a 2,3-hydrogen
shift in the intermediate 3-succinyl-CoA radical [38].
To distinguish between the fragmentation-recombination and addition-elimination mechanisms for
methylmalonyl-CoA mutase and 2-methyleneglutarate mutase, ab initio molecular orbital calculations
have been performed on the alternative reaction
pathways [39]. The results of these studies showed
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529
Fig. 5. Proposed mechanism of benzylsuccinate synthase.
a clear energetic preference for the addition-elimination pathway. It was also shown for the degenerate
rearrangement of the 2-formylethyl radical that the
cyclisation to an intermediate cyclopropyloxy radical
was facilitated by protonation of the oxygen atom.
However, with glutamate mutase, assuming that only
the fragmentation-recombination pathway (Fig. 3) is
possible, there has to be an enzymatically driven
means of lowering the activation energy of the fragmentation step. If such a mechanism exists for glutamate mutase, then why not for methylmalonylCoA mutase and 2-methyleneglutarate mutase?
The recently discovered benzylsuccinate synthase
from the denitri¢er Thauera aromatica, which catalyses the addition of toluene to fumarate [40], the ¢rst
step in anaerobic toluene oxidation, can be regarded
as a further example of the fragmentation-recombination mechanism. By sequence comparison, the enzyme is closely related to pyruvate formate lyase,
which in its active state contains a persistent protein-based glycinyl radical. It is therefore reasonable
to assume that in the ¢rst step of catalysis, a benzyl
radical (10, Fig. 5) is generated, which adds to fumarate yielding the 2-benzyl-3-succinyl radical (11).
Re-donation of the initially abstracted hydrogen
atom leads to the product (+)-benzylsuccinate. Like
the persistent radicals from glutamate and L-lysine,
the 2-benzyl-3-succinyl radical (11) is stabilised by an
K-carboxylate.
4. Umpolung via radical anions, a new concept in
enzymatic catalysis
The 2-L-lysyl and the 4-glutamyl radical described
in the previous section are both neutral radical species stabilised by an K-carboxylate and prone to
undergo rearrangement. There are numerous examples of enzymatic reactions, however, in which radical anions could be involved (Table 1). These radical
anions exhibit di¡erent properties from the radicals
implicated in lysine 2,3-aminomutase and glutamate
mutase. They may be generated by direct one-electron oxidation (H-atom abstraction, electron transfer
followed by removal of an electrophilic group, H‡ ,
CO2 , carbonyl group etc.) or by one-electron reduction. Their electron rich character enables radical
anions to react with electrophiles, to eliminate adjacent leaving groups or to introduce electrons into
aromatic systems. The paradigm for this new type
of radical anion-dependent catalysis emerges from
the study of the K,L-dehydration of (R)-2-hydroxyacyl-CoA to the corresponding enoyl-CoA [41,42].
The biosynthesis and degradation (L-oxidation) of
fatty acids always involves thiol esters of L-hydroxy
acids. This is well understood, since the acid-base
catalysed elimination of water from 3-hydroxyacylCoA is facilitated by the electron withdrawing thiol
ester carbonyl which lowers the pK of the K-hydrogen. In enoyl-CoA, the thiol ester polarises the dou-
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ble bond, which favours the addition of the hydroxyl
group at the partially positive L-carbon. The fermentation of the proteinogenous amino acids frequently
proceeds, however, via the corresponding K-hydroxy
acids to the enoates [43]. The discovery that this
dehydration also occurs at the CoA-ester level [44],
which would even impede a simple acid-base catalysed L-elimination, led to the hypothesis of an Umpolung [45] by one-electron transfer to the thiol ester
carbonyl, i.e. reduction to a ketyl radical anion
which is able to eliminate the adjacent hydroxyl
group [41].
In contrast to C. cochlearium and related Clostridia (cluster I+II [46]), which live in the soil and ferment glutamate via 3-methylaspartate, strict anaerobic organisms occurring in the guts of mammals and
birds (clostridial clusters XI, XIII, XIVa and XIX)
are able to degrade this amino acid to the same
products, but use a quite di¡erent, coenzyme B12 independent pathway [19]. The elimination of ammonia is solved in an apparently less elegant way than
that via the coenzyme B12 -dependent rearrangement.
By two consecutive redox reactions, the amino group
of glutamate is exchanged by a hydroxyl group with
inversion of con¢guration. Prior to the subsequent
elimination of water (Fig. 6), the resulting (R)-2-hydroxyglutarate is activated to (R)-2-hydroxyglutarylCoA. The decarboxylation of (E)-glutaconyl-CoA,
the product of this unusual dehydration, to crotonyl-CoA concludes the special part of this pathway.
The puri¢cation of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans (cluster
IX) led to an oxygen-sensitive enzyme exhibiting
prosthetic groups suitable for electron transfer. The
heterodimeric enzyme (K, 54 kDa; L, 42 kDa) contains 1.0 FMN, 0.3 ribo£avin and 1.0 [4Fe-4S] cluster. Furthermore, the reversible syn-dehydration of
(R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA
has to be initiated by sub-stoichiometric amounts
of ATP, high concentrations of Mg2‡ and a reducing
agent, preferentially Ti(III) citrate. This process, in
which ATP is hydrolysed, is catalysed by a separate
enzyme [47]. The £avin of the dehydratase has been
shown by EPR-spectroscopy to exist mainly as a
semiquinone and the iron-sulfur cluster has been
identi¢ed by Moëssbauer spectroscopy as a symmetric
Table 1
Potential radical anions in enzymatic catalysis
Enzyme
Mode of radical anion generation
Mode of catalysis
References
(R)-2-Hydroxyglutaryl-CoA dehydratase
(R)-Lactyl-CoA dehydratase
Chorismate synthase
One-electron reduction
One-electron reduction
One-electron reduction
K-Elimination
K-Elimination
K-Elimination
This paper
[79,80]
This paper
Benzoyl-CoA reductase
One-electron reduction
This paper
p-Hydroxybenzoyl-CoA reductase
One-electron reduction
Phthalyl-CoA decarboxylase
One-electron reduction
Protonation/electron
transfer
Protonation/electron
transfer
Protonation
Ribonucleotide reductases
H-abstraction/deprotonation
[14,74]
Diol dehydratase
Glycerol dehydratase
Ethanolamine ammonia lyase
Galactose oxidase
H-abstraction/deprotonation
H-abstraction/deprotonation
H-abstraction/deprotonation
H-abstraction/deprotonation
K-Elimination/electron
transfer
K-Elimination
K-Elimination
K-Elimination
Electron transfer
p-Hydroxyphenylacetate decarboxylase
Pyruvate formate lyase
Pyruvate ferredoxin oxidoreductase
Pyruvate dehydrogenase
4-Hydroxybutyryl-CoA dehydratase
H-abstraction and decarboxylation
One-electron oxidation/deprotonationa
One-electron oxidation
One-electron oxidation
One-electron oxidation/deprotonation
Protonation
K-Addition
Electron transfer
Electron transfer
Q-Elimination
This
This
This
This
This
a
Reaction in the reverse direction.
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[41]
B. Schink, personal
communication
[63]
[63]
[63]
[7,8]
paper
paper
paper
paper
paper
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531
Fig. 6. Proposed mechanism of the dehydration of (R)-2-hydroxyglutaryl-CoA. The dashed arrow indicates a single initiation of many
turnovers.
cubane type in which all four iron atoms are chemically indistinguishable (M. Hans, E. Bill and W.
Buckel, unpublished). There is no evidence that the
cluster contains a labile iron atom acting as a Lewis
acid as shown for aconitase [48] or L-serine dehydratase [49]. The cluster may stabilise the semiquinone
form of the £avin, as suggested for the related enzyme 4-hydroxybutyryl-CoA dehydratase [11] (see
below). The initiating enzyme has been characterised
as an extremely oxygen sensitive homodimeric and
[4Fe-4S] cluster-containing protein (Q2 , 2U27 kDa).
It probably acts as an ATP-driven electron ampli¢er
like nitrogenase reductase [50]. Interestingly, in both
enzymes the reduced [4Fe-4S]‡ cluster between the
two identical subunits shows an EPR spectrum of a
3/2 spin system (M. Hans, E. Bill and W. Buckel,
unpublished results).
The mechanism postulated for the dehydration of
2-hydroxyglutaryl-CoA involves the initial reduction
of the thiol ester carbonyl to a ketyl radical anion
(12, Fig. 6) which eliminates the adjacent hydroxyl
group. The resulting enoxy radical (13) is deprotonated to the product-related ketyl (14) which is reoxidised to the product glutaconyl-CoA [47].
Although none of these radicals have been identi¢ed,
there is some circumstantial evidence for this mechanism. The electron ampli¢er might inject the ¢rst
electron into the £avin semiquinone or directly into
the substrate in order to initiate the catalytic cycle.
The £avin in the fully reduced state could act as a
relay which saves the energised electron for the next
catalytic cycle. The electron can be trapped by oxidants such as 1 WM o-, m-, p-nitrophenol, p-nitrobenzoate or other nitro compounds such as chloramphenicol. After consumption of the oxidant, the
dehydratase activity is recovered by the residual
ATP and excess of reductant, resulting in a lag-phase
of the catalytic turnover [47].
It should be noted that besides the syn-dehydration of (R)-2-hydroxyglutaryl-CoA, the syn-dehydration of (S)-3-hydroxyglutaryl-CoA also leads to (E)glutaconyl-CoA (Fig. 7). The reaction is catalysed by
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Fig. 7. syn-Dehydration of 3-hydroxyglutaryl-CoA.
3-methylglutaconase [51], a simple homodimeric protein (K2 , 2U29 kDa), which is devoid of any prosthetic group. The enzyme was puri¢ed from cell-free
extracts of the leucine-oxidising soil bacterium Acinetobacter sp., which was originally isolated by
Hans-Guënther Schlegel, University of Goëttingen
(M. Liesert and W. Buckel, unpublished results).
The mechanism of this dehydration is presumably
very similar to that of the syn-dehydration of (S)-3hydroxyacyl-CoA to (E)-2-enoyl-CoA, i.e. the reaction is solely catalysed by a glutamate residue and a
glutamic acid residue of enoyl-CoA hydratase, the
key enzyme of the L-oxidation of fatty acids [52,53].
It has been proposed recently that the ¢rst step of
the reductions of benzoyl-CoA to cyclohexa-1,5-dienecarboxyl-CoA (Fig. 8) and of 4-hydroxybenzoyl-
CoA to benzoyl-CoA involves one-electron addition
to the thiol ester carbonyl resulting in a ketyl radical
anion (15), which is able to deliver an electron into
the aromatic ring [41]. Benzoyl-CoA reductase,
which catalyses the crucial step in the anaerobic oxidation of aromatic compounds has been puri¢ed
from T. aromatica and characterised as an FADand iron-sulfur cluster-containing enzyme complex.
Furthermore, like 2-hydroxyglutaryl-CoA dehydratase, benzoyl-CoA reductase requires hydrolysis of
ATP but in stoichiometric amounts [54]. Despite
the early proposal of this mechanistic relationship,
it came as a surprise that the amino acid sequences
of the enzymes share up to 30% identities [55,56].
Interestingly, the two smaller of the four di¡erent
subunits of benzoyl-CoA reductase are related to
Fig. 8. Proposed mechanism of benzoyl-CoA reductase.
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533
Fig. 9. Proposed mechanism of DNA-photolyase.
the electron enhancing protein of 2-hydroxyglutarylCoA dehydratase, supporting the idea of energised
electrons in both catalyses. The sequence similarity
of a dehydratase with a reductase supports the proposed redox mechanism for the L-elimination.
In the previous two examples, the reductive power
of the electron necessary to reduce the thiol ester
carbonyl to the ketyl radical anion is ampli¢ed by
hydrolysis of ATP. In the DNA photolyase from
Escherichia coli, which catalyses the repair of thymine dimers, this ampli¢cation is achieved by light
energy (V = 300^450 nm) [57]. The light is absorbed
by methylenetetrahydrofolate, which functions as a
photoantenna, and the excitation energy is transferred to the FADH3 present in the enzyme. This
`loading' of the enzyme is thought to be followed by
an e¤cient electron transfer to the thymine dimer
forming a ketyl radical anion (16, Fig. 9) which, in
a series of consecutive electron shifts, eliminates both
bases from each other.
Chorismate synthase, an essential step in the biosynthesis of aromatic amino acids in Archaea, Bacteria, fungi and plants, catalyses an intriguing 1,4elimination of phosphate from 3-enoylpyruvylshikimate 5-phosphate, in which the unactivated C-HSi bond in the L-position to the ring carboxylate has to
be cleaved (Fig. 10) [58]. The reaction seems to be
related to the dehydration of 2-hydroxyacyl-CoA derivatives, although the carboxylate is not esteri¢ed
with CoASH. The active enzyme contains reduced
FMNH2 which may transfer an electron to the double bond. The resulting radical anion (17), which is
stabilised by the carboxylate, similar to the neutral
radicals described in the previous section, may eliminate the adjacent phosphate. Deprotonation of the
C^H-bond, which is now activated by the allyl radical formed (18), and electron transfer from the radical anion (19) back to the £avin semiquinone, leads
to chorismate. For an alternative mechanistic proposal, which is based on model studies but is not
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W. Buckel, B.T. Golding / FEMS Microbiology Reviews 22 (1999) 523^541
consistent with the available data on the enzyme, see
[59].
Clostridium aminobutyricum ferments 4-aminobutyrate ammonia, acetate and butyrate. Like glutamate fermentation via 2-hydroxyglutarate, the amino
group is exchanged by a hydroxy group, the acid is
activated to the CoA-ester and the resulting 4-hydroxyglutaryl-CoA is dehydrated to crotonyl-CoA,
which disproportionates to acetate and butyrate
[43]. The oxygen sensitive 4-hydroxybutyryl-CoA dehydratase was puri¢ed and characterised as homotetrameric protein with 2 FAD in the semiquinone
form and two [4Fe-4S] clusters as prosthetic groups
by applying UV/visible, EPR, Moëssbauer and
ENDOR spectroscopy [11,60^62]. In contrast to 2hydroxyglutaryl-CoA dehydratase, the initiation of
the reaction requires one-electron oxidation of the
semiquinone. The oxidant in vitro is ferricyanide or
a short exposure to oxygen; that in vivo in the strict
anaerobic bacterium is not known. In the mechanistic scheme (Fig. 11) it is postulated that the initially
formed enolate (20) is oxidised by FAD to the enoxy
radical (21), which is deprotonated at the L-position.
The resulting ketyl radical anion (22) now eliminates
the hydroxyl group to yield the dienoxy radical (23),
which is reduced by FADH semiquinone to the dienolate (24) and protonated to crotonyl-CoA. In its
essence, this mechanism is similar to that of coenzyme B12 -dependent diol dehydratase [63]. In these
two enzymes the dehydration is enabled by removal
of a proton together with an electron. After the elimination both are returned.
A variation of the mechanism discussed above
could be the concerted addition and elimination of
H‡ and OH3 to the dienoxy radical of crotonyl-CoA
(23) and from the enoxy radical of 4-hydroxybutyrylCoA (21), respectively. In this case, a ketyl radical
anion would not be involved as a discrete intermediate, but as participant of enoxy and dienoxy radical
resonance structures. Whether this reaction is stepwise or concerted could in principle be determined
by measuring double isotope e¡ects, provided the
elimination is rate limiting, rather than the formation
and decomposition of the two radicals (cf. mecha-
Fig. 10. Proposed mechanism of chorismate synthase.
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535
Fig. 11. Proposed mechanism of 4-hydroxybutyryl-CoA dehydratase.
nism of coenzyme B12 -dependent mutases). With this
method [64,65] it was shown that the anti-elimination
of water from (S)-malate catalysed by fumarase occurs stepwise, whereas the syn-elimination of water
from (S)-3-hydroxybutyryl-CoA catalysed by crotonase is concerted [66]. Generalisation of these results
and those from other dehydratases leads to the conclusion, that the pK of the C^H-bond to be cleaved
and/or the degree of polarisation of the double bond
govern stereochemistry and mechanism. Thus, the Khydrogens in malate have a high pK v30 and fumarate is symmetrical, whereas the K-hydrogens in 3hydroxybutyryl-CoA have a much lower pK W21
[67] and the double bond of crotonyl-CoA is highly
polarised by the electron withdrawing thiol ester.
Hence, if one can apply this not yet well understood
relationship to radicals, one might predict that the
elimination of water from the enoxy radical (21) to
the dienoxy radical (23) would occur in a concerted
syn manner. In contrast, chance during enzyme evolution has also been assumed as the reason for the
occurrence of syn and anti eliminating dehydratases
[68,69].
The decarboxylation of p-hydroxyphenylacetate to
p-cresol is catalysed by a yet unidenti¢ed oxygen
sensitive enzyme from the pathogen Clostridium dif¢cile, which especially thrives in the intestine of patients treated with antibiotics [70,71]. This unusual
decarboxylase is another possible example for the
formation of a ketyl radical anion by one-electron
oxidation (Fig. 12) similar to the dehydration of
4-hydroxybutyryl-CoA, although it is not known
yet whether the enzyme contains FAD and iron-sulfur clusters. The p-phenoxyacetate radical (25) there-
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W. Buckel, B.T. Golding / FEMS Microbiology Reviews 22 (1999) 523^541
by generated is decarboxylated to the ketyl radical
anion of p-cresol (26), which is protonated to the pmethylphenoxy radical (27). The reverse of the initial
steps yields the ¢nal product.
The extremely oxygen sensitive pyruvate formate
lyase, which is considered as the key enzyme in the
`mixed acid fermentation' of E. coli and other facultative or strict anaerobic bacteria, catalyses with
CoASH the cleavage of pyruvate to acetyl-CoA
and formate. This apparently unfavourable lysis of
a carbon-carbon bond into two electrophilic carbonyls again is achieved by an Umpolung with the use of
radical intermediates. Formation of a thiosemiketal
(28, Fig. 13) from pyruvate and the cysteine residue
419 of the E. coli enzyme (29) is followed by hydrogen atom transfer from the adjacent cysteine 418 to
the persistent protein based glycinyl radical (30, residue 734) generating a thiyl radical (31) [72,73]. A
Fig. 12. Proposed mechanism of the decarboxylation of p-hydroxyphenylacetate.
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537
Fig. 13. Proposed mechanism of pyruvate formate lyase.
one-electron oxidation of the carboxylate anion by
this thiyl radical would yield a substrate-based radical (32), which is cleaved to acetyl-S-cysteine 418 and
the formyl radical anion, a ketyl (33). Protonation
and one-electron reduction yields the product formate whereby the thiyl radical (34) is regenerated.
The other product, acetyl-CoA, is formed by ace-
tyl-transfer to CoASH. This proposal involves two
radical anions, one derived from the thiosemiketal of
pyruvate (32) and the other from formate (33), which
is equivalent to that obtained by one-electron reduction of carbon dioxide. Although the reduction of a
thiyl radical by a carboxylate is thermodynamically
unfavourable by ca. 1 V, alternative mechanistic pro-
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posals also have various defects, such as the requirement to add a thiyl radical to a carbonyl group or
carboxylate [74].
5. Conclusions
Radical reactions show several distinctive features
that are illustrated by the enzymes described in this
review. Firstly, the rates of radical reactions are
often much faster than corresponding ionic processes. Indeed, for reactions via intermediate radicals
the rate limiting step may not be the interconversion(s) of the radicals, but either their formation
from a neutral precursor or their decomposition to
a neutral product. Secondly, radicals and radical
anions may have special properties useful in the context of enzymatic processes. Thus, neutral carboncentred radicals can undergo carbon-skeleton rearrangements, whereas radical anions behave as electron-rich species forcing their extra electron into an
adjacent position. This brings about elimination, reduction, C^C bond cleavage or an unusual condensation. Thirdly, Nature apparently favours radical
anions over radical cations, although the latter
have been postulated as intermediates for diol dehydratase [75] and ribonucleotide reductase [75,76].
Model reactions suggest, however, intermediate ketyl
radical anions [31,42,77]. Sir John Cornforth once
remarked that Nature in general prefers nucleophilic
over electrophilic catalysis, the only exception being
the biosynthesis of isoprenoids initiated by the allyl
cation derived by elimination of diphosphate from
dimethylallyl diphosphate (personal communication).
Several of the reaction pathways proposed in this
review require experimental veri¢cation, although it
should be su¤ciently clear with the examples given
that reactions via radicals now occupy an important
place in enzymology. For the reactions described in
this review, a major task of each protein may be to
provide an environment in which the highly reactive
intermediate radicals do not dimerise or disproportionate, do not react with groups of the enzyme and/
or coenzyme, or do other harmful things. The ability
of coenzyme B12 -dependent enzymes to avoid these
pitfalls was termed `negative catalysis' by Jaènos
Reètey [78]. However, the high reactivity of radical
species towards dioxygen has perhaps limited their
full exploitation by Nature and has tended to restrict
their role in enzymology to the world of anaerobic
organisms.
Acknowledgments
The authors thank the referees of this article for
numerous helpful comments and suggestions. They
also thank their colleagues from the DFG-Schwerpunktsprogramm `Ungewoëhnliche Reaktionen und
Katalysemechanismen in anaeroben Mikroorganismen', the EC TMR network `Chemistry and Biochemistry of B12 Coenzymes and their Enzymic Partners' and from the recently granted DFGSchwerpunktprogramm `Radikale in der enzymatischen Katalyse', as well as Professors Perry Frey,
University of Wisconsin, Madison, and JoAnne
Stubbe, Massachusetts Institute of Technology, for
helpful discussions. Work in the authors' laboratories was funded by the Deutsche Forschungsgemeinschaft (DFG), the European Commission (EC), the
Fonds der Chemischen Industrie and the Engineering and Physical Sciences Research Council.
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