Enzymatic Reactions Involving Ketyls: From a Chemical Curiosity to a

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Enzymatic reactions involving ketyls: From a chemical curiosity to a general biochemical mechanism. Wolfgang Buckel Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00171 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Biochemistry

Enzymatic reactions involving ketyls: From a chemical curiosity to a general biochemical mechanism. Wolfgang Buckel Fachbereich Biologie, Philipps-Universität, 35032 Marburg, Germany. Keywords. Radical anion, anaerobic bacteria, iron-sulfur clusters, radical enzyme, (R)-2hydroxyacyl-CoA dehydratases, benzoyl-CoA reductases, nitrogenase iron protein, electron bifurcation, 1,2-diol dehydratases, glycyl radical enzymes, coenzyme B12, ribonucleotide reductases, 4-hydroxybutyryl-CoA dehydratase.

Abstract: Ketyls are radical anions with nucleophilic properties. Ketyls obtained by enzymatic one-electron reduction of thioesters were proposed as intermediates for the dehydration of (R)-2hydroxyacyl-CoA to (E)-2-enoyl-CoA. This concept was extended to the Birch-like reduction of benzoyl-CoA to 1,5-cyclohexadienecarboxyl-CoA. Nature uses two methods to achieve the therefore required low reduction potentials of < 600 mV, either by an ATP-driven electron transfer similar to that catalyzed by the iron protein of nitrogenase or by electron bifurcation. Ketyls formed by thiyl radical-initiated oxidation of alcohols followed by deprotonation are involved in coenzyme B12-independent diol dehydratases, other glycyl radical enzymes mediating key reactions in the degradations of choline, taurine and 4-hydroxyproline, and all three classes of ribonucleotide reductases. A special case is the dehydration of 4-hydroxybutyryl-CoA to crotonyl-

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CoA, which most likely proceeds via an oxidation to an allylic ketyl, but requires neither a strong reductant nor an external radical generator. 1. KETYLS IN ORGANIC CHEMISTRY Ketyls are radical anions, which – as the name implies – are derived from ketones by insertion of one electron into the anti-bonding 2pz orbital of the carbonyl group.1 Two resonance structures contribute to the stability of a ketyl, the radical at carbon and the negative charge at oxygen and vice versa (Fig. 1). Because the oxygen is more electronegative than the carbon, the extra electron predominantly sits on the oxygen. However, the remaining negative charge at the carbon confers to ketyls nucleophilic properties, which are important for their reactivity, especially in enzymatic reactions (Fig. 1). In organic chemistry this conversion of an electrophile into a nucleophile is designated with the German word Umpolung, which in English means charge reversal.2 Ketyls can be prepared by strong reductants such as metallic sodium, which reduces benzophenone (Ph2CO) dissolved in toluene to a stable blue ketyl (Ph2CO); E°' = −2.2 V for the couple Ph2CO/Ph2CO.3 Simple ketyls are very short lived and cannot be detected by UVvisible or EPR spectroscopy unless they are additionally stabilized by conjugation with systems. In synthetic organic chemistry the reactivity of ketyls is exploited by radical dimerization, known as pinacol-coupling. A reaction applying the nucleophilicity of ketyls is the reduction of -hydroxy ketones to unsubstituted ketones by one-electron donors such as zinc in acetic acid, chromium(II), samarium(II) or dithionite. The initially formed nucleophilic ketyl expels the neighboring hydroxyl group leading to an enoxy radical, which accepts a second electron. The formed enolate is protonated to the enol, which tautomerizes to the ketone.2 Recently the one-electron reduction of 2-bromoacetophenone to bromide and acetophenoxy radical by [Ru(biphy)3]+ was reported, which proceeds via the nucleophilic ketyl of the starting ketone.4, 5 O

eX

Ketone

O

O

O X

X Ketyl

+ X

Enoxy radical

Figure 1. Formation of a ketyl and its nucleophilic property.


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Biochemistry

This review shows how the concept of ketyls explains the mechanism of many enzymatic reactions that occur in anaerobic bacteria. As stated above, in organic chemistry ketyls are generated by oneelectron reduction of ketones, which in biology are almost exclusively replaced by thioesters. The ketone-like character of thioesters was already recognized by Feodor Lynen in 1951,6 and this concept was further developed by Sir John W. Cornforth in 1959.7 Indeed, in

13C-NMR

spectroscopy the chemical shift of the thioester carbon equals that of an aldehyde (195 ppm), whereas that of an ester carbon is much lower (160 ppm).2 That there is almost no biological example of the reduction of a ketone to a ketyl, is probably due to the high reactivity of ketones in the central metabolism of all organisms. In contrast, the anaerobic reductive degradation of recalcitrant compounds such as 2-hydroxy acids and benzoic acid requires activation to the thioesters followed by reduction to ketyls. On the other hand, ketyls can also be formed by oneelectron oxidation of alcohols, which is exploited by diol dehydratases and related reactions. Finally the dehydration of 4-hydroxybutyryl-CoA presents an example, in which a thioester is converted to an allylic ketyl by two deprotonations and a one-electron oxidation.

2. KETYLS BY ENZYMATIC ONE-ELECTRON REDUCTION OF THE KETONE-LIKE CoA THIOESTERS 2.1 (R)-2-HYDROXYACYL-CoA DEHYDRATASES Clostridia from the predominantly Gram-positive phylum Firmicutes and related strictly anaerobic bacteria are able to ferment all proteinogenic and several other amino acids mainly to ammonia, CO2, acetate, short chain fatty acids and H2. There is a great variety of pathways, many of which involve unusual chemistry. Here the discussion will be limited to the dehydration of (R)2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA (HgdCAB) in the fermentation of L-glutamate by the Gram-negative Acidaminococcus fermentans (class Negativicutes of the Firmicutes), isolated from the gut of animals, and to the dehydration of (R)-2-hydroxyisocaproyl-CoA to (E)isocaprenoyl-CoA (HadIBC) in the fermentation of L-leucine by Clostridioides difficile, a human pathogen, formerly called Clostridium difficile.8 Recently the dehydration of (R)-indollactyl-CoA to the yellow indolacrylyl-CoA has also been studied in the author’s laboratory. The dehydratase is involved in the reductive pathway from tryptophan to indolpropionic acid (IPA) in Clostridium


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sporogenes.9 IPA, formed in the gut, ends up in the brain via the blood stream. Due to the propionate side chain of IPA, the indol group is able to pass the blood-brain barrier and acts inside as scavenger of free radicals which might cause Alzheimer’s disease.10 This hypothesis is supported by a recent study which revealed a more than 10-fold decrease of members of the genus Clostridium in the gut microbiomes of patients suffering from this disease.11 Furthermore, IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function.12 Enzymatic studies revealed that in C. sporogenes all three aromatic amino acids are converted to (R)-aryllactates, which are dehydrated by the same enzyme to arylacrylates (Huan Li and Wolfgang Buckel, unpublished). The dehydratase (FldBC) is part of the complex (FldABC) with a class III CoA-transferase (FldA). FldABC catalyzes the dehydration of the three (R)aryllactates via (R)-aryllactyl-CoA to the arylacrylates followed by an NADH-dependent reduction to the arylpropionates mediated by an enoate reductase.13 This important result has demonstrated for the first time that the formation of the CoA-thioester is required for the dehydration of 2hydroxyacids, because only for this reaction the CoA-derivative is synthesized. The other two dehydratases mentioned above might act adventitiously on the thioester level, because the CoA derivative is also necessary for the subsequent decarboxylation of glutaconyl-CoA to crotonylCoA14 and for the NADH-dependent reduction of isocaprenoyl-CoA to isocaproyl-CoA which is coupled to the reduction of ferredoxin by NADH via electron bifurcation.15 The dehydrations of (R)-2-hydroxyacyl-CoA are of considerable mechanistic interest, because the hydroxide has to be removed from the -position adjacent the electrophilic thioester carbon and the proton from the non-acidic -position. There is no base in an enzyme able to remove this proton with an estimated pKa ≈ 40. The dehydratases catalyze the syn-elimination of water yielding the E-isomers of the 2-enoyl-CoAs (Fig. 2). O

O

H H R

CoAS

H R + H 2O

CoAS

HO H

H

Figure 2. Reversible syn-elimination of water from (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA. R = H, methyl, isopropyl, carboxymethyl, phenyl, p-hydroxyphenyl, or indolyl.


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Biochemistry

A major breakthrough in investigating the mechanism was the detection of the activation by ATP and a reducing agent, in vitro dithionite or Ti(III)citrate and in vivo reduced ferredoxin or the hydroquinone form of flavodoxin (E°' ≈ 420 mV).16-18 A homodimeric protein (2 × 27 kDa) with one [4Fe-4S] cluster between the two subunits catalyzes the activation (Fig. 3).19 The genes coding for the activator and the 2 subunits of the dehydratases are arranged in this order and transcribed together, hgdCAB from A. fermentans and hadIBC from C. difficile.20 The structures of the activators HgdC21 and HadI22 as well as the dehydratase HadBC23 have been solved. The main structural feature of the homodimeric activators is the location of the [4Fe-4S] cluster between the two subunits. Each subunit contributes two cysteines for coordination and the -helix-5, which points with its N-terminus towards the cluster. This helix – cluster – helix motif with an angle of 105° reminded the author of an archer shooting an electron like an arrow at the dehydratase driven by hydrolysis of 2 ATP, one at each subunit of the activator. In the structure of the activator, the places at which ATP is proposed to bind, are occupied by ADP. The binding-motif is called ASKHA from acetate and sugar kinases, heat shock protein, and actin family of ATP binding proteins.24 Upon binding of ATP and interaction with the dehydratase HgdAB, the angle is thought to open to 180° in analogy to the iron protein (NifH) of nitrogenase (see below). To illustrate this, the author chose a half relief from the British Museum in London depicting the Assyrian King Ashurbanipal (668-627 BCE) as archer hunting wild asses, see Fig. 9 in ref.25 Therefore, in some publications the activator has been called ‘archerase’. However, the crystal structure of HadI in the presence and absence of ADP and the ATP analogue ADPNP did not reveal the large expected conformational changes.22 Probably the structure of the activator-dehydratase complex is required (see below).


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Figure 3. Structures of the activator (HgdC) of (R)-2-hydroxyglutaryl-CoA dehydratase from A. fermentans (left) and of the iron protein (NifH) of nitrogenase from Azotobacter vinelandii (right). The red arrows indicate the proposed conformational changes made by HgdC/NifH when they encounter the dehydratase/nitrogenase. The [4Fe-4S] clusters (red and yellow spheres) of HgdC/NifH are thought to move forward to the dehydratase/nitrogenase to inject the electron into the -[4Fe-4S] cluster/P-cluster. Parts of the bound ADP (stick model) are visible in both subunits of HgdC. Adapted from ref.26

The crystal structure of the dehydratase HadBC from C. difficile revealed one [4Fe-4S] cluster in each subunit, 12 Å apart from each other, and each cluster is ligated by three conserved cysteines. The fourth ligand of the -cluster (cluster of the B-subunit) is a hydroxyl group or a water molecule bound to Fea, whereas the fourth ligand of the -cluster (cluster of the C-subunit) is a sulfur atom of unknown origin not covalently linked to the protein (Fig. 4). Upon substrate binding to Fea, the thioester carbonyl replaces the hydroxyl group or water. The nearby carboxylate of the conserved glutamate 55 forms hydrogen bonds to the thioester and to the hydroxyl group of the conserved serine 37.23 The necessity of these residues for catalysis has been confirmed by sitedirected mutations of the conserved amino acids in the closely related (R)-aryllactyl-CoA dehydratase from C. sporogenes (C. Siegel and W. Buckel, unpublished).


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Biochemistry

(R)-2-Hydroxyisocaproyl-CoA

Cys Fe

Activator H

Cys

S

Fe

O

S

Fe

Fe

H

Fe Cys

S

HO

H+

HO -cluster SCoA

Fe

Fe

S

Fe

+ Fe

Cys

e Cys

S

Fe

e

Cys

Fe

S

Cys

S

Fe

-cluster Cys Dehydratase

O CoAS

Cys

Fe

S

2 ADP Cys

Cys

S

S

2 ATP

O CoAS H O 2

Fe+

S

Cys

HO

(E)-2-Isocaprenoyl-CoA

S

S

S

S Fe

Cys

Fe

S -cluster

S

Reduction Re-oxidation Cys Cys

++

Fe S

S

Fe

O

Fe

S

Cys

Fe

S

HO

Cys

S

OH2

CoAS

H

Fe++ S S

Fe

++

Fe

H

S

O

Fe

O

S

Fe

Allylic ketyl

CoAS

Ligand swapping

Deprotonation

++ Fe

Cys

S

O O

Cys

H

SCoA

Cys

Cys

Fe

S

Ketyl

O

H S

Cys

Fe

S

Ligand swapping Cys

Fe

++ S

Fe S

O O SCoA

Fe

Fe S

Cys

Fe Cys

H

SCoA

Cys

H

S

S

Fe

S

Fe S

Cys

Elimination of OH-

Fe Cys

H Enoxy radical

Figure 4. Proposed mechanism of the dehydration of (R)-2-hydroxyisocaproyl-CoA to (E)-2hydroxyisocaprenoyl-CoA. See text for details.

It has been proposed that the activator uses the energy of ATP hydrolysis to inject a low potential electron into the enzyme, most likely into the -cluster, which stores the electron until the substrate binds to Fea of the -cluster (Fig. 4). Upon binding, the electron tunnels through the


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protein over the 12 Å distance from the - to the -cluster, where it reduces the thioester carbonyl of the substrate (R)-2-hydroxyisocapryloyl-CoA to a ketyl. Due to the radical at the oxygen, the ketyl leaves Fea and the hydroxyl group of the substrate takes its place, just by rotation of the thioester by 120°, called 'ligand swapping'. Now the nucleophilic ketyl eliminates the adjacent hydroxyl group attached to Fea yielding an enoxy radical, in which the pKa of the 3Si-hydrogen is reduced by 26 units as compared to that of the free substrate.27 The now facile deprotonation of the 3Si-hydrogen by Fea-HO affords the allylic ketyl. In the final step, the electron is returned from the ketyl to the -cluster and the product (E)-2-isocaprenoyl-CoA is released.23 This mechanism is supported by the observation that already full activity is obtained by a dehydratase to activator ratio of 10:1. Furthermore, after removal of the activator, the activated dehydratase catalyzed about 10,000 turnovers before the electron was lost by an unspecific oxidant.28 Direct proof for this mechanism came from EPR experiments which revealed a strong signal of an organic radical with an average g value = 2.0038  0.0002 upon incubation of the dehydratase HadBC and its activator HadI with dithionite, ATP and substrate.29 Measurements with various 2H and 13Clabelled substrates indicated that the radical was indeed the allylic ketyl as expected from the proposed mechanism. Freeze quench measurements showed that the radical is 'kinetically competent', because it is formed at the same rate (140  30 s1) as substrate turnover (150 s1). Its concentration reached 2.1 % of that of the enzyme and remained constant for more than 150 ms at ambient temperature. Notably, with (R)-2-hydroxyglutaryl-CoA dehydratase no such radical could be detected. Probably the electron donating isopropyl residue of (R)-2-hydroxyisocaproyl-CoA stabilizes the radical, whereas the carboxymethyl group in (R)-2-hydroxyglutaryl-CoA has the opposite effect.1 The transient formation of ketyls in the dehydration of (R)-2-hydroxyacyl-CoA to (E)-2enoyl-CoA requires an enhancement of the reductive power of one electron by  200 mV far beyond that of reduced ferredoxin or flavodoxin (E°' = 420 mV). This is achieved by the ‘archerases’ or activators of the (R)-2-hydroxyacyl-CoA dehydratases HgdC and HadI or by BcrAD of class I benzoyl-CoA reductases (BcrABCD, see next chapter). An analogous system is the phylogenetically unrelated homodimeric NifH protein, also called iron protein of nitrogenase, which exhibits a structure similar to that of the activator (Fig. 3). In this ‘archerase’, the [4Fe-4S] cluster again sits between the two subunits, each of which binds ATP. The binding site is called ‘Walker motif’, different from that of the activators of the (R)-2-hydroxyacyl-CoA dehydratases.30


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Biochemistry

From each subunit one -helix points with its N-terminus towards the cluster, forming the helix – cluster – helix motif with an angle of about 150°.31 However, in NifH, the ATP binding domains are placed in the plane on both sides of the motif, whereas in HgdC the subunits are located above and below the plane of the motif (Fig. 3). Since nitrogen fixation requires 8 electrons per N2 reduction to 2 NH3 + H2, the amount of 2 ATP per ‘shot’ is known; probably HgdC and HadI also use 2 ATP per electron. In the presence of ADP and AlF4, a transition state analogue of ATP hydrolysis, NifH and NifDK form a tight complex which could be isolated and crystallized. Interestingly the angle of the helix – cluster – helix motif of NifH had opened from 150° to 180°. Apparently during ATP hydrolysis, the reduced cluster is shifted forward to the nitrogenase to enable the injection of the electron, analogous to the archer, who hydrolyses ATP in his muscles (see above). In the presence of ADP and AlF4, a similar complex, HadBCI2-ADP-AlF4, could be obtained. The stable complex was isolated by size exclusion chromatography, affinity chromatography, or ultrafiltration.32 Unfortunately, no crystallization trials were performed. Another similarity between NifH and HgdC/HadI is the property of their clusters. Under in vivo conditions the [4Fe-4S]2+ cluster is reduced by ferredoxin to [4Fe-4S]+ and re-oxidized in the presence of the acceptor complex, NifDK or HgdAB, concomitant with hydrolysis of ATP. The stronger reductant Ti(III)citrate (E' = −700 mV at pH 7.0)33 reduced the brown NifH and HgdC to the red all-ferrous states [4Fe-4S]0, which were characterized by EPR and Mössbauer spectroscopies.34, 35 Whether this super reduced state is just a property of these cluster types or physiologically relevant remains to be established. The activation of the nickel containing methyl coenzyme M-reductase, the key enzyme in methanogenesis also requires an extremely low reduction potential for the Ni(II)/Ni(I) couple, E°' ≈ 650 mV. Preliminary data indicate that this might be achieved by electron bifurcation36, 37 from hydrogen (E°' = 414 mV) to heterodisulfide (E°' = 140 mV) and a polyferredoxin (E°' = 500 mV), whose extra electron is further enhanced to 650 mV by ATP hydrolysis. Interestingly a NifH homologue appears to be involved in this process.38 In summary, it has been shown experimentally that under anaerobic conditions an iron– sulfur enzyme with a single electron as cofactor can generate a carbon-centered radical intermediate. Hence 2-hydroxyacyl-CoA dehydratases work with one electron as catalyst, the smallest possible cofactor, whereas other radical enzymes use the much more complex S-


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adenosylmethionine (SAM) or coenzyme B12 for this purpose. This consideration might indicate that amino acids were degraded via their corresponding 2-hydroxy acids already very early in evolution, before more sophisticated systems arose and oxygen became available. Although modern theories suggest an autotrophic origin of life, where amino acids are synthesized rather than degraded, almost simultaneously heterotrophs must have developed which had to thrive on the organic material provided by the autotrophs. Hence, fermentation of amino acids, purines and pyrimidines to ammonia, CO2, H2, acetate and short chain fatty acids might have dominated the first energy metabolism of heterotrophs.39 The detection of a single electron as catalyst in the biochemical dehydration of 2hydroxyacyl-CoA has found counterparts in organic chemistry. Armido Studer realized that many unimolecular radical substitution reactions (SRN1-chemistry) can be formulated as chain reactions with single electrons as catalysts, for which he created the term Electron Catalysis.40 An example involving a ketyl is shown in Fig. 5. The catalytic electron is introduced into the cycle by the radical initiator hypodinitrite 2. The electron reduces biphenyliodide 1 to the radical anion 3, which fragments to the biphenyl radical 4 and iodide. The biphenyl radical abstracts a hydrogen atom from lithium phenylethanolate 5 to yield biphenyl 6 and the ketyl 7, which under formation of phenylacetaldehyde 8 initiates the next catalytic cycle. In summary, an alcoholate reduces an aromatic iodide to an aromatic hydrocarbon and an iodide ion, whereby an aldehyde is formed: ArI + RCH2O  ArH + I+ RCH=O.

O N N 2

I

O

e

I

3

1 O O 7

I

8

4 O 5

6


10

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Biochemistry

Figure 5. Electron Catalysis, in which an 'energy rich' electron cycles for many turnover. The electron is introduced by a catalytic amount of 2. For details see text.

2.2 BENZOYL-CoA REDUCTASES The aerobic degradation of aromatic compounds is initiated by oxygen-dependent hydroxylations.41 In the anaerobic world most aromatic compounds are converted to benzoyl-CoA, whose 6-system is broken by a Birch-like reduction. Thereby two electrons and two protons are introduced one by one via the thioester carbonyl, yielding 1,5-cyclohexadienecarboxyl-CoA.2, 42 The reduction potential of 1,5-cyclohexadienecarboxyl-CoA/benzoyl-CoA, E°' = 622 mV (see below), is probably similar to that obtained by ATP-hydrolysis by the ‘archerases’ as shown above. Surprisingly, nature developed two independent and completely different enzyme complexes (class I and II) to achieve this low reduction potential. Class I uses the hydrolysis of ATP whereas class II, the ATP-independent benzoyl-CoA reductases, most likely employs electron bifurcation36, 37

It has been postulated that in both classes the first electron reduces the thioester carbonyl of

benzoyl-CoA to a resonance stabilized ketyl, which is conjugated with the aromatic ring, followed by protonation at the para-position. Then the second electron enters the thioester and protonation occurs from the opposite side of the ring at the meta-position (Fig. 6).43 CoAS

O

CoAS Fd-

2 ATP Benzoyl-CoA

O

Fd

O

CoAS

CoAS Fd-

H+

O

Fd

CoAS

O

H+ H

2 ADP Ketyl

H

H

H Cyclohexadiene carboxyl-CoA

Figure 6. Postulated mechanism of class I benzoyl-CoA reductases. Fd = ferredoxin, Fd = reduced ferredoxin. Class II enzymes work in the same manner, but do not require ATP. The possible electron donor for class II benzoyl-CoA reductases is tungsten, W(V), whose E’ ca. 650 mV is most likely achieved by electron bifurcation.


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The ATP-dependent benzoyl-CoA reductase (class I) is phylogenetically and functionally related to the 2-hydroxyacyl-CoA dehydratases. The enzyme from Thauera aromatica comprises a tight heterotetrameric complex (BcrABCD) composed of a heterodimeric ATP-dependent electron amplifier (BcrAD) with one [4Fe-4S] cluster in both subunits related to the activator and a heterodimeric reductase (BcrBC) with one [4Fe-4S] cluster in each subunit related to the of 2hydroxyacyl-CoA dehydratases. Each subunit of the amplifier, though of different size, contains the ATP-binding sequence ASKHA motif. Hence, class I most likely functions like the dehydratase in the first step. An electron driven by hydrolysis of both ATPs enters benzoyl-CoA and reduces it to a ketyl, whereas the second electron converts the radical to the dienolate anion for which it probably needs no ATP hydrolysis.43 The other two subunits of the hetero tetrameric benzoyl-CoA reductase complex catalyze the reduction of benzoyl-CoA. Probably the electron enters one [4Fe4S] cluster and remains there until binding of benzoyl-CoA to the other cluster. Then the electron is transferred via this cluster to the thioester carbonyl. The main difference between class I benzoyl-CoA reductase and 2-hydroxyacyl-CoA dehydratases is the consumption of ATP. Whereas class I benzoyl-CoA reductase requires two ATP for each turnover, the 2-hydroxyacylCoA dehydratases only need two ATP to initiate the first turnover, then the electron is recycled in all subsequent turnovers without further ATP hydrolysis. The comparison with NifH of nitrogenase raises the question why benzoyl-CoA reductase only needs 2 ATP for every turnover. If benzoyl-CoA reductase would work like NifH, 2 ATP for each electron, 4 ATP would be required for every turnover. Hence two possibilities arise, either each electron requires 1 ATP, as suggested by Boll and Fuchs,44 or 2 ATP for the first electron and none for the second, as proposed above. The ATP binding subunits of benzoyl-CoA reductase (BcrAD), however, are not identical (48 + 30 kDa, T. aromatica) as in NifH (2 × 30 kDa) and the activators of the dehydratases (2 × 27 kDa). Furthermore, the four benzoyl-CoA reductase subunits (BcrABCD) form a tight complex, whereas NifH and the activators are free enzymes. Thus BcrAD of benzoyl-CoA reductase could interact differently with the 2 reduced ferredoxins, which deliver the electrons. Recently two proteins present in many anaerobic bacteria have been characterized, which according to their activities can be regarded as primordial nitrogenase (in German ‘Urnitrogenase’).45 One of the proteins contains a double-cubane [8Fe-9S]-cluster (DCCP) and acts as electron acceptor, which is reduced by ferredoxin (in vitro Ti(III)citrate or dithionite) mediated by the other protein DCCP-R with one [4Fe-4S]-cluster driven by ATP hydrolysis.


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Biochemistry

Interestingly DCCP together with DCCP-R catalyzes the ATP-dependent reduction of acetylene to ethylene as well as azide and hydrazine to ammonia, but not of nitrogen. As the specific activities are only in the range of 100 nmol min1 mg1 protein, the real substrate remains to be detected. Both proteins stem from Carboxydothermus hydrogenoformans, have been produced in Escherichia coli, and are considered as the evolutionary precursors of the 2-hydroxyacyl-CoA dehydratases (HadAB + HadI) and class I benzoyl-CoA reductases (BcrABCD). The homodimeric DCCP with the [8Fe-9S]-cluster is thought to have given rise to the heterodimeric HadAB with one [4Fe-4S]-cluster in each subunit, which in turn acted as the precursor of BcrBC. Most likely the homodimeric DCCP-R evolved in parallel to the homodimeric activator (HadI2) and further to the heterodimeric reductase (BcrAD). The ATP-independent benzoyl-CoA reductase (class II) from Geobacter metallireducens represents a much larger enzyme complex than that of class I, to which it is not related. Recently, a soluble one mega-Dalton (MDa) protein was isolated comprising 8 different subunits arranged as Bam[(BC)2DEFGHI]2 harboring 4 tungsten, 4 calcium, 4 zinc, 2 selenocysteines, 6 FAD, 46 [4Fe-4S] cluster and 8 [2Fe-2S] cluster.46 Though it could not be shown that the whole complex catalyzed the reduction of benzoyl-CoA, the subcomplex (BamBC)2 mediated the irreversible oxidation of 1,5-cyclohexadienecarboxyl-CoA to benzoyl-CoA with methyl viologen (E°' = 448 mV) as electron acceptor. Using the 4,4′-dimethyl derivative of the propylene bridged 2,2′biypridyl as electron donor/acceptor (E°' = 674 mV), the reaction became reversible and allowed the determination of the reduction potential as E°' = 622  16 mV.47 BamB contains the tungstopterin and 1 [4Fe-4S]-cluster, whereas BamC harbors 3 [4Fe-4S]-cluster. The crystal structure of (BamBC)2 is related to that of aldehyde oxidoreductases (AOR), but the subcomplex does not catalyze the reversible reduction of carboxylic acids to aldehydes.48 The BamDEF subunits of the 1 MDa complex are related to the Hdr proteins present in the electron bifurcating heterodisulfide reductase from methanogenic archaea and the BamGHI subunits are similar to the Nuo proteins of complex I of the respiratory chain. Most likely these 6 subunits function to lower the reduction potential of ferredoxin from 420 mV to ca. 650 mV, which could be achieved by electron bifurcation.36, 37 Immuno gold labelling experiments indicated that the 1 MDa complex is localized at the cytoplasmic membrane.46 Therefore, in addition menaquinone is most likely involved in the


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Page 14 of 49

electron bifurcation leading to the reduction potential of 622 mV. However, a bifurcation similar to the FixABCX system from A. vinelandii49 from NADH (E°' = 320 mV) via A-FAD to menaquinone (MK; E°' = 70 mV) could lead to the reduction of an iron-sulfur cluster with a reduction potential of 570 mV, which is not low enough for the reduction of benzoyl-CoA. Hence, a double bifurcation has been proposed (Fig. 7).46 In the second step the bifurcation could start with the iron-sulfur cluster, which reduces W(VI) to W(IV) followed by bifurcation to the higher potential FAD and to the lower potential W(V), which reduces benzoyl-CoA. The involvement of tungsten (W) in electron bifurcation has been already proposed by Nitschke and Russel50, 37 This proposal accounts for the presence of two FAD in class II benzoyl-CoA reductases, but ony 1 FAD in other enzymes related to heterodisulfide reductase.46 A problem is the required two-electron reduction of W(VI) to W(IV) by iron sulfur clusters, which act as single electron carriers. Probably this is overcome by the non-cubane iron-sulfur clusters present in the 1 MDa complex, similar to those in heterodisulfide reductase, in which the bifurcating FAD must be reduced by two electrons to the hydroquinone, FADH.51

Figure 7. Possible scheme of the double electron bifurcation (1 and 2, yellow) proposed for class II benzoylCoA reductases. B-FAD (bifurcating) and A-FAD (non-bifurcating) are the two FADs present only in class II benzoyl-CoA reductases; CHC-CoA = 2,5-cyclohexadieneoyl-CoA; FexSy = non-cubane iron sulfur cluster; 4 × e = 4 × 1-electron transfer; 2e = 2-electron transfer; MK = menaquinone; red arrows = electron transfer; blue arrows = reactions.


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Biochemistry

It should be mentioned at this place that the reduction of the C17=C18 double bond in the D-ring of the aromatic 22- system of protochlorophyllide also requires energy (Fig. 8). Presumably, the ketone, conjugated via the C-ring to the 22- system, is transiently reduced to a ketyl, like the thioester of benzoyl-CoA. Whereas angiosperm plants take NADPH and light (lightdependent protochlorophyllide oxido-reductase, LPOR), all other chlorophyll-synthesizing organisms use reduced ferredoxin and ATP. This dark operating protochlorophyllide oxidoreductase (DPOR) is related to nitrogenase (see above) rather than to class I benzoyl-CoA reductase. It is composed of a dimeric NifH-like Fe-protein (BchL)2 and a NifDK-like MoFeprotein BchNB, in which the P-cluster and the Mo-cofactor are replaced by two [4Fe-4S]clusters.52, 53

A

N

N

B NADPH + H + h

Mg D

N

O COO-

N

+

N

-

Mg

+

2 Fd + 2 H + 2 ATP

N

C

O

O O CH3

N

-

COO

N

O O CH3

Chlorophyllide

Protochlorophyllide

Figure 8. Reaction catalyzed by the protochlorophyllide reductases. Class I uses NADPH and light (LPOR), whereas class II takes reduced ferredoxin and ATP (DPOR). The red double bond of ring D is reduced during the reaction. Presumably, the blue carbonyl group is transiently reduced to a ketyl. This would be the only example in biology of the reduction of a ketone rather than a thioester to a ketyl.


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3. KETYLS BY ONE-ELECTRON OXIDATION OF ALCOHOLS 3.1. 1,2-DIOL DEHYDRATASES The dehydration of 1,2-diols to aldehydes are catalyzed by two different classes of enzymes, class I is coenzyme B12 dependent and class II belongs to the glycyl radical enzymes. The substrates are ethylene glycol, (R)- and (S)-propane 1,2-diol or glycerol which are converted to acetaldehyde, propanal or 3-hydroxypropanal, respectively. Both classes abstract the non-acidic hydrogen at C1, either by the 5'-deoxyadenosyl radical generated from coenzyme B12 (Fig. 9)54 or by the thiyl radical formed upon substrate binding in glycyl radical enzymes (Fig. 10).55 These substratederived radicals are easily deprotonated to the ketyls, because the pK of the OH group at C1 is lowered by about 5 units to pK ≈ 12.56 In 1996 the author of this review proposed that the ketyl might eliminate the neighboring OH group at C2 to yield an enoxy radical, to which the initially abstracted hydrogen atom is returned yielding the aldehyde (Fig. 10).57 Though this mechanism appeared plausible, especially as compared to that of the dehydration of 2-hydroxyacyl-CoA, it was not accepted at the time of publication, when only the class I enzymes were known. Earlier experiments were conducted with (R)- and (S)- propane-1,2diols, labeled with

18O

at positions C1 and C2, using a cell extract from Aerobacter aerogenes

containing a coenzyme B12-dependent propane-1,2-diol dehydratase. The formed propanal was reduced to 1-propanol with NADH and alcohol dehydrogenase. With (S)-[1-18O]propane-1,2-diol, 88% of

18O

were retained in the 1-propanol, with the (R)-enantiomer 8%, and with (RS)-[2-

18O]propane-1,2-diol

43%. From these results it was concluded that the OH group at C2 migrates

intramolecularly to C1. The formed 1,1-gem-diol-2-radical re-abstracts the hydrogen from 5'deoxyadenosine to regenerate the 5'-deoxyadenosyl radical and to afford the product propane-1,1diol, which stereospecifically eliminates H2O to yield propanal.58,

59

Repetition of these

experiments by Levin and Balskus with the purified enzyme from Klebsiella oxytoca and with all four enantio-pure substrates led to 50% 18O-retention in all cases. This result also agreed well with the intramolecular O-shift, but without stereospecific elimination of water from the gem-diol.60 Since propane-1,1-diol is prochiral like citrate, an enzyme catalyzing this stereospecific elimination could have been present in the cell extract. However, in the formulated mechanisms the gem-diol is dehydrated before it is released from the enzyme and therefore should dehydrate in a stereospecific manner; a problem to be resolved.54, 60 The discovery by Toraya et al. that class


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Biochemistry

I diol dehydratases are metalloenzymes containing tightly bound calcium ions, makes the intramolecular hydroxyl shift much better understandable (Fig. 9).61

HO

OH 1

Ca2+ HO OH

Ca2+ HO OH

Ca2+ HO O

4

3

2 H

H

-H -H++

Ca2+ HO O

5

O Ado

11

H

H 2O

HO

OH 10

H

AdoH

Ca2+ HO 9

OH

Ca2+ HO

OH

+H+

8

Ca2+ HO O 7

HO

Ca2+ O 6

H

Figure 9. Proposed mechanism of class I 1,2-diol dehydratases. Whereas Toraya54 and Balskus60 formulate a direct rearrangement of radical 3 to radical 8, this conversion could also occur via the ketyls 4-7. Ado = 5'-deoxyadenosyl radical derived from coenzyme B12. The dehydration is a stereospecific process: With (S)-propane 1,2-diol the 1-Si-hydrogen is abstracted and vice versa.60

In the strict anaerobe Clostridium butyricum, a coenzyme B12-independent but extremely oxygen-sensitive glycerol dehydratase was discovered in 2003.62 This class II dehydratase was characterized as glycyl radical enzyme, which is activated by hydrogen abstraction from the conserved Gly763 by the 5'-deoxyadenosyl radical generated by reductive cleavage of Sadenosylmethionine catalyzed by a specific activase. The crystal structure of the non-activated glycerol dehydratase revealed an active site that perfectly fitted to the ketyl mechanism proposed in 1996.63, 57After substrate binding, the glycyl radical transforms Cys433 to a thiyl radical, which is located close to C1 and C2 of glycerol, whereas Glu435 forms a hydrogen bond to the OH group at C1.63 Here Levin and Balskus60 as well as the author propose that after abstraction of one hydrogen at C1 by the thiyl radical, Glu435 removes the proton from the hydroxyl group at C1. The formed nucleophilic ketyl eliminates the hydroxyl group at C2 aided by protonation from His281. Finally the transient Cys433 donates the hydrogen atom back to the enoxy radical yielding the product. This mechanism is supported by calculations, which indicate that the resonancestabilized enoxy radical is able to remove the hydrogen atom from Cys433, but unable from 5'deoxyadenosine as does the 1,1-gem-diol-2-radical in class I enzymes.64, 65 The experiments with


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(R)- and (S)-1,2-[1- and 2-18O]propane-1,2-diols (see above) were repeated with pure class II propane-1,2-diol dehydratase and its activator from Roseburia inulinivorans isolated from the human gut. Whereas the label at C2 of both enantiomers was almost completely lost (2.5% retention), the label at C1 was retained (92%). The results clearly show that the OH-group at C2 is eliminated and the OH-group at C1 remains at its position,60 which is in complete agreement with the ketyl mechanism (Fig. 10). OH

OH

O

H OH

OH C-S

H+

O

OH

C-SH OH

O

O

O OH

H

Figure 10. Proposed ketyl mechanism of class II 1,2-diol dehydratases. C-SH is the cysteine residue in the glycyl radical enzymes, to which upon substrate binding the radical is transferred. The histidine and glutamate residues acting as acid and base, respectively, are omitted. The dehydration is a stereospecific process: With (S)-propane 1,2-diol the 1-Si-hydrogen is abstracted and vice versa.60

Glycerol dehydratases are of great technical importance because glycerol can be converted via 3-hydroxypropanal reductively to 1,3-propanediol for polymers or oxidatively to 3hydroxypropionate (3HP) for acrylates In this procedure the glycyl radical enzyme will replace the established coenzyme B12-dependent enzyme, because it requires no supplementation with the precious vitamin B12. Currently, glycerol is a cheap feedstock, because it is a byproduct of the conversion of plant oils to fatty acid methyl esters (FAME) for use as fuels.

3.2 GLYCYL RADICAL ENZYMES INVOLVED IN THE DEGRADATION OF CHOLINE, TAURINE AND 4-HYDROXYPROLINE Emily Balskus and coworkers detected in the human gut microbiome besides propane-1,2-diol dehydratase (see above) three more glycyl radical enzymes (Fig. 11): choline trimethylamine


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Biochemistry

lyase,66 isethionate sulfite lyase involved in taurine catabolism,67 and 4-hydroxyproline dehydratase.68,

69

Choline trimethylamine lyase catalyzes the radical cleavage of choline (2-

trimethylammoniumethanol) to trimethylamine and acetaldehyde. The reaction is similar to that of 1,2-diol dehydratases, but instead of water trimethylamine is eliminated. The combined action of the thiyl radical and a glutamate as base convert the hydroxymethyl part of choline to a ketyl, which expels the adjacent trimethylammonium. The formed enoxy radical regenerates the thiyl radical and tautomerizes to acetaldehyde. Isethionate is derived from taurine (2-sulfoethylamine) by exchange of the amino group by a hydroxyl group via transamination to 2-oxoglutarate and reduction to the alcohol. A glycyl radical enzyme again converts the alcohol to a ketyl which eliminates sulfite. Though trans-4-hydroxy-L-proline produced by hydrolysis of collagen can be degraded by non-radical pathways,70 the strictly anaerobic environment of the gut apparently favors bacteria that express the genes encoding the glycyl radical enzyme 4-hydroxyproline dehydratase and its activator. Since a hydroxyl group adjacent to 4-OH is absent, the ketyl mechanism sensu stricto is not applicable. However, hydrogen abstraction at C5 and proton abstraction from the imino group could yield an imino radical anion ('iminyl') which reacts like a ketyl and expels the OH group at C4, finally yielding 1-pyrroline carboxylate followed by reduction with NADH to proline. N(CH3)3

H+ N

HO

N

O C-S

O C-SH

C-SH SO3

H+ SO32-

HO

C-S

C-S

2-

SO32-

O

O

O

C-SH H

HO

O

C-SH

C-S

OH-

+

HO NADH

N

COO-

N C-S

H

C-SH

COO-

OH-

2'

HO

OH

N C-S

N

COO-

COO-

H

O

O

3'

COOC-SH

H+

O

N

O

O TR

O C-S

C-SH

OH

2'

O

O C-SH

HO

C-S


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Page 20 of 49

Figure 11. Eliminations catalyzed by glycyl radical enzymes and ribonucleotide reductases, which are proposed to proceed via ketyls. The mechanisms are short versions of that shown in Fig. 10. 1st line: choline trimethylamine lyase; 2nd line: isethionate sulfite lyase involved in taurine degradation; 3rd line: 4hydroxyproline dehydratase with subsequent reduction of the product 1-pyrrolidine-5-carboxylate to proline; 4th line: all 4 classes of ribonucleotide reductase; thioredoxin (TR) is the reductant of class I. C-S, conserved cysteine radical.

3.3 RIBONUCLEOTIDE REDUCTASES. Ribonucleotide reductases (RNR) are radical enzymes that catalyze the replacement of the 2'hydroxyl group at the ribose moiety by hydrogen yielding 2'-deoxyribonucleotides. The mechanism involves four steps, (i) generation of the thiyl radical, (ii) elimination of the 2'-OH group, (iii) reduction of the formed enoxy radical (3'-keto radical) to the 3'-hydroxy radical, (iv) regeneration of the thiyl radical. The enzymes are classified according to the source of the thiyl radical at the active site.71 Class I RNRs are composed of two subunits; in subunit R1 oxygen generates a radical, which is transported over a distance of about 35 Å to subunit R2 where the conserved cysteine residue is converted to the thiyl radical and the actual ribonucleotide reduction takes place.72 Thus the oxygen dependent radical generator is separated of form the oxygensensitive thiyl radical. Class I is divided into 5 subclasses (a – e), which differ in the kind of the primary radical and its formation. Most class I members store the radical as a stable tyrosyl radical near a diferric cluster, others use manganese instead, and the recently discovered class Ie converts the conserved tyrosine into a dihydroxyphenylalanine radical.73, 74 Coenzyme B12 serves as radical generator for class II, whereas class III reductases belong to the family of glycyl radical enzymes (see above).71 In all three classes, the mechanism of the elimination of the 2'-OH group is identical and very similar to that of the coenzyme B12-independent diol dehydratases (Figs. 10 and 11). The reaction starts with the thiyl radical abstracting the hydrogen atom at the 3'-carbon of the ribonucleotide. Like in diol dehydratases, the pK of the proton at the 3'-OH group is lowered to about 12, which facilitates deprotonation by a conserved glutamate residue. The formed 3'-ketyl eliminates the 2'-OH group generating the enoxy radical (keto radical), which oxidizes either a dithiol to a disulfide (all classes) or formate to CO2 (class III only).57 The obtained highly reactive 2'-deoxyribonucleotide-3'-radical regains the initially abstracted hydrogen from the cysteine and


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Biochemistry

affords the product 2'-deoxyribonucleotide, whereby the thiyl radical is regenerated.71 Finally, in the case of class I enzymes, the radical travels back from the thiyl residue to the site of generation, completing the whole catalytic cycle.72 4.

4-HYDROXYBUTYRYL-COA

DEHYDRATASE:

KETYL

FORMATION

BY

OXIDATION AND DEPROTONATION OF A THIOESTER ENOLATE 4-Hydroxybutyryl-CoA dehydratase is involved in the anaerobic fermentation of the inhibitory neurotransmitter 4-aminobutyrate (-aminobutyrate or GABA) to ammonia, acetate and butyrate in Clostridium aminobutyricum.75, 76 The enzyme also participates in the reduction of succinate via succinate semialdehyde and 4-hydroxybutyryl-CoA to butyrate in Clostridium kluyveri.77 Anaerobic degradation of 4-aminobutyrate was also reported from an anaerobe in the human gut.78 Another occurrence of 4-hydroxybutyryl-CoA dehydratase is the CO2 fixation cycle in the archaeal aerobes Metallosphaera sedula79 and Nitrosopumilus maritimus80, in which it connects lipid to carbohydrate metabolism. Surprisingly, under air the half-life of purified 4-hydroxybutyryl-CoA dehydratase from the anaerobe C. aminobutyricum is 14 min, whereas the enzyme from the aerobe N. maritimus is almost two orders of magnitude more stable, retaining 50% of its activity even after 46 h of incubation.81 H H CoAS

OH

H

H H

H

CoAS

O H H 4-Hydroxybutyryl-CoA

H H

+ H 2O

O H Crotonyl-CoA

Figure 12. The stereochemistry of the reaction catalyzed by 4-hydroxybutyryl-CoA dehydratase. The blue hydrogen stems from the solvent.82 The stereochemistry of the hydrogens at C2 and C3 is identical to that observed with the related butyryl-CoA dehydrogenase.59

The reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA [(E)-but-2-enoyl-CoA] bears the same problem as the dehydration of 2-hydroxyacyl-CoA, because the proton at C3 is not activated (pKa ≈ 40) (Fig. 12). Therefore a dehydration to vinylacetyl-CoA (but-3-enoyl-CoA) followed by isomerization to crotonyl-CoA appears unlikely, though 4-hydroxybutyryl-CoA


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Page 22 of 49

dehydratase from C. aminobutyricum exhibits a vinylacetyl-CoA -isomerase activity.76 During the reaction, the 2Re- and the 3Si-protons (red) are removed from 4-hydroxybutyryl-CoA and the replacement of the OH group by hydrogen (blue) occurs with retention of configuration (Fig. 12).82 The dehydratase has been purified from C. aminobutyricum and characterized as an enzyme containing 1 [4Fe-4S]2+-cluster and 1 FAD. An external radical generator required for ketyl generation has not been found. To get maximum activity, the cofactors of the enzyme have to be in the oxidized state. A brief exposure to air ( 1min) is necessary to fully activate the anaerobically purified enzyme, whereas the assay is conducted under anaerobic conditions.76 Larger amounts of the dehydratase prepared for spectroscopy and crystallography were oxidized with 5 mM K3[Fe(CN)6] followed by removal of the excess oxidant under anaerobic conditions. The crystal structure revealed a homotetrameric protein with one [4Fe-4S]2+-cluster and one FAD in each subunit, forming a cleft between the cofactors into which the 4-hydroxybutyryl moiety of the substrate could be modelled (Fig. 13).83 The [4Fe-4S]2+ cluster is coordinated by 3 cysteines and histidine 292, though the N2-Fe1 bond length of 2.4  0.1 Å between histidine and cluster exceeds the usual value by 0.4 Å. Whether the structure represents a mixture of His at the cluster (His-on) and His removed from the cluster (His-off) is not known. Tyrosine 296 is located near the N2Fe1 bond and 6.6 Å apart from FAD (Y-OH – N5 distance). Threonine 190 is connected by hydrogen bonds to N5 of FAD at 3.3 Å and to glutamate 257 at 3.0 Å. Glutamate 455 forms a hydrogen bond to the OH group of the substrate (3.6 Å) modelled into the structure. The 2Rehydrogen of the substrate is connected to N1 of histidine 292 (3.4 Å) and the 3Si-hydrogen to N5 of FAD (2.7 Å). Importantly, the carbonyl group of the thioester forms hydrogen bonds to 2'-OH of the ribitol moiety of FAD and to NH of alanine 460. This hydrogen bond net reduces the pKa of the protons at C2 and thus facilitates the abstraction of the 2Re-hydrogen by histidine 292.83, 84 However, oxidizing 4-hydroxybutyryl-CoA like butyryl-CoA would lead to the dead-end product 4-hydroxycrotonyl-CoA. Stopped flow measurements have shown that binding of 4hydroxybutyryl-CoA to the dehydratase leads within milliseconds to formation of intensive UVvis and EPR signals, long before the reaction starts, probably due to the formation of a neutral FAD semiquinone (absorption at 590 nm) interacting with the [4Fe-4S]2+-cluster.85 Site directed mutagenesis showed that the replacements C99A, C103A, C299A (the 3 cysteines coordinating the cluster), H292C/E, E257Q, and E455Q were completely inactive, while Y296F and T190V exhibited 0.7% and 0.5% residual activities, respectively (Fig. 13). However, upon addition of the


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Biochemistry

substrate to the E455Q mutant, the UV-vis and EPR signals were identical to those of the wild type, whereas the E257Q and Y296F mutants remained silent. Hence the substrate might induce an electron transfer from tyrosine296 to N5 of FAD over 6.6 Å aided by glutamate 257 via threonine 190 as base at N5 of FAD to yield the neutral FAD semiquinone, visible at 590 nm. The dehydration of 4-hydroxybutyryl-CoA is proposed to start with abstraction of the 2Re-proton by H292 and the 3Si-hydrogen by the FAD semiquinone yielding an allylic ketyl that eliminates the hydroxyl group assisted by E455 (Fig. 14). The formed dienoxy radical regenerates the FAD semiquinone and is protonated by E455 to afford the product crotonyl-CoA.85 The isomerization of vinylacetyl-CoA to crotonyl-CoA most likely proceeds via removal of the 2Re-proton by His292 and protonation by E455 at C4.

Figure. 13. Structure of the active site of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum into which 4-hydroxybutyryl-CoA (C2, C3, C4) was modelled. Carbons in gray, oxygens in red, nitrogens in blue, sulfurs in yellow and irons in brown. The isoalloxazine ring of FAD is shown in the lower part; the [4Fe-4S]-cluster is located in the upper part. Adapted from ref.85


23

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O

Page 24 of 49

O

H H OH

CoAS

O N

H H 4-Hydroxybutyryl-CoA

N

Fe1 of [4Fe-4S] O

N N

R

H H

CoAS

Y296

H292

O FAD

E455

OH

H

CoAS T190 E257

H H

H Crotonyl-CoA

O

H292

H

+

H

O

N H H

H Enolate

T190 E257

H

CoAS

N

N

R H

O

N

H

N

O Fe1

R

H

N

H O Fe1

CoAS

O

Vinylacetyl-CoA

O H O Fe1

O

CoAS

H

FADH

E455

H+

.

O

O

H

CoAS H Dienolate

H

N

O O

N

FADH-

H

CoAS

H Allylic ketyl

H Dienoxyradical

H292

H+

H2O + Fe1

Fig. 14. Prosed mechanism of 4-hydroxybutyryl-CoA dehydratase. Upon substrate binding a neutral FAD semiquinone (FADH) is generated; the electron is thought to come from Y296. During turnover FADH is transiently reduced to the hydroquinone, FADH. The hydroxyl group of 4-hydroxybutyryl-CoA probably binds to Fe1 of the [4Fe-4S] cluster which is not coordinated by a cysteine. The italicized amino acids are proposed to act as base (blue) and as acid (red), whereas Y296 most likely is oxidized to a tyrosyl radical. Adapted from ref.85


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Biochemistry

5. REACTIONS THAT DO NOT INVOLVE KETYL INTERMEDIATES. Over 25 years ago the author of this review postulated that the reductions of benzoyl-CoA to 2,5cyclohexadiene dicarboxylic acid and 4-hydroxybenzoyl-CoA to benzoyl-CoA and as well as the oxidation of phenylacetyl-CoA to mandelyl-CoA (phenylglycolyl-CoA) involve ketyls.2 Apparently, only with benzoyl-CoA he was right, though the radical has not been identified yet. The biochemical and structural characterization of 4-hydroxybenzoyl-CoA reductase revealed, however, that the reaction required ferredoxin as reductant but no ATP.86 Furthermore, in the structure of the enzyme, the 4-hydroxy group was coordinated to the molybdate of the molybdopterin cofactor but not the thioester carbonyl to a [4Fe-4S]-cluster as detected in the 2hydroxisocaproyl-CoA dehydratase (Fig. 4). Therefore it appears unlikely that this reductive dehydration involves a ketyl, though such a mechanism can be easily formulated. Unfortunately the other enzyme, a quinone containing phenylacetyl-CoA oxidase, catalyzes the oxidation of the substrate to phenylglyoxylate without mandelate or mandelyl-CoA as intermediate.41 A mechanism via a ketyl could have been the addition of OH to the enoxy radical followed by oxidation to mandelyl-CoA.

6. CONCLUSION In 1996, Bernard T. Golding and the author of this review published an article entitled: “Glutamate and 2-methyleneglutarate mutase: From microbial curiosities to paradigms for coenzyme B12dependent enzymes”.87 We wrote about Horace Albert Barker (University of California, Berkeley), who working with the anaerobe Clostridium tetanomorphum isolated from soil, unraveled the coenzyme form of vitamin B12 as 5'-deoxyadenosylcobalamin. Later it turned out that the homolytic cleavage of the cobalt-carbon bond of this coenzyme generated the radical for class I diol dehydratases, class II ribonucleotide reductases and unusual carbon skeleton rearrangements like that of (R)-methylmalonyl-CoA to succinyl-CoA catalyzed by a human mutase. A similar curiosity was the proposal of a ketyl involved in the dehydration of 2-hydroxyacyl-CoA to enoylCoA in anaerobic bacteria from the gut of animals.2 Now this intermediate appears to occur in such diverse enzymes as benzoyl-CoA reductases, several glycyl radical enzymes like glycerol dehydratase, all four classes of ribonucleotide reductase and 4-hydroxybutyryl-CoA dehydratase,


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which is involved in the anaerobic degradation of the neurotransmitter GABA in the human gut and in the aerobic CO2 fixation cycle of Nitrosopumilus maritimus, one of the most abundant microorganisms of the world.

ACKNOWLEDGEMENTS The author thanks all coworkers for performing the experiments as well as Professor Matthias Boll (Universität Freiburg, Germany), Professor Holger Dobbek (Humboldt Universität, Berlin, Germany), Professor Bernard T. Golding (University of Newcastle upon Tyne, UK), Professor Reinhard Keese (Universität Bern, Switzerland), Dr. Berta M. Martins (Humboldt Universität, Berlin, Germany), Professor Antonio J. Pierik, (Universität Kaiserslautern, Germany), Professor János Rétey (Universität Karlsruhe, Germany), and Professor Rudolf K. Thauer (Max-PlankInstitut für terrestrische Mikrobiologie, Marburg, Germany) for many helpful discussions.

AUTHOR INFORMATION Corresponding author: Prof. Dr. Wolfgang Buckel, E-mail: [email protected] Funding: The author is without funding since 2017 Note: The author declares no competing financial interest

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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Biochemistry



Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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Biochemistry



Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10 338x190mm (96 x 96 DPI)


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Biochemistry

Figure 11 338x190mm (96 x 96 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 12 338x190mm (96 x 96 DPI)


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Biochemistry

Figure 13 338x190mm (96 x 96 DPI)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 14 338x190mm (96 x 96 DPI)


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Biochemistry

Graphic for Table of Content (TOC) 338x190mm (96 x 96 DPI)


Enzymatic Reactions Involving Ketyls: From a Chemical Curiosity to a

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