Tài liệu Báo cáo khoa học: Reaction mechanisms of thiamin diphosphate enzymes: redox reactions pdf

Keywords electron transfer; flavin; intermediate; iron-sulfur cluster; lipoamide; oxidative decarboxylation; phosphorolysis; pyruvate; radical; X-ray crystallography Correspondence K.. T

Reaction mechanisms of thiamin diphosphate enzymes: redox reactions

Kai Tittmann

Albrecht-von-Haller-Institut fu¨r Pflanzenwissenschaften und Go¨ttinger Zentrum fu¨r Molekulare Biowissenschaften, Georg-August-Universita¨t Go¨ttingen, Germany

Introduction

The oxidative decarboxylation of 2-keto acids, such

as pyruvate, branched-chain keto acids and

ketoglu-tarate, is a key reaction of intermediary metabolism

in virtually all organisms and is catalyzed by thiamin

diphosphate (ThDP)-dependent enzymes [1] In view

of the central metabolic role of pyruvate, the various

biochemical reactions involving pyruvate are the

most intensely studied and are well understood

Thus, they serve as prototypical reactions for the

enzymic oxidative conversion of 2-keto acids Hence,

the present review mainly focuses on the reaction

mechanisms of ThDP enzymes that directly oxidize

pyruvate Special emphasis is devoted to the nature and reactivity of transient intermediates, the coupling

of oxidation–reduction and acyl group transfer and electron transfer between cofactors The review includes a discussion of general aspects of enzyme catalyzed pyruvate oxidation, in addition to individ-ual sections on the different ThDP enzymes that act

on pyruvate

Pathways of pyruvate oxidation by ThDP enzymes

Generally, there are at least four major different path-ways of ThDP enzyme catalyzed pyruvate oxidation

Keywords

electron transfer; flavin; intermediate;

iron-sulfur cluster; lipoamide; oxidative

decarboxylation; phosphorolysis; pyruvate;

radical; X-ray crystallography

Correspondence

K Tittmann, Albrecht-von-Haller-Institut fu¨r

Pflanzenwissenschaften und Go¨ttinger

Zentrum fu¨r Molekulare Biowissenschaften,

Georg-August-Universita¨t Go¨ttingen,

Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11,

D-37077 Go¨ttingen, Germany

Fax: +49 551 39 5749

Tel: +49 551 39 14430

E-mail: ktittma@gwdg.de

(Received 7 November 2008, revised 3

February 2009, accepted 13 February 2009)

doi:10.1111/j.1742-4658.2009.06966.x

Amongst a wide variety of different biochemical reactions in cellular car-bon metabolism, thiamin diphosphate-dependent enzymes catalyze the oxi-dative decarboxylation of 2-keto acids This type of reaction typically involves redox coupled acyl transfer to CoA or phosphate and is mediated

by additional cofactors, such as flavins, iron-sulfur clusters or lipoamide swinging arms, which transmit the reducing equivalents that arise during keto acid oxidation to a final electron acceptor EPR spectroscopic and kinetic studies have implicated the intermediacy of radical cofactor intermediates in pyruvate:ferredoxin oxidoreductase and an acetyl phos-phate-producing pyruvate oxidase, whereas the occurrence of transient on-pathway radicals in other enzymes is less clear The structures of pyru-vate:ferredoxin oxidoreductase and pyruvate oxidase with different enzymic reaction intermediates along the pathway including a radical intermediate were determined by cryo-crystallography and used to infer electron tunnel-ing pathways and the potential roles of CoA and phosphate for an intimate coupling of electron and acyl group transfer Viable mechanisms of reduc-tive acetylation in pyruvate dehydrogenase multi-enzyme complex, and of electron transfer in the peripheral membrane enzyme pyruvate oxidase from Escherichia coli, are also discussed

Abbreviations

AcThDP, 2-acetyl-ThDP; HEThDP, 2-(1-hydroxyethyl)-ThDP; PDHc, pyruvate dehydrogenase multi-enzyme complex; PFOR,

pyruvate:ferredoxin oxidoreductase; POX, pyruvate oxidase; Q8,ubiquinone 8; ThDP, thiamin diphosphate.

Pyruvate dehydrogenase multi-enzyme complex

In mitochondria and respiratory eubacteria, the

pyru-vate dehydrogenase multi-enzyme complex (PDHc)

catalyzes the essentially irreversible conversion of

pyru-vate, CoA and NAD+ into CO2, NADH and

acetyl-CoA (Eqn 1) [2] The latter is utilized as a precursor

for the Krebs cycle and the biosynthesis of fatty acids

and steroids, whereas NADH feeds the reducing

equiv-alents into the respiratory chain for oxidative

phos-phorylation (i.e ATP synthesis)

PDHc:pyruvateþ CoA + NADþ! acetyl-CoA+CO2

PDHc is the largest molecular machine known (Mr

of  106–107) and is composed of multiple copies of

three enzyme components: a ThDP-dependent

pyru-vate dehydrogenase (termed the E1 component), a

dihydrolipoamide transacetylase (E2 component),

which carries lipoyl groups covalently attached to

lysine residues [N6-(lipoyl)lysine, lipoamide], and

lipoa-mide dehydrogenase (E3 component) with a

nonco-valently yet tightly bound FAD cofactor [3] In

mammals, PDHc contains an additional E3 binding

protein and specific kinases and phosphatases, which

control the activity of the complex by reversible

phos-phorylation⁄ dephosphorylation of serine side chains in

E1 [4] Initially, E1 catalyzes the irreversible

decarbox-ylation of pyruvate and the subsequent reductive

acet-ylation of an N6-(lipoyl)lysine in E2 E2 itself catalyzes

acyl group transfer from the reduced

S-acety-ldihydrolipoyl-lysine to CoA Finally, E3 regenerates

the oxidized form of lipoamide and transfers the two

reducing equivalents to NAD+

Pyruvate:ferredoxin oxidoreductase

In anaerobic organisms, acetyl-CoA is synthesized by

the enzyme pyruvate:ferredoxin oxidoreductase

(PFOR), which may contain one or multiple

[Fe4S4]2+clusters in addition to ThDP [5] The two

electrons, which arise during oxidation of pyruvate at

the ThDP site, are sequentially transferred via the

iron-sulfur cluster(s) to final electron acceptors ferredoxin

(Fd) or flavodoxin (Eqn 2) [6] Unlike PDHc, PFOR

also carries out the reverse reaction, namely the

reduc-tive carboxylation of acetyl-CoA to yield pyruvate

PFOR: pyruvateþ CoA + 2Fdox! acetyl - CoA

þ CO2þ2Fdred ð2Þ

The low-potential electrons of Fdred formed in the forward direction (Eo of pyruvate oxidation

)540 mV) are used to drive several low-potential transformations such as CO reduction or hydrogen formation [7] The reverse synthase reaction (pyruvate formation) is central to CO2 fixation in acetogenic and green photosynthetic bacteria [8]

Acetyl phosphate-producing pyruvate oxidases

In Lactobacillae such as Lactobacillus plantarum or Lactobacillus delbrueckii, which are unable to synthe-size hemes and thus lack a respiratory chain for oxi-dative phosphorylation, ATP is mainly generated by fermentation of carbohydrates with lactic acid as a final product Under aerobic growth conditions, some Lactobacillae convert carbohydrates to the high-energy metabolite acetyl phosphate, which in turn is used for ATP synthesis A key reaction of this path-way is the oxidative decarboxylation of pyruvate by the enzyme pyruvate oxidase (POX) that requires ThDP, Mg2+ and FAD as cofactors [9,10] After binding and decarboxylation of pyruvate, the reduc-ing equivalents are transferred to the neighborreduc-ing FAD cofactor The flavin is then reoxidized by the final electron acceptor dioxygen to yield hydrogen peroxide (Eqn 3)

POX : pyruvateþ phosphate + oxygen + Hþ

! acetyl phosphate + CO2+ H2O2 ð3Þ

Phosphate-independent pyruvate oxidase from

E coli

In E coli, a related ThDP and FAD-dependent pyru-vate oxidase (EcPOX) feeds electrons from the cytosol directly into the respiratory chain at the membrane [11,12] EcPOX is considered to be a backup system to PDHc and was shown to be important for aerobic growth of E coli Unlike POX from Lactobacillae, EcPOX produces acetate rather than acetyl phosphate and its reduced flavin is unreactive towards oxygen Two-electron reduction of the flavin has been sug-gested to induce a structural rearrangement of the enzyme that exposes a lipid-binding site at the C-termi-nus After binding to the membrane, the flavin reduces the membrane-bound mobile electron carrier ubiqui-none 8 (Q8) (Eqn 4)

EcPOX : pyruvate + Q8+ OH! acetate + CO2+ Q8H2

ð4Þ

Reaction intermediates in the ThDP

catalyzed oxidation of pyruvate

The oxidative decarboxylation of pyruvate in PDHc,

PFOR and POX involves a series of covalent ThDP

intermediates and analogous elementary reactions

(Fig 1) [13] In pioneering studies on models, Breslow

[14] identified C2 of the ThDP thiazolium as the

reactive center that, in its carbanionic form, attacks

the substrate carbonyl yielding, in the case of

pyru-vate, the tetrahedral pre-decarboxylation intermediate

2-lactyl-ThDP Decarboxylation of the latter gives the

resonant a-carbanion⁄ enamine forms of

2-(1-hydroxy-ethyl)-ThDP (HEThDP) The enamine is sometimes

(and more accurately) referred to as

2-(1-hydroxye-thylidene)-ThDP and formally represents the

C2a-deprotonated conjugate base of HEThDP in a

resonance stabilized form Essentially, all steps

encompassing binding and decarboxylation of

pyru-vate are common to PDHc, PFOR and POX Reac-tion sequences diverge at the HEThDP carbanion⁄ enamine intermediate, which is highly reducing and may undergo one-electron or two-elec-tron oxidation by proximal redox cofactors The [Fe4S4] clusters in PFOR are exclusive one-electron acceptors, whereas the flavin in POX may function as

a one-electron and two-electron capacitor Model studies suggest that reduction of the redox active dithiolane moiety of lipoic acid is a two-electron process linked to atom transfer [15]

One-electron oxidation of the HEThDP carban-ion⁄ enamine results in the formation of a ThDP cat-ion radical (termed the HEThDP radical) with different resonance contributors (the most relevant ones are shown in Fig 1) In principle, the unpaired spin may be delocalized over the hydroxyethyl and thiazolium moieties but it appears likely that the active sites in enzymes are poised to stabilize just one

Fig 1 Intermediates in the ThDP-catalyzed oxidative decarboxylation of pyruvate.

or few electronic states If electron transfer is coupled

to proton transfer (i.e deprotonation of the Ca-OH),

the neutral 2-acetyl-ThDP (AcThDP)-type radical will

be formed with a set of resonant forms similar to

those described for the HEThDP radical

Two-elec-tron reduction or stepwise one-elecTwo-elec-tron reduction

yields the AcThDP intermediate that exists in three

distinct forms: the keto form, the hydrate and the

tri-cyclic carbinolamine [16] The keto form undergoes

acyl transfer to nucleophilic acceptors or is

hydroly-sed with the geminal diol (hydrate) as a transitory

state

The occurrence of 2-lactyl-ThDP, HEThDP and

AcThDP as reaction intermediates in ThDP enzymes

has been confirmed by 1H NMR spectroscopy after

acid quench isolation [17] EPR spectroscopy was

employed to detect radical ThDP intermediates [18]

Thermodynamic aspects of pyruvate

oxidation

In PDHc, PFOR and POX, the thermodynamically

favorable oxidation of pyruvate is coupled to

forma-tion of the ‘energy-rich’ metabolites acetyl-CoA and

acetyl-phosphate, which serve either as chemically

acti-vated building blocks in anabolic pathways, or for

ATP synthesis because the group transfer potential of

the acetyl-CoA thioester (DG¢=)35.7 kJÆmol)1) and

the acetyl phosphate acid anhydride (DG¢= )44.8

kJÆmol)1) exceeds that of ATP (DG¢= 31.8 kJÆmol)1)

[19]

Electrochemical studies on thiazolium models

revealed very low subsequent one-electron oxidation

potentials of the presumed pyruvate-derived enamine

(Eox(1)=)0.96 V and Eox(2)=)0.73 V versus

satu-rated calomel electrode; Eox(1)=)0.72 V and

Eox(2)=)0.49 V versus standard hydrogen electrode)

[20] Thus, the high reducing power of the enamine

intermediate is by far sufficient to initiate downhill

transfer of the reducing equivalents to the final

elec-tron acceptors ferredoxin (E¢ [Fe++⁄ Fe+++]

)0.4 V versus standard hydrogen electrode at pH 7),

NAD+ (E¢ [NADH, H+⁄ NAD+, 2 H+] =

)0.32 V), ubiquinone (E¢ [dihydroquinone⁄ quinone,

2 H+] = +0.10 V) or oxygen (E¢= +0.29 V for

the O2⁄ H2O2 couple) The redox potentials of

additional cofactors directing electron transfer from

the ThDP enamine onto final electron acceptors may

be modulated to some degree by the protein

environ-ment but are suspected to lie in between Redox

potentials of [Fe4S4] clusters in PFOR and FAD in

POX will be discussed in the sections on the different

enzymes

Reaction mechanism of pyruvate:

ferredoxin oxidoreductase

Evidence for a free radical mechanism

In the early 1980s, Oesterhelt et al discovered that mixing PFOR from Halobacterium halobium with its substrate pyruvate led to the formation of an organic free radical that gives rise to a continuous wave X-band EPR signal centered at g = 2.006 [21] The radical was reported to be stable even at room temper-ature, but was readily depleted upon addition of the second substrate CoA Quantitative analysis of sub-strate turnover revealed that two moles of final one-electron acceptor ferredoxin are reduced per mole of pyruvate in the presence of CoA There are several lines of evidence indicating that the organic radical is a HEThDP radical resulting from one-electron oxidation

of the HEThDP carbanion⁄ enamine intermediate by the neighboring FeS cluster (i.e this PFOR contains a single [Fe4S4] cluster) At first, additional experiments with selectively 14C-labeled pyruvate revealed that radioactivity remained tightly bound to the enzyme when PFOR was reacted with [3-14C]pyruvate, whereas

no label could be detected after addition of [1-14C]pyruvate, clearly suggesting the radical to be formed after decarboxylation [18] Second, the hyper-fine splitting of the radical EPR signal was shown to

be dependent on the chemical nature of the substrate methyl substituent (i.e the number of nuclear spins at C3 of pyruvate) [5] When the EPR spectra were recorded at temperatures below 20 K, spin coupling between the ThDP-derived radical and the reduced [Fe4S4]1+cluster was observable, indicating that the two paramagnetic centers are located at a distance of approximately 1 nm or less [18]

Subsequently, kinetic and spectroscopic studies on PFORs from different organisms including Desulfovib-rio africanus and Clostridium thermoaceticum suggested

a common reaction mechanism with an obligate tran-sient ThDP-based radical, the lifetime of which criti-cally depends on the presence of CoA [22] Unlike the archetypical PFOR from H halobium, these PFORs contain three [Fe4S4] clusters with slightly different midpoint potentials (E1 =)540 mV, E2=)515 mV,

E3=)390 mV) [23] Thermodynamics suggest an elec-tron transfer chain from the thiamin radical to the final electron acceptor ferredoxin via the three iron-sulfur clusters involving the initial reduction of the lowest-potential iron-sulfur cluster (suspected to be located in close proximity to the ThDP cofactor), fol-lowed by sequential reduction of the other two clusters leading towards the surface of the protein, where

ferredoxin will be reduced Remarkably, in the absence

of CoA, addition of pyruvate to D africanus PFOR

eventually resulted in the reduction of only the highest

potential [Fe4S4] cluster (E3 =)390 mV) No

mag-netic interaction between this cluster and the ThDP

radical was detectable, and it was concluded that the

reduced cluster is distant from the thiamin binding site

[23] In the presence of pyruvate and CoA, all three

clusters become reduced This exciting discovery on

different PFORs pinpointed a crucial role of CoA for

facilitating transfer of the second electron from the

ThDP radical to the iron-sulfur clusters

Structural studies on PFOR and its ThDP radical

intermediate

In 1999, Fontecilla-Camps et al solved the X-ray

crys-tallographic structure of the homodimeric PFOR from

D africanus in the resting state at 2.3 A˚ resolution

[24] The ThDP cofactor is deeply buried within the

protein, and its reactive center, the thiazolium nucleus

of ThDP, is located approximately 10 A˚ from the most

proximal [Fe4S4] cluster (referred to as cluster A,

prox-imal) (Fig 2) Clusters A, B (medial) and C (distal) of

each subunit are separated by approximately 10–12 A˚,

thus allowing for facile electron transfer in a suitably

organized redox chain (Dutton’s empirical analysis [25]

predicts electron tunneling to take place when

donor⁄ acceptor pairs are within 14 A˚ edge-to-edge distance) Cluster C is located close to the enzyme surface, where the final electron acceptor ferredoxin

is suspected to bind Electron tunneling is likely to proceed dominantly in a through-bond mechanism involving the backbone and coordinating cysteinyl ligands of the iron-sulfur clusters Clusters B and C are covalently linked to each other via the protein, and only few gaps with edge-to-edge distances close to van-der-Waals distance (< 4 A˚) exist between ThDP and cluster B, such that through-space tunneling will be scarcely required

Fontecilla-Camps et al then reported the high-reso-lution X-ray structure of the free AcThDP radical trapped at the active center of PFOR from D afric-anus [26] The electron density maps suggested the thiazolium moiety of the cofactor intermediate to be markedly puckered, a structural feature that indicates reduction or even loss of aromaticity Therefore, the thiazolium ring was proposed to adopt an unprece-dented tautomeric form, in which a proton from the 4-methyl group has undergone transfer to C5 (Fig 3A) Also, the C2-C2a bond that connects C2 of ThDP with the substrate C2 was reported to be excep-tionally long (1.86 A˚) prompting the authors to suggest a r⁄ n-type AcThDP radical in which the unpaired spin is mostly confined to the acetyl moiety and, to a lesser degree, to C2 of the cofactor [26]

Fig 2 Stereo drawing of PFOR structure (Protein Data Bank code: 1kek) in transpar-ent surface represtranspar-entation The ThDP radical and the three [Fe4S4] clusters are shown as sticks Edge-to-edge distances between all cofactors are indicated.

After fragmentation of the r⁄ n-type cation radical and

formation of an acetyl radical, radical recombination

with a CoA thiyl radical was proposed to occur By

contrast to p-type radicals with extensive delocalization

of the unpaired spin over aromatic systems, the

pro-posed r⁄ n-type AcThDP radical must be regarded,

especially in view of the tenuously bonded acetyl

moi-ety, as an unstable high-energy intermediate and, thus,

its long lifetime, as demonstrated experimentally both

in the crystalline phase and in solution, is seemingly

counterintuitive

EPR studies on the free radical in PFOR and role

of CoA for electron transfer

Ragsdale and Reed [27] thoroughly examined the

HEThDP radical in PFOR from Moorella thermoacetica

by X-band and D-band EPR spectroscopy EPR

spec-tra of PFOR were recorded for different combinations

of native and isotopically labeled cofactor ([2-13C],

[3-15N]) and substrate ([3-2H3], [2-13C], [3-13C]), and

further analyzed by spectral simulations The obtained

g-values and 14N⁄15N hyperfine-splittings are in good

agreement with a planar p-type radical and extensive

delocalization of the unpaired spin over the thiazolium

ring The EPR spectra and associated simulations on

simplified thiazolium models are not consistent with a

r⁄ n-type AcThDP radical proposed on the basis of

pure structural data [26] Although which protonation

state pertains to the radical intermediate cannot be

clarified unambiguously, the observed 1H- and

13C-hyperfine splittings of the C2ß protons and C2

and C2a carbons would best correspond to an

interme-diate state between C2a O-protonated (HEThDP radi-cal) and O-deprotonated (AcThDP radiradi-cal) forms The close proximity of the cofactor’s exocyclic 4¢-amino group demonstrated in the X-ray structure favors a hydrogen-bonding interaction between C2a-O and N4¢ (Fig 3B)

As noted above, addition of pyruvate to PFOR gen-erates a stable ThDP radical and one reduced [Fe4S4] cluster, which was more recently demonstrated to be the medial cluster [28] Rapid depletion of the thiamin radical and reduction of all clusters is only achieved after addition of CoA What is the special role of CoA for propagation of the second electron and the associ-ated 105-fold rate enhancement of electron transfer? In pursuit of this question, Ragsdale proposed several viable mechanisms [7,29]

At first, he considered that CoA itself could comprise part of the electron transfer chain by wiring the HEThDP radical and one iron-sulfur cluster, followed by nucleo-philic attack of the AcThDP formed in that process and eventual release of acetyl-CoA The observation that the CoA analogue desulfo-CoA induces no rate enhancement

of electron transfer, despite only marginally compro-mised binding energy compared to CoA, renders the above-considered mechanism unlikely In line with that argument, if indeed CoA were to lend its orbitals for bridging the pathway and effective through-bond tunnel-ing, why then does transfer of the first electron proceed

so facilely from the HEThDP enamine to cluster B via cluster A in the absence of CoA?

Second, a biradical mechanism was proposed that involves one-electron reduction of one iron-sulfur cluster by CoA and subsequent recombination of the

Fig 3 Chemical structures of the

HEThDP ⁄ AcThDP radical in PFOR proposed

on the basis of structural data (A) [26] or

EPR spectroscopic analysis (B) [27].

resultant CoA thiyl radical and the HEThDP radical

to form acetyl-CoA Support for this comes from the

observation that CoA reduced one [Fe4S4] cluster in

PFOR from C thermoaceticum, even in the absence of

pyruvate [22] However, such behavior has not been

reported for all PFORs and there was no EPR

spectro-scopic evidence for the putative CoA sulfur-based thiyl

radical An additional intricacy of this mechanism is

the necessity of a structural rearrangement of CoA in

the course of catalysis: initially, the reactive thiol

group of CoA must be positioned proximal to an

iron-sulfur cluster and distant to ThDP but, after

oxida-tion–reduction, it would have to swing closer to ThDP

Although a simple bond rotation could account for

such conformational transition, diffusion of the CoA

radical out of the active site and abortive side

reactions of the highly reactive thiyl radical could

successfully compete with radical recombination

Furthermore, direct access to the clusters is sterically

occluded by different loops, so the structural

confine-ments of the active site channel render the proposed

double duty of CoA (cluster reduction and radical

recombination) unlikely, unless binding of CoA would

enforce large structural rearrangements of the protein

Third, it was proposed that the rate enhancement of

electron transfer by CoA could result from a chemical

and kinetic coupling of oxidation–reduction and acyl

group transfer [7] This mechanism would generate a

covalent adduct between the AcThDP-type radical and

CoA to form an anion radical, the reducing power of

which can be anticipated to be much higher than of a

charge-neutral AcThDP radical, thus increasing the

driving force of the redox reaction As noted earlier

above, in model studies, it was established that the

potential of the thiazolium enamine⁄ radical couple

(Eox(1)=)0.72 V versus standard hydrogen

elec-trode) is more negative than that of the radical⁄ acetyl

couple (Eox(2)=)0.49 V) It is conceivable that the

redox potential of the former is low enough to reduce

the lowest potential PFOR cluster (E1=)0.54 V),

whereas the reducing ability of the latter might be

insufficient in that concern Thus, conclusively, the

formation of a low potential anion radical may be

thermodynamically mandatory to drive re-reduction of

the lowest potential clusters in PFOR

Mechanism of pyruvate oxidation in

pyruvate dehydrogenase multi-enzyme

complex

As noted earlier, oxidation of pyruvate in the E1

com-ponent of PDHc is coupled to reductive acetylation of

lipoic acid covalently attached in amide linkage to the

e-amino function of a lysine in the E2 component By contrast to iron-sulfur clusters in PFOR or FAD in POX, the N6-(lipoyl)lysine conjugate is structurally flexible, a ‘swinging arm’ that permits active site cou-pling between E1, E2 and E3 components by rotation

of the lipoyl moiety itself and by additional movement

of the whole protein domain (‘swinging domain’) that carries the lipoyl-lysine, thus providing a ‘super arm’ that is capable to span the gaps between the active centers on the different components [2,30]

Oxidation–reduction chemistry of lipoic acid in models and implications for reductive acetylation

in pyruvate dehydrogenase Lipoic acid exists in an oxidized disulfide form with a slightly strained five-membered dithiolane ring (LipS2) and in the two-electron reduced acyclic dithiol form (dihydrolipoic acid, Lip(SH)2) The standard redox potential of the Lip(SH2)⁄ LipS2 couple has been determined by polarographic analysis to be approxi-mately )0.32 V (pH 7) and is thus more positive than the two subsequent one-electron oxidation potentials

of thiazolium enamine models, making oxidation of the enamine by LipS2 thermodynamically favorable [31] However, LipS2 will be electrochemically reduced only at extremely negative potentials in acetonitril solution ()1.92 V versus saturated calomel electrode) and it even resists reduction in water [15] Low-poten-tial single-electron reductants such as reduced methyl viologen do not undergo oxidation–reduction with LipS2, clearly indicating that sequential one-electron reduction is an unlikely mechanism for two-electron reduction of LipS2 [15] A lipoic acid disulfide anion radical can be generated by one-electron reduction using hydrated electrons as reductants, but the reduc-ing power of the disulfide radical is much higher than that of the HEThDP enamine, disfavoring its one-electron oxidation by LipS2 [32] Because the complete reduction of LipS2 was easily achieved by reaction with molecular hydrogen, it was concluded that reduction and concomitant cleavage of the disulfide linkage must be coupled to atom (proton) transfer [15]

In line with these early investigations, Jordan et al observed that, in chemical models, reductive acetyla-tion of lipoic acid by thiazolium enamine occurs extremely slowly and requires the addition of a mercury trapping reagent [33] Subsequently, the same laboratory used S-methylated lipoic acid [LipS(SCH3)+] as a viable chemical model for the S-protonated form of LipS2 [34] MS analysis revealed the existence of a tetrahedral adduct with an S-C

linkage formed between lipoic acid and the thiazolium

C2a [34] Very remarkably, LipS(SCH3)+ easily

oxi-dizes thiazolium enamine models with second-order

rate constants that, in view of the effective molarity

of the lipoyl-lysine in the multi-enzyme complex, can

account for the observed turnover number of PDHc

This intriguing observation suggests that reductive

acetylation in PDHc requires an acid⁄ base catalyst to

protonate the dithiolane part of lipoamide Two

dif-ferent mechanisms were envisioned to explain the

cat-alytic role of a proton donor At first, the ThDP

enamine might attack at one of the sulfurs to form

the tetrahedral adduct, and the free thiolate anion

would then be protonated by a proximal proton

source Alternatively, LipS2 could be protonated in a

pre-equilibrium to give a highly reactive thiolanium

cation LipS2H+, followed by nucleophilic attack by

the enamine and concomitant cleavage of the disulfide

bond An important factor concerning the latter

mechanism is the low pKa of the thiolanium cation,

so that only minor fractions will be present in

equili-brium under physiological conditions

Mechanistic analysis of reductive acetylation

in PDHc

A key question related to the mechanism of reductive

acetylation in PDHc is whether enzyme-bound

dihydrolipoamide is acetylated at S6 or S8 by the

HEThDP enamine It has been impossible, thus far, to

directly test the two alternatives or to disprove one of

them for the reconstituted multi-enzyme complex;

however, elaborate studies conducted by Frey et al on

the E2-catalyzed acetylation of free dihydrolipoamide

by acetyl-CoA clearly revealed formation of the 8-S

isomer, followed by non-enzymatic isomerization and

formation of the 6-S isomer [35] By invoking the

prin-ciple of microscopic reversibility,

8-(S)-acetyl-dihydrolipoylamide is the chemically (and kinetically)

competent isomer for the physiologically relevant

for-ward reaction of E2 (i.e the formation of acetyl-CoA)

and this must be formed in the preceding reductive

acetylation at E1

A further compelling question concerns a possible

coupling of oxidation–reduction and acyl group

trans-fer In principle, the two elementary reactions of

reduc-tive acetylation could occur simultaneously in a

tightly-coupled mechanism or, alternatively, in a

step-wise manner Both mechanisms would involve the

tetrahedral adduct between reduced lipoamide and

AcThDP; however, AcThDP would be a compulsory

on-pathway intermediate only in the stepwise

mecha-nism Frey et al could isolate AcThDP in the steady

state of the overall reaction of PDHc by acid quench trapping [36] This finding is consistent with a stepwise mechanism of oxidation–reduction and acyl group transfer; however, it cannot disprove a coupled mecha-nism because AcThDP could be generated from the tetrahedral thiamin-lipoamide adduct in an equilibrium side reaction

Further support for a stepwise mechanism comes from the observation that E1-bound AcThDP (formed

by enzymic conversion of 3-flouropyruvate) is a chemi-cally competent acyl group donor to externally added dihydrolipoamide [37] In search of putative free radi-cal intermediates that could be transiently formed in the course of sequential one-electron transfer from the enamine to oxidized lipoyl-lysine, a p-type HEThDP radical could be detected by EPR spectroscopic studies

on PDHc from different organisms [13,38] There was, however, no spectroscopic evidence for a correspond-ing lipoamide sulfur-centered thiyl radical, and the for-mation of the ThDP-based radical appeared to result from an oxygenase side reaction of the HEThDP enamine with dioxygen rather than from on-pathway oxidation by lipoamide

X-ray crystallographic studies on E1 from different dehydrogenase complexes have provided the structural framework for our mechanistic understanding of reductive acetylation and active site coupling between E1 and E2 [39,40] The active center of E1 with the ThDP cofactor is located at the bottom of a long fun-nel-shaped substrate channel, which is suitable orga-nized to accommodate a flexible E2-linked lipoamide swinging arm for chemical coupling and intermediate channeling As noted earlier above, reductive acetyla-tion is likely to involve acid⁄ base chemistry from the protein and⁄ or ThDP cofactor General acid catalysis

is required for protonation of the lipoamide disulfide, and a general base must be involved to deprotonate the a-OH of the HEThDP enamine Structural and functional data implicate highly-conserved His side chains at E1 to fulfil this function Some of the His residues such as His407 in E coli E1 are located in loops that are flexible in the resting state but become ordered upon binding and processing of pyruvate [41,42] A probable (and partially modeled) structural snapshot of catalysis showing E2-bound lipoamide prior to reaction with the planar HEThDP enamine intermediate (atomic coordinates of HEThDP enamine taken from POX) at the active center of E1 from

E coli is illustrated in Fig 4 The lipoamide molecule was modeled into the substrate channel of E1 such that (a) formation of 8-(S)-acetyl-dihydrolipoylamide

is more likely than of the 6-S isomer and (b) protonation of the lipoamide dithiolane by His407

may occur (on the basis of structural considerations

and previously available functional data [41]) The

resultant model invokes active center residue His640 to

be important for deprotonation of the a-OH of the

HEThDP enamine

Reaction mechanism of acetyl

phosphate-producing pyruvate oxidases

Chemical considerations of acetyl phosphate

formation by pyruvate oxidases

In phosphate-dependent pyruvate oxidases, such as

that from L plantarum (LpPOX), thermodynamically

favorable oxidation of pyruvate is coupled to

forma-tion of the ‘energy-rich’ metabolite acetyl phosphate

that carries an acid anhydride linkage [10] Owing to

its high group transfer potential, acetyl phosphate

may undergo favorable phosphotransfer to ADP to

give ATP, a process that is catalyzed by the enzyme

acetate kinase [19] Besides ThDP and a divalent

cat-ion (Mg2+) required for anchoring the former,

pyru-vate oxidases contain FAD as an additional cofactor,

of which the apparent catalytic role is to oxidize the

HEThDP carbanion⁄ enamine formed after binding

and decarboxylation of pyruvate at the thiamin site

Two-electron oxidation of the HEThDP

carban-ion⁄ enamine by FAD gives AcThDP, an intermediate

that was initially suspected to be highly unstable but,

in chemical models (AcThDP and 2-acetyl-thiazolium

salts), was shown to be relatively stable at low hydroxide ion concentrations or in the absence of trapping nucleophiles [16,43] Water and other less basic nucleophiles, such as the phosphate dianion, were demonstrated to add to 2-acetyl-thiazolium salts and result in tetrahedral adducts; however, only the water adduct underwent further decomposition to acetate, whereas reactions with phosphate gave back the starting material rather than acylated phosphates [43] This is because phosphate is a better leaving group than the thiazolium ylid, and electron donation

to C2 in the tetrahedral phospho adduct is not as extensive as in the water adduct to enable expulsion

of the thiazolium ylide In the case of AcThDP, phosphate did not even form a tetrahedral addition compound [16] Conclusively, these model studies indicate that acetyl phosphate-producing pyruvate oxidase may not utilize simple oxidation–reduction chemistry followed by acyl transfer to phosphate Besides overcoming the large barrier for expelling acetyl phosphate from the tetrahedral phospho adduct, the enzyme must also suppress hydrolytic cleavage of the presumed AcThDP intermediate to avoid decoupling of oxidation and acid anhydride bond formation

Molecular architecture of phosphate-dependent pyruvate oxidases and implications for electron transfer

The X-ray crystal structure of the homotetrameric LpPOX was solved by Muller and Schulz [44] in the early 1990s and serves as the structural prototype for acetyl phosphate-producing POXs As in all ThDP-dependent enzymes that have been structurally charac-terized to date, the active site is located at the interface

of two corresponding subunits constituting the cata-lytic dimer (Fig 5) The thiazolium of ThDP and the redox active isoalloxazine of FAD are bound at approximately 7 A˚ edge-to-edge distance, with the dimethylbenzene part of FAD pointing directly towards the thiazolium The flavin isoalloxazine is markedly bent over the N5–N10 axis ( 10–15), which is a structural feature that increases the driving force of oxidation–reduction because the distorted con-formation resembles the reduced state of the flavin, thus increasing its oxidizing power The widely-accepted theoretical framework for biological electron transfer (Dutton’s ruler) predicts that pure electron (quantum-chemical) tunneling between both cofac-tors in POX would occur extremely rapidly (ktheo 108s)1) when assuming ‘normal’ free and reorganization energies [25]; however, the packing

Fig 4 Putative structure of E2-bound lipoamide attacking the

HET-hDP enamine at the active center of E1 from E coli in stereo view.

A lipoamide molecule in the oxidized state was modeled into the

substrate channel of PDHc-E1 from E coli (Protein Data Bank code:

2g25), thus representing the catalytic state prior to reductive

acety-lation To adequately illustrate the confinements of the substrate

channel, the protein is shown in surface representation with a

sliced active center pocket and substrate funnel The HEThDP

enamine (modeled according to [47]) and selected His residues

implicated as participating in general acid ⁄ base catalysis are shown

in a stick representation.

density (i.e a measure of the volume in the

inter-reac-tant space occupied by protein atoms) between the

thiazolium and the isoalloxazine is very small so that

electron transfer would mostly occur as through-space

tunneling Because of this structural observation, it has

been alternatively suggested that electron tunneling

might involve the side chains of two Phe residues

(Phe479 and Phe121) contributed by different

mono-mers as way stations for electron transfer in a

com-bined through-space⁄ through-bond mechanism [44] In

support of this proposal, an arginine side chain sitting

atop Phe479 could partially offset the transiently

formed negative charge at the phenyl ring These

dif-ferent possible modes notwithstanding, theoretical

treatment of oxidation–reduction between the

HET-hDP enamine and FAD might not be as

straightfor-ward as in other systems because electron transfer in

POX is definitely coupled to proton transfer (i.e

deprotonation of C2a-OH of HEThDP and

proton-ation of FAD at N5 and N1) The tight and rigid

binding of both cofactors excludes a direct carbanion

mechanism with covalent linkage between C2a of the

HEThDP enamine and C4a of FAD, as suggested for

FAD-catalyzed oxidation of other organic substrates

(e.g amino acids)

Besides the different hydrophobic active center

resi-dues considered above as being involved in electron

transfer, there are a few polar side chains (Glu, Gln)

in close vicinity to the ThDP cofactor, which are likely

to play important roles for catalysis and binding of

phosphate This initially premature functional

assign-ment has been corroborated by kinetic and structural

analysis of different LpPOX variants (G Wille and

K Tittmann, unpublished results)

Kinetic and spectroscopic analysis of oxidation–reduction in pyruvate oxidase

As considered above, the structural confinements of the active site and the long distance between the two cofactors ( 11 A˚ between ThDP-C2 and FAD-N5 or FAD-C4a) relegate a direct carbanion mechanism with covalent HEThDP-FAD linkage or an alternative hydride transfer mechanism to minor probability Fur-ther experimental evidence arguing against a hydride transfer mechanism comes from the finding that no oxidation–reduction can be detected in LpPOX recon-stituted with 5-deaza-5-carba-FAD, which is a FAD analogue that functions as a good hydride acceptor but does not catalyze single electron transfer [45] Fur-thermore, FAD reduction kinetics exhibits no kinetic solvent isotope effect Conclusively, two-electron reduction of the flavin by the HEThDP carban-ion⁄ enamine should take place in two sequential one-electron transfer steps coupled to proton transfer Stopped-flow kinetics and spectroscopic analysis of the reductive half-reaction (i.e single turnover reduction

of the flavin under anaerobic conditions) could not demonstrate transient formation of flavin radicals [45,46] Two-electron reduction of the flavin occurred with a kobs of approximately 102s)1 at saturating pyruvate concentrations The inability to observe radi-cal intermediates cannot, however, rule out a two-step sequential electron transfer mechanism because a kinetic stabilization of radical intermediates requires the transfer of the second electron (kred2) to proceed at

a comparable rate or slower than that which occurs for the first electron (kred1) No transient radicals will

be kinetically stabilized when kred2» kred1 Initial

Fig 5 Stereo drawing of the active site of

pyruvate oxidase from L plantarum (Protein

Data Bank code: 1pox) showing the

cofac-tors ThDP and FAD and selected proximal

amino acid residues The amino acid

resi-dues contributed by the corresponding

subunits are colored individually (green or

pink) The two Phe residues suggested to

be involved in electron transfer are

indicated.