Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum. Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

Abstract

The thiamin diphosphate (ThDP)-and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of R-hydroxyethylThDP to the adjacent flavin cofactor over a distance of approximately 7 Å, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1 H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s -1 and is kinetically gated by the preceding formation of the tetrahedral substrateThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s -1 and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD ox /FAD sq (E 1 ) -37 mV) and FAD sq /FAD red (E 2 ) -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3′-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10 2 -10 3 -fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosporolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.Pyruvate oxidase from Lactobacillus plantarum (LpPOX, 1 EC 1.2.3.3) belongs to a superfamily of enzymes that utilize the cofactor thiamin diphosphate (ThDP), the biologically active derivative of vitamin B 1 . In addition to ThDP, LpPOX contains a flavin adenine dinucleotide (FAD) that is positioned at a distance of approximately 7 Å from the thiamin cofactor (Figure 1) (1).Pyruvate oxidase (POX) catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to the high-energy metabolite acetyl phosphate, carbon dioxide, and hydrogen peroxide (2). Catalysis can be assumed to follow the typical Breslow mechanism of ThDP enzymes (Scheme 1). After ionization of the acidic C2-H of the thiazolium ring, pyruvate enters the active site where it reacts with the C2

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

“…In contrast, the side chain of F 465 in EcPOX points away from the active site with a displacement of Ϸ6 Å compared with F 479 in LpPOX. As the estimated rate constants of ET between the thiamin and flavin cofactor differ so substantially (k obs Ϸ3 s Ϫ1 in EcPOX and Ϸ400 s Ϫ1 in LpPOX) (19,37,38), the orientation of this Phe residue might be central to controlling the rate of inter-cofactor ET.…”

EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase

“…One-electron oxidation of such a radical anion would lead to formation of acetyl-CoA and release the second reducing equivalent into the electron transfer chain (16). An analogous scenario involving a radical anion intermediate has been suggested for the reaction of POX from Lactobacillus plantarum where P i is the second substrate and acetyl-phosphate is the ultimate product (37). Equally plausible is CoA-(P i -) gated oxidation of the HE-TPP radical, in which binding of a negatively charged thiolate (phosphate) in the active site could trigger the second electron transfer step in PFOR (POX) (16).…”

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

“…15 This potential is significantly more negative than that for the HE-TPP radical/acetyl-TPP couple, which has been estimated to be -487 mV. 15 This enhanced reducing power would provide a strong driving force for reduction of cluster A (E 0 = -540 mV). Therefore, this kinetic coupling mechanism would allow the protein to generate a stable radical that only undergoes electron transfer when the co-substrate is present.…”

“…4,15 For PFOR, the kinetic coupling mechanism would generate an anion radical adduct between CoA and the HE-TPP radical, which is estimated to have a negative redox potential of about -0.76 V (vs NHE). 15 This potential is significantly more negative than that for the HE-TPP radical/acetyl-TPP couple, which has been estimated to be -487 mV. 15 This enhanced reducing power would provide a strong driving force for reduction of cluster A (E 0 = -540 mV).…”

“…This scenario would facilitate the next electron-transfer reaction. However, in both PFOR 4 and PO, 15 the HE-TPP radical is relatively stable unless their co-substrates, CoA (PFOR) or phosphate (PO), are present. The rate enhancement by the co-substrate in each of the two systems indicates a similar mechanism for enhancing the reactivity of the radical in PFOR and PO.…”