title: 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 alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H 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 substrate-ThDP 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) (E1 = -37 mV) and FAD(sq)/FAD(red) (E2 = -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 phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.
authors.affiliation: Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany. kai@bc.biochemtech.uni-halle.de
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.forename: Georg
authors.initials: G
authors.lastname: Wille
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Annett
authors.initials: A
authors.lastname: Weidner
authors.forename: Sandro
authors.initials: S
authors.lastname: Ghisla
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hübner
mesh_terms: D002384:Catalysis; D004583:Electrons; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005609:Free Radicals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D015233:Models, Statistical; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011771:Pyruvate Oxidase / Q000737:chemistry*; D019289:Pyruvic Acid / Q000737:chemistry; D012997:Solvents; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D013997:Time Factors
chemical_list: D005415:Flavins; D005609:Free Radicals; D010710:Phosphates; D012997:Solvents; D005182:Flavin-Adenine Dinucleotide; C047467:2-(1-hydroxyethyl)thiamine pyrophosphate; D019289:Pyruvic Acid; D011771:Pyruvate Oxidase; D013835:Thiamine Pyrophosphate; D010100:Oxygen