On the Transhydrogenase Activity of Baker's Yeast Flavocytochrome b₂

Abstract

It is shown that, when baker's yeast flavocytochrome b, is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction.It is further shown that reduced flavocytochrome b, can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control.Determinations of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the a-hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion.It was recently demonstrated that bromopyruvate inactivates flavocytochrome b, (or L-lactate dehydrogenase) from baker's yeast in a reaction which displays the kinetic characteristics of affinity labeling [I -31. Flavocytochrome b, is known under two forms, the intact one and the nicked form, both of which are tetramers and possess one heme and one FMN per subunit. They differ in a number of physicochemical properties [4] ; in particular, significant differences were demonstrated between the two forms with respect to substrateenzyme interactions and flavin reduction rate [4-61. Bromopyruvate inactivated both enzyme forms in the oxidized state [I]. In the reduced state, the nicked enzyme appeared completely protected from inactivation while the intact form was still inactivated, but more slowly, because lactate acted as a competitive inhibitor [l]. The labeling stoichiometry and the localization of modified residues were studied with the nicked enzyme only [2,3]. Further studies with intact flavocytochrome h,, however, showed an unexpected destruction of bromopyruvate when it was incubated with the enzyme in the presence of lactate as reducing substrate. The reaction was finally ascribed to a flavocytochrome-b,-catalyzed transhydrogenation reaction between lactate and bromopyruvate 171. We report in this paper the full details concerning this reaction, which was also studied using other hydroxy acidlketo acid couples, in particular fluoro and chloro substrates. We provide evidence that the transhydrogenation reaction is under thermodynamic control and bypasses the heme cofactor. For the sake of convenience, these studies were carried out with the nicked enzyme, since it is nearly completely resistant to inactivation by bromopyruvate when reduced. MATERIALS AND METHODS EnzymeNicked enzyme was prepared from lyophilized commercial baker's yeast according to [S]. It was stored as an ammonium sulfate precipitate, and working solutions were prepared in the standard buffer (0.1 M N a + / K...

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

“…The reduction of fluoropyruvate at the expense of 2-hydroxy-3-butynoate was followed spectrophotometrically by monitoring at 3 15 nm the formation of the glutathione-ketobutynoate adduct, as described before (Pompon & Lederer, 1982;Urban et al, 1983). The method was modified by the addition of an oxygen scavenging system (Whittaker et al, 1990;Harpel et al, 1993;Cross et al, 1994).…”

“…The column was eluted with a linear gradient of formic acid (0 to 0.8 M, 800 mL total volume). The elution profile was monitored by measuring total cpm by liquid scintillation counting (LSC 3000, Hewlett-Packard) and determining the amount of fluorolactate formed by titration with excess ferricyanide in the presence of flavocytochrome b2 (Urban et al, 1983). The latter determination is used for calculating the extent of conversion of fluoropyruvate to fluorolactate and make sure that the reaction was stopped well before reaching equilibrium.…”

“…As a consequence of the high isotope discrimination factor (Urban et al, 1983;Rouvikre-Fourmy et al, 1994), the residual fluorolactate would become enriched in tritium but, nevertheless, some isotope would be lost to the solvent and the bulk radioactivity in the fluorolactate fraction would decrease; this would artifactually decrease the calculated value of r . There is an approximate 60 mV difference between the midpoint redox potentials of the lactate/pyruvate and fluorolactate/fluoropyruvate couples (-190 mV and --130 mV, respectively) (Urban et al, 1983 100 at equilibrium. The experimental ratios at the end of the incubations were lying between 0.2 and less than 10, except in two cases (15 and 28% for 15 mM initial fluoropyruvate in 0.2 and 0.5 M buffer, respectively).…”

“…Kinetic parameters can be determined for the reverse reaction by using saturating lactate concentrations and varying concentrations of the keto acid substrate, such as a halogenopyruvate (Urban et al, 1983). Furthermore, when [2-'Hllactate is used, the tritium liberated by the substrate is found both in the solvent and in the hydroxy acid product of the reverse reaction.…”

Inactivation of flavocytochrome b2 with fluoropyruvate

“…The reaction was observed during studies of the transhydrogenation reaction catalyzed by the enzyme, using fluoropyruvate as a substrate [6, 15,161. Considering the well known inertness of the C-F bond to nucleophilic substitution [17], it was thought worthwhile to study this unexpected reaction.…”