Flavocytochrome b2 (Baker's Yeast). Deuterium Isotope Effect Studied by Rapid-Kinetic Methods as a Probe for the Mechanism of Electron Transfer

Abstract

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 'C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J . Biochem. 46, 393-3991. We report here studies carried out at 5°C with 1~-[2-~H]lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the a-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme.The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillere-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J . Biochem. 54, 549-5661, An alternative model based on computer-simulation studies will be presented in a paper in this journal. . This result suggested that the rate-determining step was more precisely the abstraction of the a-hydrogen, before electron transfer to flavin. Since, however, the magnitude of an isotope effect depends on the extent of proton transfer in the transition state [3], a value of 5 did not preclude that some other step was partially rate-limiting; consequently it appeared of interest to carry out a study of prosthetic group reduction by deuterolac-* Deceased.Enzyme. L-Lactate : cytochrome c oxidoreductase (EC 1 .I .2.3).tate, using rapid kinetic methods. This study is the subject of the present paper. Design of the experiments and their interpretation were facilitated by a recent investigation which led the authors to propose a detailed kinetic scheme for the series of electron transfers leading from oxidized enzyme and lactate to fully reduced enzyme and pyruvate [4,5]. While that investigation provided guidelines for the present work, the use of [2-2H]lactate conversely appeared to offer a possible means of verifying the scheme since the replacement of hydrogen by deuterium would selectively modify the rate of certain steps.The present study was carried out with L -[~-~H ] -lactate, instead of the racemic lactate which had been used previously [2]. Moreover, the behaviour of both forms of flavocytochrome 62 was examined. The intact enzyme and the cleaved one differ by a number of steady-state kinetic parameters [6,7]. In particular, the intact enzyme specific activity is 2.5-fold higher than that of the cleaved one [7], the substrate K, is about 3-fold smaller ...

Probing the active site of flavocytochrome b2 by site‐directed mutagenesis

“…In fact k,,, is reduced almost 50-fold (Table 1) whereas the K,,, for lactate is identical within experimental error in the wildtype and mutant enzymes. For the wild-type enzyme the steady-state K,,, value is believed to be close to that of K, [8]. The results therefore suggest at first sight that Tyr2" is important not in substrate binding but in transition-state stabilization, i. e. it makes no interaction with the substrate hydroxyl group in the Michaelis complex.…”

“…e) Ala306 is located some distance from the active site for lactate oxidation within a disordered loop region. The mobility of this region appears to be important for substrate oxidation [8].…”

“…e) Ala306 is located some distance from the active site for lactate oxidation within a disordered loop region. The mobility of this region appears to be important for substrate oxidation [8].In this paper we describe the generation of four mutant forms of flavocytochrome b2 by site-directed mutagenesis and the kinetic properties of these enzymes with particular regard to lactate oxidation. …”

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

“…Substrate solutions were prepared in Tris/HCl buffer, and titrated with 0.25 M NaOH to pH 7.5 before use; they contained glycollic acid (Aldrich), L-lactate (lithium salt; Sigma) and the following enantiomerically pure long-chain 2- Kinetic isotope effects (KIEs) were measured as previously reported [13], using L-[2-2H]lactate prepared as described in [14]. The flavin-binding domain of flavocytochrome b2 is closely related to several other 2-hydroxy acid dehydrogenases with different substrate specificities.…”

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

“…This question has been discussed before (Urban & Lederer, 1985;Balme & Lederer, 1994). On the basis of our measurements of the deuterium isotope effect in the forward and the reverse reaction, and of the discrimination against tritium in the forward reaction (Pompon et al, 1980), it is reasonable to assume that the discrimination factor against tritium could be maximal, Le.,[17][18][19][20] If there were no discrimination at all in the exchange reaction, the calculated pK, would be higher by log 20, i.e., higher by 1.3 pH unit. This can be considered as an extreme and unlikely case.…”