Flavocytochrome b 2 : Simulation Studies of the Electron‐Transfer Reactions among the Prosthetic Groups

102

The reduction by L-lactate of the prosthetic groups of flavocytochrome b2 (L-lactate cytochrome c oxidoreductase from aerobic yeast, a tetrameric molecule containing one haem and one flavin mononucleotide per protomer) was reinvestigated. It was confirmed that the enzyme ultimately takes up 3 electrons per protomer from this 2-electron donor. Stopped-flow absorbance data at an haem isosbestic point to follow the oxidized flavin and in a haem band indicate that, under the conditions used, haem and flavin reduction time courses are indistinguishable, both being biphasic (phases I and 11). Comparison with electron paramagnetic resonance data (Fe3 + haem and flavosemiquinone signals) led to a complete description at 24 "C of the time courses of the various reduction states of the prosthetic groups. It has been previously demonstrated (Morton and Sturtevant, 1964) that, after the formation of the enzyme-substrate complex, the electron transfer to the enzyme takes place as the first and rate-limiting step of the turnover.In the present study, an initial burst of fully reduced flavin, of small amplitude, is detected at the very beginning of phase I (before 6 ms). The redox forms which accumulate thereafter till the end of phase I (30-35 ms) are the reduced haem (up to 80 %), the flavin semiquinone (up to 50 %) and the fully reduced flavin (from 25 % up to 35 %); the total of electrons distributed at the end of phase I is about 2 per protomer meaning that, in this phase, each enzyme site acts as a 2-electron and not a 3-electron acceptor. A 2-electron flow as the limiting step during phase I with the rate constant k, accounts for the steady-state electron flow during catalysis. Phase I is followed by the much slower phase 11 which corresponds to the entry of the third electron and cannot be involved in the turnover.The interpretation of the results are given as a scheme, with the proper rate constants, allowing a satisfactory fitting of experimental data by simulation. Among the elementary steps required are a rapid distribution of one electron from reduced flavin to the haem, a rapid interprotomers dismutation between couples of flavin semiquinone regenerating two oxidized flavin per tetramer. The very low reactivity of the latter for the entry of the third electron per protomer is tentatively explained by the occurrence of a slow additional step limiting the final reduction reaction.It was observed that, over phase I and the beginning of phase 11, from 15 to 200 ms, all the redox species remain apparently under equilibrium conditions. Parallel studies (titrations of flavocytochrome b2 by L-lactate) showed that the set of equilibrium parameters relative to haem and flavin species is significantly different in the "final" equilibrium (after 30 s) from that in the time interval 15 -200 ms. Such an anomaly suggests a conformation change takes place very slowly in the molecule after the acceptance of the first two electrons per protomer. Eur. J. Biochem. 54 (1975)

Section: The Entry Of the Third Electron Is Very Slowmentioning

confidence: 99%

“…7. A detailed critical presentation of the simulation will be given in a following paper [50]. The kinetic arguments taken into consideration are standard ones in the theory of multi step sequential reversible processes (see reaction scheme) [55].…”

Section: Comments On The"simp1est" Reaction Scheme Proposedmentioning

confidence: 99%

Flavocytochrome b₂: Kinetic Studies by Absorbance and Electron‐Paramagnetic‐Resonance Spectroscopy of Electron Distribution among Prosthetic Groups

Capeilléere-Blandin

Iwatsubo

Labeyrie

et al. 1975

102

“…It has been shown that in the tetramer there are privileged heme-flavin pairs, between which electron transfer is rapid [22, Electron transfer between any member of one pair to any member of another pair is supposed to be slow, with rates lower than 10 s-' [23]. The rate constant for lactate to flavin transfer, which is the slow step in the forward reaction, was estimated by simulation studies to be 120 s-' at 24 "C [20,22]. Furthermore, the measured k,,, for the reverse reaction found in this work is 3.6 s -I ; this low rate cannot be due to slow bromolactate dissociation, for the following reason.…”

Section: Oxidation State Of the Heme During Turnovermentioning

confidence: 99%

On the Transhydrogenase Activity of Baker's Yeast Flavocytochrome b₂

Urban

Lederer

Alliel³

1983

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...

“…New kinetic schemes capable of explaining data obtained with deuterated lactate are proposed. These new schemes differ from that of Capeillere-Blandin in that : (a) the hypothesis of simultaneous prosthetic group reduction for the two protomers of one given dimer is abandoned; (b) the limiting step of the slow phases of heme and flavin reduction is a slow interprotomer electron exchange between a heme pair, a flavin pair or heme and flavin; (c) a rather fast conformational change controlled by the redox state of heme or flavin of one protomer can modulate the rate of electron transfer in another protomer.These new kinetic schemes allow us to determine the rate of intraprotomer and interprotomer electron transfer and to decide precisely how these steps are modified by the proteolytic cleavage of 'intact' enzyme to 'cleaved' enzyme.The mechanism of electron transfers in flavocytochrome h2 was reinvestigated by Capeillere-Blandin et al [1,2] who proposed a 'minimum' mechanism capable of explaining with sufficient accuracy all data available at the time. The proposed mechanism includes three kinds of electron transfer: substrate, i.e.…”

mentioning

confidence: 99%

“…The mechanism of electron transfers in flavocytochrome h2 was reinvestigated by Capeillere-Blandin et al [1,2] who proposed a 'minimum' mechanism capable of explaining with sufficient accuracy all data available at the time. The proposed mechanism includes three kinds of electron transfer: substrate, i.e.…”

mentioning

confidence: 99%

Flavocytochrome b₂ from Baker's Yeast

Pompon

1980

It has been shown recently that the use of ~-(+)-[2-~H]lactate as substrate instead of unlabeled L-(+)-lactate induces a lowering of the flavin reduction rate of cytochrome b2 by a factor of 8 [D. Pompon, M. Iwatsubo, and F. Lederer (1980) Eur. J. Biochem. 104,479-4881. This high isotope effect enabled us to study electron transfer between prosthetic groups at a very low rate of electron entry. The kinetic scheme for electron transfer in cytochrome h2 proposed by Capeillere-Blandin [Eur. J . Biochem. 56, 91 -101 (1975)l is examined here in the light of the kinetic data reported in our preceding paper. This study indicates some disagreements, particularly at a low rate of electron entry. New kinetic schemes capable of explaining data obtained with deuterated lactate are proposed. These new schemes differ from that of Capeillere-Blandin in that : (a) the hypothesis of simultaneous prosthetic group reduction for the two protomers of one given dimer is abandoned; (b) the limiting step of the slow phases of heme and flavin reduction is a slow interprotomer electron exchange between a heme pair, a flavin pair or heme and flavin; (c) a rather fast conformational change controlled by the redox state of heme or flavin of one protomer can modulate the rate of electron transfer in another protomer.These new kinetic schemes allow us to determine the rate of intraprotomer and interprotomer electron transfer and to decide precisely how these steps are modified by the proteolytic cleavage of 'intact' enzyme to 'cleaved' enzyme.The mechanism of electron transfers in flavocytochrome h2 was reinvestigated by Capeillere-Blandin et al. [1,2] who proposed a 'minimum' mechanism capable of explaining with sufficient accuracy all data available at the time. The proposed mechanism includes three kinds of electron transfer: substrate, i.e. L-(+)-lactate, to FMN, FMN to protoheme IX of the same protomer and FMN to FMN between two protomers. Steady-state kinetic studies [4,5] and stoppedflow experiments [2,3] show the main limiting step of flavin and heme reduction to be at the level of electron transfers between substrate and flavin. We have pointed out in our preceding paper [ 3 ] that the use of ~-(+)-[2-'H]lactate instead of the normal un, labeled L-(+)-lactate as substrate causes a strong decrease in the rate of this first step. This high isotope effect allowed us to study flavin and heme reduction at an electron input rate lower that the rate of the limiting step of the slow phase proposed by CapeillereBlandin.