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 ...

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)

Titration of native methionyl‐tRNA synthetase from Escherichia coli with tRNAfMet and tRNAmMet leads to 37% and 38% quenching of the enzyme fluorescence respectively. The quenching curves for the two tRNAs are not additive. Analysis of the binding data indicates the presence of a single site for tRNA per mole enzyme, with a three‐fold higher affinity for tRNAmMet. The tRNA binding parameters do not vary significantly in the presence of a saturating concentration of methionine. On the other hand, titration of the enzyme · methioninyl‐adenylate complex (two moles of the ligand bound/mole enzyme) with tRNAfMet and tRNAmMet, leads to an extensive decrease of fluorescence, from an initial level of 134% to 40% and 35%, respectively, indicative of a major structural or conformational change of the enzyme. Analysis of the quenching curves reveals two distinct binding processes, corresponding to occupation of two tRNA sites on the enzyme. Requirements for optimal interaction between enzyme and tRNA were investigated. Magnesium ions are essential for complex formation. Maximal interaction is attained at 1 mM and 4 mM MgCl2, respectively, for tRNAmMet and tRNAfMet. However, close to 40 mM MgCl2 are required to reach equivalence of the two tRNA sites which are available in the presence of methioninyl adenylate. Both spermine and spermidine are able to simulate the role of magnesium in the binding of tRNAMet to the free enzyme, but not in the occupation of the second tRNA site on the enzyme · methioninyl‐adenylate complex. Addition of KCl results in a decrease of the fluorescence quenching produced by tRNA and in a lowering of the affinity for tRNA, both in the presence of MgCl2 and spermidine. Essentially similar quenching levels and affinities are obtained when titration experiments are performed in imidazole‐HCl or Tris‐HCl buffer in the pH range of 6.3 to 8.7. In contrast, in potassium phosphate buffer which inhibits the enzyme‐tRNA interaction, raising the pH leads to a progressive decrease of the fluorescence quenching caused by tRNA, with concomitant lowering of its affinity for the enzyme. For comparative purposes the interaction of tRNAMet with trypsin‐modified methionyl‐tRNA synthetase was investigated. Analysis of the binding data indicates the presence of a single tRNA site per mole enzyme, even in the presence of saturating amounts of methioninyl adenylate (one mole of this ligand bound per mole enzyme). Furthermore, the magnesium ion concentration required to reach maximal quenching is the same (i.e. 4 mM) in the presence or absence of methionyl adenylate.

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