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)

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Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b,) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements.In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18°C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.Flavoenzymes differ from other redox proteins of oxidative chains in that their prosthetic group does not shuttle between two redox states, as is the case of NAD(P)H, ironsulfur centers and iron porphyrin, but between three redox states: oxidized, semiquinone and fully reduced, i. e. hydroquinone. These three states, as underlined by Hemmerich and Massey 111, are not all involved in the normal mechanism of each flavoprotein; they are thus in the class of dehydrogenases/one-electron transferases in which the coupling between 2e-donors and le-acceptors requires the active role of the semiquinone intermediate in the r e d o n sequence during turnover.We suggest that a general procedure of feedback control in dehydrogenases/le-transferases might involve modulation of the redox parameters defining equilibrium proportions of the three flavin states by preferential binding of the reaction product to one of them. Indeed, as shown in the present paper, we observed in a yeast mitochondria1 flavocytochrome enzyme, L-lactate : cytochrome c oxidoreductase, that pyruvate, the reaction product, binds preferentially to the semiquinone form with concomitant large shifts of the midpoint potentials corresponding to the two one-electron flavin couples. This leads to a strong increase in the level of flavosemiquinone and inhibition of activity. This effect of pyruvate on the equilibrium proportion of flavin semiquinone and also on individual rate constants in the intramolecular electron exchange was first detected in the course of a temperature-jump investigation in collaboration with Brunori and Silvestrini [2].Two other similar observations have been previously reported concerning the behaviour of succinate dehydrogenase in the presence of oxaloacetate [3, 41 and of NADH:cytochrome b, reductase in the presence of NAD' [S, 61. In these flavoenzymes, the 'product' binding ...

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