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

Abstract

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)

(Photo)chemistry of 5‐Deazaflavin

“…(12) Apart from the initial breakage of a substrate R-H bond, the dissociation of the product R + from the flavin as well as the reshuffling of the active site required for radical stabilization and, concomitantly, flavin-acceptor e-transfer might all be partially ratelimiting. This would explain the sequence of steps as, for example, found recently for flavocytochrome bz from baker's yeast [51,52].…”

Flavocytochrome b2: Simulation Studies of the Electron‐Transfer Reactions among the Prosthetic Groups

“…2C). k , , values lower than 400 s-' are irrelevant: they imply a lag time in the Hred and Fl,, simulated time courses higher than 1 ms which is not experimentally detected [1,8]. In the 4OO-sC' and 5O0-sC1 range of k + , , the lag time becomes lower than 1 ms, but the simulated curves are still not consistent with the experimental ones: a close agreement is only obtained for k + , values higher than 500 s-'.…”

“…Since this step is the initial limiting one, it controls all the following ones [6] (Table 1). Due to the large difference in the redox potentials of the substrate and the enzyme systems (140 mV [1,7]) this principal step of lactate dehydrogenation is considered as irreversible.…”

Cellobiose oxidase from Phanerochaete chrysosporium Stopped‐flow spectrophotometric analysis of pH‐dependent reduction

“…This observation indicates that the electron transfer mechanism for cellobiose oxidation by CBO could be rather complicated. This could involve inter-molecular electron transfer between CBO molecules, as is the case in yeast flavocytochrome b2 [12]. Further experiments are still ongoing to elucidate the electron transfer mechanisms in CBO in more detail.…”