Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies

Abstract

The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques. The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-[2H]lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested. The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited. Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate. In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II. Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide. These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described. Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor). The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion. The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C. Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide. Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide. When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, [2H]lactate and ferricyanide concentrations. Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2. The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

“…This is in marked contrast with the results of previous work on wild-type enzyme in which biphasic kinetics were observed for FMN reduction, at least at high lactate concentrations [12,[15][16][17]. For wild-type enzyme, it is known that the rapid phase corresponds to initial reduction of FMN and haem (with the slow phase being kinetically irrelevant in catalytic turnover of the enzyme) [12,[15][16][17]. The effect of the hinge-swap mutation on the rate of FMN reduction is small, with kcat for the mutant enzyme being only 2-to 3-fold lower than that for the wild-type enzyme.…”

“…A number of workers have previously described the origin of this biphasic behaviour which arises because each flavocytochrome b2 protomer requires three electrons for full reduction (i.e. each tetramer requires 12 electrons from six lactate molecules for full reduction) [12,[15][16][17]. These workers have shown that the rapid phase corresponds to the initial reduction of flavin and haem groups and the slow phase (which is kinetically irrelevant during catalytic turnover of the enzyme) corresponds to the entry of the third electron which is permitted because of interprotomer electron transfer [12,[15][16][17].…”

Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

“…The reduction of the FMN and haem groups of wild-type and Y143F flavocytochromes b2 by L-lactate and L-[2-2H]lactate was monitored by stopped-flow spectrophotometry as described in the Materials and methods section. As noted in previous work [10,17,18], at high lactate concentrations biphasic traces were observed with the rapid phase corresponding to at least 80 % of the absorbance change (monophasic traces were observed at low lactate concentrations, as described in the Materials and methods section). It has been well documented that the rapid phase corresponds to the initial reduction of FMN and haem groups by one L-lactate molecule, whereas the slow phase corresponds to entry of a third electron into the flavocytochrome b2 promoter [10,18,19].…”

Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

“…characteristics of the isolated flavocytochrome b2 flavin domain and the intact, native enzyme is seen in stopped-flow experiments. FMN in the isolated domain is reduced as a single exponential process, as found also in the dehaemo-enzyme [14], whereas FMN reduction in the intact holoenzyme is biphasic [15,16,18,19]. The slow phase has been attributed to an intramolecular redistribution of electrons after haem reduction (Figure 3a).…”

“…Although monophasic kinetics were observed for fully active flavin domain we found that enzyme which had lost activity exhibited biphasic behaviour; however, this is most likely due to heterogeneity in the enzyme. The monophasic reduction of fully active enzyme contrasts dramatically with the behaviour of wild-type flavocytochrome b2 which exhibits biphasic FMN reduction, notably at high concentrations of lactate [15,16,18,19]. The fast phase corresponds to the initial two-electron reduction of FMN but subsequent reduction of the haem and interprotomer electron transfer Table 2 Steady-state kinetc parametws and 2H kinetic Isotope effects for the Isolated flavin domain and intact, wild-type flavocytoochrome b2…”