Potentiometric and further kinetic characterization of the flavin-binding domain of Saccharomyces cerevisiae flavocytochrome b2. Inhibition by anions binding in the active site.

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind to the oxidized and the two-electron-reduced forms of the FDH domain, and that excess L-lactate also binds to the two-electron-reduced form. These findings point to the existence of a common or strongly overlapping binding site. A comparison of the effect of the anions on WT and R289K holo-flavocytochromes b2 indicates that invariant R289 belongs to this site. According to literature data, it must also be present in other members of the family of L-2-hydroxy acid-oxidizing enzymes.

Another biological effect of tosylphenylalanylchloromethane (TPCK): it prevents p47phox phosphorylation and translocation upon neutrophil stimulation.

TPCK (tosylphenylalanylchloromethane), first discovered as a serine protease inhibitor, has been described to affect in diverse systems a number of physiological events probably unrelated to its antiprotease effect, such as proliferation, apoptosis and tumour formation. In the present study, we focus on its inhibition of the neutrophil respiratory burst, an important element of non-specific immunological defence. The superoxide anion-producing enzyme, NADPH oxidase, is quiescent in resting cells. Upon cell stimulation, the redox component, membrane-bound flavocytochrome b558, is activated when the cytosolic factors (p47phox, p67phox and p40phox, as well as the small GTPase Rac) associate with it after translocating to the membrane. This requires the phosphorylation of several p47phox serine residues. The signal transduction events leading to enzyme activation are not completely understood. In the past, the use of diverse protease inhibitors suggested that proteases were involved in NADPH oxidase activation. We suggested previously that TPCK could prevent enzyme activation by the phorbol ester PMA, not due to inhibition of a protease, but possibly to inhibition of the cytosolic factor translocation [Chollet-Przednowed and Lederer (1993) Eur. J. Biochem. 218, 83-93]. In the present work, we show that TPCK, when added to cells before PMA, prevents p47phox phosphorylation and hence its translocation; moreover, when PMA-stimulated cells are incubated with TPCK, p47phox is dephosphorylated and dissociates from the membrane. These results are in line with previous suggestions that the respiratory burst is the result of a series of continuous phosphorylation and dephosphorylation events. They suggest that TPCK leads indirectly to activation of a phosphatase or inactivation of a kinase, and provide the first clue towards understanding the steps leading to its inhibition of NADPH oxidase activation.