Web Release Date: March 21,
Potentiometric and Further Kinetic Characterization of the Flavin-Binding Domain
of Saccharomyces cerevisiae Flavocytochrome b2. Inhibition by Anions Binding in
the Active Site
énas,
and
CNRS FRE2930, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France, and Institute of Biochemistry, Mokslininku 12, Vilnius 2600, Lithuania
Received December 22, 2006
Revised Manuscript Received February 7, 2007
Abstract:
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
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.
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