CHAPTER 10
Epitope Mapping for the Monoclonal Antibody that
Inhibits Intramolecular Flavin to Heme Electron Transfer
in Flavocytochrome b 2 from Baker's Yeast
(L-Lactate Dehydrogenase)
K. H. DIEP LE', M. MAYER' and F. LEDERER'
1
Introduction
Flavocytochrome b2 is a homotetramer, each subunit of which carries one FMN
and one protoheme IX. The FMN oxidizes lactate to pyruvate in a reaction
involving the transfer of two reducing equivalents. These are then transferred in
two successive steps to heme b2 in the same subunit. Heme b2 in turn is reoxidized by cytochrome c, the physiological acceptor, in the intermembrane space of
yeast mitochondria. The enzyme enables yeast to grow on lactate as sole carbon
source (for review, see Lederer 1991).
The enzyme can now be expressed under a recombinant form in E. coli (Black
et al. 1989). Its crystal structure has been refined to 2.4 A resolution (Xia and
Mathews 1990). The asymmetric unit is composed of two subunits. Subunit Sl
shows two domains (Fig. 10.1); residues 1 to 99 encompass the heme-binding
domain. Its fold is similar to that of cytochrome bs, as predicted from sequence
data (Guiard et al. 1974). Residues 100 to 511 encompass the flavodehydrogenase
domain (FDH). It presents the typical ~8a8 barrel fold, also present in glycolate
oxidase, another member of the family of the FMN-dependent a-hydroxy acid
oxidizing enzymes (Lindqvist et al. 1991). The planes of the two prosthetic
groups are nearly parallel to each other, and the distance between flavin N5 and
the closest porphyrin ring atom is 10 A. There is no intervening protein matter
between flavin N5 and the opening of the heme crevice. In contrast, subunit S2
only shows the FDH domain, with pyruvate, the reaction product, bound at the
active site.
Thus, the crystal structure shows evidence for mobility of the heme domain in
the crystal. Subsequently, NMR data suggested that it is also mobile in solution
(Labeyrie et al. 1988). The interdomain contacts comprise a number of hydrophobic interactions, a single electrostatic interaction between K296 and a heme
propionate carboxylate, as well as a single direct hydrogen bond between Y143
and the other heme propionate carboxylate, and a few water-mediated hydrogen
bonds (Xia and Mathews 1990). Altogether, these interactions do not provide
strong cohesive forces, since, once the covalent bond between the domains is broken, the domains do not recognize each other and are incapable of exchanging
1 Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique,
91198 Gif-sur-Yvette Cedex, France.
Epitope Mapping for the Monoclonal Antibody that
Inhibits Intramolecular Flavin to Heme Electron Transfer
in Flavocytochrome b 2 from Baker's Yeast
(L-Lactate Dehydrogenase)
K. H. DIEP LE', M. MAYER' and F. LEDERER'
1
Introduction
Flavocytochrome b2 is a homotetramer, each subunit of which carries one FMN
and one protoheme IX. The FMN oxidizes lactate to pyruvate in a reaction
involving the transfer of two reducing equivalents. These are then transferred in
two successive steps to heme b2 in the same subunit. Heme b2 in turn is reoxidized by cytochrome c, the physiological acceptor, in the intermembrane space of
yeast mitochondria. The enzyme enables yeast to grow on lactate as sole carbon
source (for review, see Lederer 1991).
The enzyme can now be expressed under a recombinant form in E. coli (Black
et al. 1989). Its crystal structure has been refined to 2.4 A resolution (Xia and
Mathews 1990). The asymmetric unit is composed of two subunits. Subunit Sl
shows two domains (Fig. 10.1); residues 1 to 99 encompass the heme-binding
domain. Its fold is similar to that of cytochrome bs, as predicted from sequence
data (Guiard et al. 1974). Residues 100 to 511 encompass the flavodehydrogenase
domain (FDH). It presents the typical ~8a8 barrel fold, also present in glycolate
oxidase, another member of the family of the FMN-dependent a-hydroxy acid
oxidizing enzymes (Lindqvist et al. 1991). The planes of the two prosthetic
groups are nearly parallel to each other, and the distance between flavin N5 and
the closest porphyrin ring atom is 10 A. There is no intervening protein matter
between flavin N5 and the opening of the heme crevice. In contrast, subunit S2
only shows the FDH domain, with pyruvate, the reaction product, bound at the
active site.
Thus, the crystal structure shows evidence for mobility of the heme domain in
the crystal. Subsequently, NMR data suggested that it is also mobile in solution
(Labeyrie et al. 1988). The interdomain contacts comprise a number of hydrophobic interactions, a single electrostatic interaction between K296 and a heme
propionate carboxylate, as well as a single direct hydrogen bond between Y143
and the other heme propionate carboxylate, and a few water-mediated hydrogen
bonds (Xia and Mathews 1990). Altogether, these interactions do not provide
strong cohesive forces, since, once the covalent bond between the domains is broken, the domains do not recognize each other and are incapable of exchanging
1 Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique,
91198 Gif-sur-Yvette Cedex, France.