Probing intramolecular electron transfer within flavocytochrome b2 with a monoclonal antibody.

Flavocytochrome b2 or L-lactate dehydrogenase from yeast is a tetrameric enzyme which oxidizes lactate at the expense of cytochrome c or artificial electron acceptors. The prosthetic group FMN is reduced by the substrate and then transfers sequentially the reducing equivalents to heme b2 in the same subunit. The latter is reoxidized by cytochrome c. The crystal structure of the enzyme indicates that each subunit is composed of a flavodehydrogenase domain (FDH) and a cytochrome b2 domain; the latter, which encompasses the first 99 residues of the peptide chain, is mobile relative to the tetrameric FDH assembly. We describe here the properties of a monoclonal antibody elicited against the holoenzyme. It only recognizes the heme-binding domain, with a Kd lower than 10(-7) M, and its epitope is conformational. In the enzyme-IgG complex, flavin is reduced normally and can be reoxidized by ferricyanide, but no longer by heme b2. Stopped-flow experiments in the absence of electron acceptors give no indication of flavin to heme electron transfer in the enzyme-antibody complex. In other words, the two domains are functionally uncoupled. The binding stoichiometry is 1/1 for the Fab fragment with respect to the isolated, monomeric, heme-binding domain, but 2/4 with respect to the enzyme tetramer; furthermore, binding of two Fab fragments per tetramer is sufficient to cause inhibition of intra-subunit flavin to heme electron transfer in all four subunits. Altogether these results can only be rationalized by considering that mobility of the cytochrome domain with respect to the FDH is an essential component of the catalytic cycle. The first experiment designed to locate the epitope shows it does not encompass the interdomain peptide linker (so-called hinge region, centered on residues 99-100).