PROBING INTRAMOLECULAR ELECTRON-TRANSFER WITHIN FLAVOCYTOCHROME B(2) WITH A MONOCLONAL-ANTIBODY

Flavocytochrome b(2) or L-lactate dehydrogenase from yeast is a tetram eric enzyme which oxidizes lactate at the expense of cytochrome c or a rtificial electron accepters. The prosthetic group FMN is reduced by t he substrate and then transfers sequentially the reducing equivalents to heme b(2) in the same subunit. The latter is reoxidized by cytochro me c. The crystal structure of the enzyme indicates that each subunit is composed of a flavodehydrogenase domain (FDH) and a cytochrome b(2) domain; the latter, which encompasses the first 99 residues of the pe ptide chain, is mobile relative to the tetrameric FDH assembly. We des cribe here the properties of a monoclonal antibody elicited against th e holoenzyme. It only recognizes the heme-binding domain, with a K-d l ower than 10(-7) M, and its epitope is conformational. In the enzyme-I gG complex, flavin is reduced normally and can be reoxidized by ferric yanide, but no longer by heme b(2). Stopped-flow experiments in the ab sence of electron accepters give no indication of flavin to heme elect ron transfer in the enzyme-antibody complex. In other words, the two d omains are functionally uncoupled. The binding stoichiometry is 1/1 fo r the Fab fragment with respect to the isolated, monomeric, heme-bindi ng domain, but 2/4 with respect to the enzyme tetramer; furthermore, b inding of two Fab fragments per tetramer is sufficient to cause inhibi tion of intra-subunit flavin to heme electron transfer in all four sub units. Altogether these results can only be rationalized by considerin g that mobility of the cytochrome domain with respect to the FDH is an essential component of the catalytic cycle. The first experiment desi gned to locate the epitope shows it does not encompass the interdomain peptide linker (so-called hinge region, centered on residues 99-100).