title: Interdomain contacts in flavocytochrome b(2), a mutational analysis.
abstract: Each flavocytochrome b(2) (l-lactate cytochrome c oxidoreductase) subunit consists of an N-terminal cytochrome domain and a C-terminal flavodehydrogenase (FDH) domain. In the enzyme crystal structure, only two heme domains are visible per enzyme tetramer, because of the mobility of the other two heme domains relative to the FDH domains. Evidence was subsequently provided that this mobility also exists in solution. Numerous kinetic studies showed that, during the catalytic cycle, electrons are transferred one by one from the reduced flavin to heme b(2) in the same subunit. In previous work, we provided evidence that a monoclonal antibody that abolishes flavin to heme electron transfer uses part of the interdomain interface for binding to its antigen, the native heme domain. In this work, we use a number of heme domain side chain substitutions in and around the interface to probe their effect on flavin to heme electron transfer. Using steady-state and pre-steady-state kinetics, as well as redox potential determinations and EPR measurements, we define several hydrophobic interactions and van der Waals contacts that are important for a catalytically competent docking of the heme domain onto the FDH domain. In addition, with several extremely slow mutant enzymes, we propose an isosbestic wavelength between oxidized and reduced heme for specifically following the kinetics of flavosemiquinone formation from two-electron reduced flavin.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR9063, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: K H Diêp
authors.initials: KH
authors.lastname: Lê
authors.forename: Alain
authors.initials: A
authors.lastname: Boussac
authors.forename: Bettina
authors.initials: B
authors.lastname: Frangioni
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
authors.forename: Florence
authors.initials: F
authors.lastname: Lederer
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
chemical_list: D005415:Flavins; D042844:L-Lactate Dehydrogenase (Cytochrome)