Inhibition of L-lactate: cytochrome-c reductase (flavocytochrome b2) by product binding to the semiquinone transient. Loss of reactivity towards monoelectronic acceptors.
Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39-45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J. M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491-503]. In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs-pyruvate complex is actually much more stable than the Fo-pyruvate and Fh-pyruvate complexes. The inhibition produced by pyruvate has been characterized under steady-state conditions using both ferricytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead-end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.
Tegoni
M
M
Centre National de la Recherche Scientifique (Groupe d'Enzymologie Physicochimique), Université Pierre et Marie Curie, Gif-sur-Yvette, France.
Janot
J M
JM
Labeyrie
F
F
D003574:Cytochrome c Group / Q000032:analysis; D004579:Electron Transport; D005292:Ferricyanides / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000032:analysis; D008433:Mathematics; D011773:Pyruvates / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D013816:Thermodynamics
D003574:Cytochrome c Group; D005292:Ferricyanides; D005415:Flavins; D007773:Lactates; D011773:Pyruvates; C007931:hexacyanoferrate III; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)