Fluorescence studies on the interaction between two cytochromes extracted from the yeast, Hansenula anomala

Interaction between Cytochrome b2 Core and Flavodehydrogenase from the Yeast Hansenula anomala

“…The concentration of FMN was determined spectrophotometrically using an extinction coefficient of 12.5 mM-' cm-I at 450 nm (oxidized form) (23). Cytochrome b, core was prepared from flavocytochrome b, by proteolysis with trypsin as described by Gervais et al (24) with the modifications detailed by Albani (8). The hemoprotein domain thus obtained corresponded to a cytochrome form with a molecular mass equal to 14 kDa without any bound FMN.…”

“…Flavocytochrome b2 catalyzes the oxidation of L-lactate to pyruvate with subsequent electron transfer to cytochrome c. Many studies have been performed to understand the role of the two domains in the electron transfer between flavocytochrome b, and cytochrome c and in the stability of the complex (2,4,(6)(7)(8)(9)(30)(31)(32)(33)(34). Cytochrome c binds to isolated cytochrome b2 core with a dissociation constant equal to 6 FM (8), to flavocytochrome b2 (2,7,9) and to isolated FDH (9) with a dissociation constant equal to 0.1 pM.…”

“…Cytochrome c binds to isolated cytochrome b2 core with a dissociation constant equal to 6 FM (8), to flavocytochrome b2 (2,7,9) and to isolated FDH (9) with a dissociation constant equal to 0.1 pM. The product of charges intervening in the proteins interaction was found to be equal to 5.85 2 0.25 for the cytochrome c-flavocytochrome b2 complex (4,7) and 2.25 2 0.35 for the cytochrome c-cytochrome b2 core complex (6,8). Therefore, the presence of FDH increases the contribution of electrostatic interactions in the control of cytochrome b2 reactivity toward cytochrome c and stabilizes the cytochrome c-flavocytochrome b2 complex.…”

“…Each protomer of the flavoprotein contains one FMN. Interaction studies between the cytochrome c from one part and the flavocytochrome bZ, the FDH and the cytochrome b, core from the other part have been carried out by equilibrium dialysis ( 2 ) , stoppedflow kinetics (4)(5)(6) and fluorescence spectroscopy (7)(8)(9). The purpose of these studies was to understand the mechanism and the role played by each domain in the cytochrome cflavocytochrome b, interaction.…”

“…The fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was largely used to study interactions between proteins and proteins with their ligands (8,9,(11)(12)(13) and to follow the local and global motions in proteins (14)(15)(16)(17)(18). The present work deals with the interaction between cytochrome b2 core and FDH obtained from H. anomala flavocytochrome b2 using TNS as a probe.…”