title: Transient kinetics of intracomplex electron transfer in the human cytochrome b5 reductase-cytochrome b5 system: NAD+ modulates protein-protein binding and electron transfer.
abstract: Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 3.1 x 10(8) M-1 s-1. When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k2 = 3.4 x 10(7) M-1 s-1). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k2 < 1 x 10(7) M-1 s-1). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s-1, respectively. In the presence of NAD+, the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s-1 were obtained at both I = 56 mM and I = 96 mM and 235 s-1 at I = 120 mM. Thus, NAD+ appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD+ is to appreciably slow autoxidation and disproportionation of the FAD semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Arizona, Tucson 85721, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Meyer
authors.forename: K
authors.initials: K
authors.lastname: Shirabe
authors.forename: T
authors.initials: T
authors.lastname: Yubisui
authors.forename: M
authors.initials: M
authors.lastname: Takeshita
authors.forename: M T
authors.initials: MT
authors.lastname: Bes
authors.forename: M A
authors.initials: MA
authors.lastname: Cusanovich
authors.forename: G
authors.initials: G
authors.lastname: Tollin
mesh_terms: D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D009243:NAD / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010782:Photolysis; D011485:Protein Binding; D012256:Riboflavin / Q000031:analogs & derivatives / Q000494:pharmacology; D013053:Spectrophotometry
chemical_list: D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C007228:5-deazariboflavin; D006418:Heme; D015786:Cytochromes b5; D004492:Edetic Acid; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D012256:Riboflavin