Transient Kinetics of Intracomplex Electron-Transfer in the Human Cytochrome b5 Reductase-Cytochrome b5 System: NAD+ Modulates Protein-Protein Binding and Electron Transfer

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 × 108 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 × 108 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 × 107 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 × 107 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 μM. 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. Cytochrome b5 also increases the binding constant for NAD+ in the ternary complex.