Binding and Electron Transfer between Cytochrome b5 and the Hemoglobin α- and β-Subunits through the Use of [Zn, Fe] Hybrids

Abstract

We have measured the binding affinity (KA) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b5 (b5), using triplet quenching titrations of mixed-metal [ZnM, Fe3+(N3-)] Hb hybrids and of fully substituted Zn−mesoporphyrin (ZnM)Hb by b5 (trypsin-solubilized, bovine) (pH values 6.0 and 7.0). The use of the mixed-metal Hb hybrids with Zn in one chain type allows us to selectively monitor the 3ZnP → Fe3+P ET reaction of Fe3+b5 with either the α-chains or the β-chains. The self-consistent analysis of the results for the mixed-metal hybrids and those for the (ZnM)Hb allows us to determine the reactivity and affinity constants for the interactions of b5 with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b5 and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the α-chains (Kα ≈ 2.0 × 104 M-1) is ∼4-fold larger than that of the β-chains (Kβ = 4.9 × 103 M-1); the intracomplex ET rate constant of the α-chains (kα ≈ 1500 s-1) is ∼2-fold larger than that of the β-chains (kβ ≈ 850 s-1). The binding affinity and ET rate constant of the α-chains both decrease as the pH is increased from 6.0 to 7.0; the binding affinity of the β-chains is essentially the same at pH 6.0 and 7.0, while the ET reactivity decreases. The kinetic results are consistent with a docking model in which each subunit binds a molecule of b5. However, they permit an alternative in which b5 reacts with the α-chains by binding at a site which spans the α1β2 dimer interface.