We have measured the binding affinity (K(A) 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 (K(α) ≃ 2.0 x 104 M- 1) is ~4-fold larger than that of the β-chains (K(β) = 4.9 x 103 M- 1); the intracomplex ET rate constant of the α-chains (k(α) ≃ 1500 s- 1) is ~2-fold larger than that of the β-chains (k(β) ≃ 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 α1/β2 dimer interface.
ASJC Scopus subject areas
- Colloid and Surface Chemistry