We have measured the binding affinity (K-A) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b(5) (b(5)), using triplet quenching titrations of mixed-metal [ZnM, Fe3+(N-3(-))] Hb hybrids and of fully substituted Zn-mesoporphyrin (ZnM)Hb by b(5) (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 (ZnP)-Zn-3 --> Fe3+P ET reaction of Fe(3+)b(5) with either the alpha-chains or the beta-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 b(5) with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b(5) and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the alpha-chains (K-a approximate to 2.0 x 10(4) M-1) is similar to 4-fold larger than that of the beta-chains (K-beta = 4.9 x 10(3) M-1); the intracomplex ET rate constant of the alpha-chains (k(alpha) approximate to 1500 s(-1)) is similar to 2-fold larger than that of the beta-chains (k(beta) approximate to 850 s(-1)). The binding affinity and ET rate constant of the alpha-chains both decrease as the pH is increased from 6.0 to 7.0; the binding affinity of the beta-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 b(5). However, they permit an alternative in which b(5) reacts with the alpha-chains by binding at a site which spans the a(1)beta(2) dimer interface.