Binding and Electron Transfer between Cytochrome <i>b</i>₅ and the Hemoglobin α- and β-Subunits through the Use of [Zn, Fe] Hybrids

Abstract

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, Fe 3+ (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 3 ZnP → Fe 3+ P ET reaction of Fe 3+ b 5 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 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 α-chains ( K α ≈ 2.0 × 10 4 M - 1 ) is ∼4-fold larger than that of the β-chains ( K β = 4.9 × 10 3 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 b 5 . However, they permit an alternative in which b 5 reacts with the α-chains by binding at a site which spans the α 1 β 2 dimer interface.