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 (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 f Fe 3+ P ET reaction of Fe 3+ b 5 with either the R-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 R-chains (K R ≈ 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 R-chains (k R ≈ 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 R-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 R-chains by binding at a site which spans the R 1 β 2 dimer interface.

An Antiapoptotic Neuroprotective Role for Neuroglobin

“…The high rate of intermolecular electron exchange (approx 2,000 s −1 ) is in the range reported for other biologically significant protein-protein redox reactions such as cytochrome c reduction of cytochrome c oxidase [46] or the reduction of hemoglobin or cytochrome c by cytochrome b 5 [47,48]. This reaction appears to be composed of two steps, namely complex formation followed by electron exchange [49,50].…”

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin ?1?1half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

“…Zn 2+ ‐substituted hemes, which closely mimic the structure of native Fe 2+ hemes80, 81 but do not bind O 2 , have been successfully employed as analogs for deoxy hemesites in a number of Hb studies 82–86. The eight deoxy tetrameric hybrids of human Hb (comprised of subunits containing Zn 2+ ‐substituted hemes in combination with native deoxy Fe 2+ hemes) were determined to have assembly free energies that were indistinguishable from that of native deoxy Fe 2+ Hb (i.e., −14.3 ± .2 kcal/mol) under conditions of this study 27.…”

Determination of the Hemoglobin Surface Domains That React with Cytochrome b5

“…The kinetic results, however, also permitted an alternative model in which b 5 reacts with a chain by binding at sites which span the dimer-dimer interfaces (7). A possible R 1 R 2 -site was noted in which Lys residues on both R-chains provide binding contacts as represented in Figure 1A′.…”