We have compared the photoinitiated electron-transfer (ET) reaction between cytochrome b5 (b5) and zinc mesoporphyrin-substituted hemoglobin [(ZnM)Hb] and Hb variants in order to determine whether b5 binds to the subunit surface of either or both Hb chains, or to sites which span the dimer-dimer interface. Because the dimer-dimer interface would be disrupted for monomers or αβ dimers, we studied the reaction of b5 with αZnM chains and (ZnM)Hb βW37E, which exists as αβ dimers in solution. Triplet quenching titrations of the ZnHb proteins with Fe3+b5 show that the binding affinity and ET rate constants for the α-chains are the same when they are incorporated into a Hb tetramer or dimer, or exist as monomers. Likewise, the parameters for β-chains in tetramers and dimers differ minimally. In parallel, we have modified the surface of the Hb chains by neutralizing the heme propionates through the preparation of zinc deuterioporphyrin dimethyl ester hemoglobin, (ZnD-DME)Hb. The charge neutralization increases the ET rate constants 100-fold for the α-chains and 40-fold for the β-chains (but has has little effect on the affinity of either chain type for b5, similar to earlier results for myoglobin). Together, these results indicate that b5 binds to sites at the subunit surface of each chain rather than to sites which span the dimer-dimer interface. The charge-neutralization results further suggest that b5 binds over a broad area of the subunit face, but reacts only in a minority population of binding geometries.
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