Determination of the hemoglobin surface domains that react with cytochrome b5.
We have compared the photoinitiated electron-transfer (ET) reaction between cytochrome b(5) (b(5)) and zinc mesoporphyrin-substituted hemoglobin [(ZnM)Hb] and Hb variants in order to determine whether b(5) 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 alpha beta dimers, we studied the reaction of b(5) with alpha ZnM chains and (ZnM)Hb beta W37E, which exists as alpha beta dimers in solution. Triplet quenching titrations of the ZnHb proteins with Fe(3+)b(5) show that the binding affinity and ET rate constants for the alpha-chains are the same when they are incorporated into a Hb tetramer or dimer, or exist as monomers. Likewise, the parameters for beta-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 alpha-chains and 40-fold for the beta-chains (but has has little effect on the affinity of either chain type for b(5), similar to earlier results for myoglobin). Together, these results indicate that b(5) 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 b(5) binds over a broad area of the subunit face, but reacts only in a minority population of binding geometries.
Naito
N R
NR
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Hui
H L
HL
Noble
R W
RW
Hoffman
B M
BM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015786:Cytochromes b5 / Q000737:chemistry*; D003905:Deuteroporphyrins / Q000737:chemistry; D019281:Dimerization; D004579:Electron Transport; D004951:Esterification; D005290:Ferric Compounds / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008665:Metalloporphyrins / Q000737:chemistry; D010782:Photolysis; D017434:Protein Structure, Tertiary; D017186:Titrimetry; D015032:Zinc / Q000737:chemistry
D003905:Deuteroporphyrins; D005290:Ferric Compounds; D006454:Hemoglobins; D008565:Membrane Proteins; D008665:Metalloporphyrins; D015786:Cytochromes b5; C058069:zinc mesoporphyrin; D015032:Zinc