Rate constants for hemin dissociation from the ␣ and ␤ subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37°C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated ␣ and ␤ chains and for recombinant hemoglobin tetramers stabilized by ␣ subunit fusion. The rate constant for hemin dissociation from ␤ subunits in native hemoglobin increases from 1.5 h ؊1 in tetramers at high protein concentration to 15 h ؊1 in dimers at low concentrations. The rate of hemin dissociation from ␣ subunits in native hemoglobin is significantly smaller (0.3-0.6 h ؊1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-␣ subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric ␣ and ␤ chains are much greater, 12 and 40 h ؊1 , respectively, at pH 7, 37°C. Aggregation of monomers to form ␣ 1 ␤ 1 dimers greatly stabilizes bound hemin in ␣ chains, decreasing its rate of hemin loss ϳ20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to ␤ subunits. A significant reduction in the rate of hemin loss from ␤ subunits does occur after formation of the ␣ 1 ␤ 2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.

All globins consist of eight helices and interconnecting loops except alpha hemoglobin subunits which lack the D-helix due to deletion of five consecutive residues. Previous site-directed mutagenesis work suggested that this deletion is a neutral modification in hemoglobin with respect to equilibrium O2 binding [Komiyama, N. H., Shih, T.-B., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 349-351]. To examine the role of the D-helix in myoglobin, we have measured the O2 and CO binding and hemin dissociation properties of recombinant sperm whale myoglobin mutants in which residues 52-56 have been deleted, Mb(-D), replaced by five alanines, Mb(Ala52-56), and substituted with four alanines and a methionine, Mb(Ala52-55Met56). Crystal structures of aquometMb(-D) and aquometMb(Ala52-55Met56) were determined to 2.0 A resolution and show that the conformation of the distal pocket is little affected by removal of the D-helix or mutations in this region. As a result, these mutations have little effect on O2 and CO binding. Diffuse electron density is observed in the region between the C- and E-helices of Mb(-D), indicating a highly mobile or heterogeneous conformation in this portion of the tertiary structure. This flexibility provides an explanation for the 50-fold higher rate of hemin loss from Mb(-D) as compared to that from wild-type myoglobin. Hemin loss from Mb(Ala52-56) is also rapid. In contrast, Mb(Ala52-55Met56) shows a well-defined D-helix and has a rate of hemin loss identical to that of wild-type holoprotein [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)

Lineweaver-Burk plots, secondary replots, and 31 P NMRbased titration curves (34 pages). Ordering information is given on any current masthead page.