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)