title: The D-helix in myoglobin and in the beta subunit of hemoglobin is required for the retention of heme.
abstract: 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)
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Whitaker
authors.forename: M B
authors.initials: MB
authors.lastname: Berry
authors.forename: E L
authors.initials: EL
authors.lastname: Ho
authors.forename: M S
authors.initials: MS
authors.lastname: Hargrove
authors.forename: G N
authors.initials: GN
authors.lastname: Phillips
authors.forename: N H
authors.initials: NH
authors.lastname: Komiyama
authors.forename: K
authors.initials: K
authors.lastname: Nagai
authors.forename: J S
authors.initials: JS
authors.lastname: Olson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014907:Whales
chemical_list: D006454:Hemoglobins; D046911:Macromolecular Substances; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme