title: Structure-function studies of the Vitreoscilla hemoglobin D-region.
abstract: The D-region connecting helices C and E of Vitreoscilla hemoglobin (VHb) appears disordered in the crystal structure. Six site-directed mutants in this region were made to investigate its possible functions. The mutant VHb's were analyzed using UV-visible and FTIR spectroscopy, using primarily the CO liganded forms, and their heme/protein ratios were determined. The results implicate Asp44, Arg47, and Glu49 as especially important in heme-globin interactions and ligand binding, and enabled construction of a model in which the D-region forms a loop that protrudes upward over the heme. Interactions between VHb (wild type and the D-region mutants) with the flavin domain of 2,4-DNT dioxygenase from Burkholderia were tested using bacterial two-hybrid screening. There was a correlation between the extent of the D-loop perturbation predicted for each mutant and the amount of the reduction in VHb-flavin domain interaction, suggesting that this region may be more generally involved in binding of VHb to flavoproteins.
authors.affiliation: Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Stark
authors.forename: Dale A
authors.initials: DA
authors.lastname: Webster
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D019117:Burkholderia / Q000201:enzymology*; D003198:Computer Simulation; D006454:Hemoglobins / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D054793:Truncated Hemoglobins
chemical_list: D001426:Bacterial Proteins; D006454:Hemoglobins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D010105:Oxygenases; C100896:2,4-dinitrotoluene dioxygenase