Structure-function studies of the Vitreoscilla hemoglobin D-region.
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.
Lee
Sang Yeol
SY
Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Stark
Benjamin C
BC
Webster
Dale A
DA
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
D001426:Bacterial Proteins; D006454:Hemoglobins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D010105:Oxygenases; C100896:2,4-dinitrotoluene dioxygenase