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. (C) 2004 Elsevier Inc. All rights reserved.