| The research presented here is a study of specific mutants of Vitreoscilla hemoglobin (VHb) to learn the importance of specific amino acid residues in VHb which are unique to this protein and may play an important role in its function. For this reason mutations at the E7, E8 and H12 positions were created. In wild type VHb, the Gln residue at the E7 position is out of the heme pocket so that it can not ligand the bound oxygen, Pro at the E8 position prevents the E7–E10 residues of VHb from adapting a regular α-helix and Tyr at the H12 position is believed to participate in the hydrogen bonding network which is necessary for the structure of the proximal pocket.; Six recombinant strains, E. coli DH5α[pMS1], E. coli DH5α[pMS2], E. coli DH5α[pMS3], E. coli DH5α[pMS4], E. coli DH5α[pMS6], and E. coli DH5α[pMS7] were constructed by transformation of plasmids pMS1, pMS2, pMS3, pMS4, pMS6, and pMS7, respectively, into E. coli DH5α.; Each plasmid in the pMS series carries the Vitreoscilla hemoglobin gene in vector pQE-80 and was constructed from the corresponding plasmid in the pNKD series, pNKD-1 carries wild type vgb, each of which carries the Vitreoscilla hemoglobin gene in the Bluescript vector. Vgb in the remaining pNKD plasmids was altered by site directed mutagenesis. pNKD-2/pMS2 carry vgb with vector pQE-80 pNKD-3/pMS3, pNKD-4/pMS4, pNKD-6/pMS6 and pNKD-7/pMS7.; Each mutant VHb was purified and examined by CO-difference and EXAFS spectroscopy. In addition, the results were compared with previous measurements of the oxygen dissociation constants and some IR spectra of mutant and wild type VHb. The results from the EXAFS experiments indicate that except for PMS3, mutant VHbs and wild type VHb appear virtually identical. This is in contrast to differences in CO-difference spectra and oxygen Kd values which are most pronounced between PMS2 and PMS6. The reason might be that the mutations we have created do not affect the heme pocket but rather change the ligand binding properties in some way.; We can suggest that new alterations in other regions of VHb may be required to create a “super VHb”, that is one which has improved ability to stimulate growth and productivity of heterologous strains. |
