George C. Blouin, John S. Olson, Angela R. Hight Walker


The truncated hemoglobin from Bacillus anthracis (BaTrHb) has an ultra-high oxygen affinity when compared to the homologous mammalian protein. The oxygen dissociation rate from the heme iron of hemoglobins can be lowered by hydrogen bonding between protein residues and bound O2. While there is one amino acid that can act as a hydrogen bond donor in the binding pocket of most mammalian hemoglobins the distal histidine, there are two in BaTrHb a tryptophan and a glutamine residue. The strength of hydrogen bond donation from this Trp-Gln combination is assessed using Raman spectroscopy and the reporter ligand CO. The stretching frequency of the Fe-CO bond can vary by 10s of cm-1 depending on the polarity within the binding pocket, and has been shown to correlate tightly with the O2 dissociation rate for most globins studied to date. Raman spectra are reported for CO bound to wild-type BaTrHb, to the binding pocket mutant Trp(G8)->Leu(G8), and to horse and sperm whale myoglobins (Mb) as controls. The Raman spectrum for BaTrHbO2 is also reported and compared to published data for wild type and mutant MbO2.