Vitreoscilla is a genus of Gram-negative aerobic bacterium which has the capability to synthesis a soluble, homodimertic hemoglobin,... Show moreVitreoscilla is a genus of Gram-negative aerobic bacterium which has the capability to synthesis a soluble, homodimertic hemoglobin, Vitreoscilla hemoglobin (VHb). The Vitreoscilla hemoglobin was the first bacterial hemoglobin discovered, and has a wide range of biological and biotechnological applications. The distal site is one of the hot spots in VHb studies because of its unique structure. The Tyrosine residue at B10 and its hydrogen bonded Proline at E8 were considered as the ligand binding functional sites in distal space of VHb according to the previous study. In this study, two single mutated and one double mutated Vitreoscilla hemoglobin at position B10 and E8 were constructed and purified. In the two single mutants, the Tyr at B10 and the Pro at E8 were mutated to Ala. In the double mutant, both of the sites were mutated to Ala. The CO di↵erence spectrum data of the mutants indicate that the ligand binding ability of the Vitreoscilla hemoglobin was not neutralized by the mutations at ProE8 and TyrB10. Circular dichroism spectrum data of the mutants is similar to the wild type Vitreoscilla hemoglobin, which means the globin secondary structure is conserved. However the micro-environment in the distal sites is changed: the IR spectrum of the carbonyl stretch bond red-shifted in the CO bound VHb double mutant. A molecular dynamic simulation was introduced in the study to o↵er some guidance for future research plans. The simulation results showed that the B10 and E8 residue mutated to Ala might reduce the flexibility of the D-region, because of the more completed C and E helix. The volume of cavity where the heme group inserts changes significantly in various mutant models, which may provide a rough explanation of the change in carbonyl stretch bond IR spectrum. Additionally, an interesting conformation of Gln E7 was found in the simulation of double mutant model. M.S. in Biology, May 2015 Show less
