Studies On The Relationship Of Vitreoscilla Hemoglobin Structure And Peroxidase Activity

Vitreoscilla hemoglobin (VHb) is a bacterial globin originated from theobligatory aerobic bacterium Vitreoscilla. The VHb has been thought to maintain anessential level of intracellular dissolved oxygen. Nowadays, it has become a versatiletool in current biotechnology, especially for large-scale fermentation, since shortageof oxygen supply during cultivation can be overcome by heterologous expression ofVHb. Cell growth and productivity are enhanced particularly under oxygen-depletedconditions. Thus, VHb technology has been applied for industrial scale production ofmany valuable compounds. Beside its role in O2delivery, VHb has been involved invarious cellular activities, such as protein translation efficiency, cellular metabolism,and alleviation of nitrosative stress.The Vitreoscilla hemoglobin was the first bacterial hemoglobin discovered.Therefore, its structure and function have been extensively investigated. It wasinitially proposed that, under oxygen limiting conditions, VHb is induced in order tobind the oxygen and deliver it to the terminal respiratory oxidase(s) to maintainaerobic respiration at a high level under these conditions. Recently, peroxidaseactivity has been disclosed in VHb. The steadystate kinetic study has revealed thatVHb catalyzes the oxidation of various aromatic substrates, as normally found inhorseradish peroxidase (HRP). Thus, VHb as the newly discovered peroxidase has agreater value and is considered to be used in industrial applications. However, itscatalytic activity seems to be limited, possibly as a result of the improper nature of itsactive site. HRP is a commonly used peroxidase with very high peroxidase activity.Compared with HRP, the distal histidine of catalytically important residues is missingin VHb, which may account for the low activity. In this study, we constructed the expression system of VHb in E. coli to study themechanisms of VHb regulated by oxygen. The expression of VHb was induced at lowoxygen concentration and purified. Constructing the VHb expression system includedlinking the promoter to the target gene and reconstructing the recombinant plasmid.The wild-type gene of VHb and its natural promoter was connected to the expressionvectors pUC19without histidine-tagged. Only the natural promoter of VHb couldopen the transcription and translation process of VHb. Without histidine tag and anymutation could guarantee the physical environment of VHb. The recombinant plasmidwas transformed into E. coli BL21(DE3). A single band of SDS-PAGE (about16KD)and the Rz>3indicated the purity of our target protein.pH-dependent (pH6.0-8.0) quaternary structural changes of ferric Vitreoscillahemoglobin has been investigated using dynamic light scattering. The experimentalresults show that the molecular size of VHb is4.1nm under the neutral conditions,the molecular size is5.9nm in acidic conditions, and the molecule size of the underalkaline condition becomes6.8nm. Therefore, the VHb exhibits a monomeric stateunder neutral conditions at pH7.0, while the protein forms distinct homodimericspecies at pH6.0and8.0, respectively. The dissociation constant obtained using theBio-Layer Interferometry technology indicate that, at pH7.0, the monomer-monomerdissociation of VHb is about6-fold or5-fold higher (KD=6.34μM) compared withthat at slightly acidic pH (KD=1.05μM) or slightly alkaline pH (KD=1.22μM). ThepH-dependent absorption spectra demonstrate that the heme microenvironment ofVHb is sensitive to the changes of pH value. The maximum absorption band of hemegroup of VHb shifts from402nm to407nm when pH changes from6.0to8.0. Inaddition, the fluorescence emission spectra of VHb, taken at excitation wavelength of295nm, suggest that the single Trp122fluorescence quantum yields in VHb aredecreased due to the formation of the homodimeric species. However, the circulardichroism spectra data display that the secondary structures of VHb are little affectedby pH transitions. The pH-dependent peroxidase activity of VHb was also investigated in this study. The optimum pH for VHb using2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as substrate is7.0, which impliesthat the monomer state of VHb would exhibit better peroxidase activity than thehomodimeric species of VHb at pH6.0and8.0.Peroxidase is known for its capacity to decolorize textile dye. Peroxidase activityof Vitreoscilla hemoglobin has been disclosed recently. In addition, VHb has thecapacity to decolorize textile dyes. In order to investigate the decolorization ability ofVHb as a peroxidase,2catalytically conserved residues (histidine and cysteine) foundin the distal pocket of peroxidases were introduced into that of VHb. The mutantQ53H/P54C showed the highest removal efficiency of dye decolorization; theremoval efficiency was70%within5min of contact time. UV-spectra studiesrevealed that the porphyrin environment is changed in mutant Q53H/P54C (λSoretat413nm), in which the amino acids at positions53and54are substituted by histidineand cysteine, respectively. Spectroscopy data showed that the mutant Q53H/P54C hasmore closely protein structure. Differential scanning calorimetry data indicate that theprotein structure of Q53H/P54C is more stable. In addition, spectroscopy studies wereconducted to explain the reasons of the increased peroxidase activity of Q53H/P54C,and the results indicated that the mutant Q53H/P54C has a more stable proteinstructure (CD data show increase in helix; DSC data show that the meltingtemperature is higher than90°C). Therefore, we selected an excellent enzyme that hasa higher catalytic ability for dye decolorization and a more stable structure underhigh-temperature conditions.