Structural And Functional Studies On Vitreoscilla Hemoglobin

Oxygen-based metabolism is highly adaptive performance in biological evolution. In the process of evolution, the emergence of heme proteins makes the aerobic metabolism no longer subject to the restrictions of oxygen solubility in water. In vivo, heme proteins not only act as oxygen storager and transporter but also involve in the process of electron transport and signal transduction. Hemoglobin is an important oxygen binding protein, almost ubiquitous in animals, plants and microorganisms. In 1970's, American scientists Tyree et al. found a hemoglobin in aerobic bacteria Vitreoscilla named Vitreoscilla hemoglobin (VHb). In 1974, Webster et al. purified VHb from Vitreoscilla sp. for the first time, In 1986, Wakabayashi S firstly determined the amino acid sequence of VHb. In 1988, Webster DA encoded the gene of VHb, about 500 bp for the complete length, and successfully implemented the heterologous expression of VHb in E. coli. In 1989, Khosla C and Bailey JE found that the expression of VHb in E. coli was regulated by oxygen and by cAMP-CAP. In 1994, Zhu Jia provided a supplementary for the mechanism of VHb regulation by oxygen, he proposed that regulatory proteins FNR had some relevant with such regulation. Till now, the mechanism of VHb facilitating bacteria to survive in hypoxic conditions is not clear and still under the state of hypothesis. In 1966, Wittenberg C provided the hypothesis of diffusion. In 1990, Khosla C proposed the hypothesis of redox condition in cytoplasmic. In 1997, Wu proposed the hypothesis of ending receptor.However, most evidence suggests that VHb combines with oxygen to form oxygenated state then participating the oxygen metabolism, transferring oxygen to the respiratory chain, adjusting the activity of terminaloxidase, changing the efficiency of phosphorylation, ultimately affecting the gene translation and protein expression. So far, in vivo investigating the mechanisms of physiological function and regulation of VHb under micro-oxygen conditions is still a hot issue.We constructed the expression system of VHb in E. coli to study the mechanisms of VHb regulated by oxygen. The expression of VHb was induced at low oxygen concentration and purified. Both lipid bound VHb and no lipid bound VHb was identified for the first time and using spectroscopy technology, lipid bound VHb was found to have a higher catalase activity than that of no lipid bound VHb. In 2006, Rinaldi found that non lipid bound VHb had stronger oxygen binding capacity 20 folds than that of lipid bound VHb which suggested that the physiological function of VHb could be regulated by lipid.Constructing the VHb expression system included linking 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 expression vectors pUC19 without histidine-tagged. Only the natural promoter of VHb could open the transcription and translation process of VHb. Without histidine tag and any mutation could guarantee the physical environment of VHb. Double digestion and sequencing results identified the correction of our recombinant plasmid.The recombinant plasmid was transformed into E. coli BL21 (DE3). A single band of SDS-PAGE (about 16 KD) indicated the purity of our target protein. However, two different Soret bands (406 nm and 402 nm) of our target protein were identified. N-terminal analysis showed that both of their N-terminal is Met which was consistent with the reported N-terminal of natural VHb. UV-visible absorption spectra showed that both of VHb (402) and VHb (406) contained heme, and had a very high structural similarity which indicated the VHb (402) and VHb (406) were the same protein but in two different forms.The relationship between structure and function of VHb (406) and VHb (402) was studied using UV-visible absorption spectroscopy at the wavelength of 240-750 nm. VHb (406) had a feature absorption peak at 626 nm and Rz ratio 3.30, while VHb (402) had characteristic absorption peak at 644 nm and Rz ratio 3.15, which indicated that the two samples had no significant loss of heme and could be tested for further structural and functional studies. In 2006, Rinaldi identified that the two forms of VHb were lipid bound VHb (406) and no lipid bound VHb (402).Combined with circular dichroism and Raman spectroscopy we found that the lipid bound VHb (406) had more a-helical structure than that of no lipid bound VHb (402). Both of the two samples had a strong Raman band at 1357 cm-1 indicated that the heme group was ferric oxidation state. The 1631 cm-1 band of the two samples indicated the v (C=C) vibration mode which suggested the vinyl group of heme is at cis state.We used L-dopa and H2O2 as substrates of VHb to explore its activity of catalase, the results showed that the lipid bound VHb had a higher catalase activity than that of no lipid bound VHb. In 1992, Dikshit KL and Webster DA identified that, VHb (Fe2+) catalyzed to form hydrogen peroxide in the normal physiological conditions, which could improve the host cell oxidative respiratory rate. However, Anand (in 2010) pointed out, when VHb in host cells induced to generate hydrogen peroxide, the expression of VHb was inhibited, that it to say, produced hydrogen peroxide was not the normal intracellular physiological function. The difference between the two models may be derived from two states of VHb with lipid-bound and lipid-free, they were different on the reactive capacity with oxygen, which was the main source of differences between two models.VHb had a higher affinity for L-dopa than that of hydrogen peroxide. At 25℃, pH 7.5 VHb had 10 times specific activity than Mb using L-dopa and hydrogen peroxide as the substrates. When using L-dopa as the substrate, at 60℃pH 7.5, VHb exhibited the maximum peroxidase activity and the active half-life was about 30 hours. When the temperature was higher than 60℃, VHb lost the peroxidase activity rapidly in half an hour. The DSC experiments showed that the phase transition temperature of VHb was 82℃which indicated that the loss of peroxidase activity had nothing to do with the protein denaturation. UV-visible absorption spectroscopy and Raman spectroscopy studies had shown that VHb peroxidase activity should come from its six-iron heme coordinated low-spin state and the twisted conformation of the porphyrin ring plane. From the three-dimensional structure of VHb (PDB as 1 VHB) we could deduce that the propionic acid side chain of heme in VHb was pointing to D-loop region of the protein. Combined with the temperature-dependent activity of VHb research and DSC results we could speculate that the D-loop region 43-53 amino acid peptide within the state was not in complete freedom but existence a stabilizer which connected to the acid side chain of heme and amino acid residues for stabilizing the distortion of porphyrin conformation and maintaining its iron atom in oxidation state of the six-coordinated low-spin state. Such an interaction was damaged at 60℃although the three-dimensional structure of VHb was still in the state of considerable integrity at this temperature.In our research, we focused on constructing VHb expression vector, purification, structure and function of lipid-bound and lipid-free VHb, and the chemical and physical factors effective on the activity of VHb. It provided technical support and theoretical basis for the VHb's further explain the true intracellular physiological function, molecular mechanism and its structure and function.