Research On Vibrio Cholera HutX Protein Purification And The Combination With Heme

The ability of capturing iron plays an important role to the survival and multiplicationof Vibrio Cholera. The content of iron in environment is very rich, but because of itsinstability, iron is easy to have an oxidation-reduction reaction with water and oxygen innature and the product is hard to be utilized by microorganism. In human body, largeamount of iron exists in the form of a combination with protein and the amount of free ironis measured not to reach the level which meets suvival, growth and multiplication ofVibrio Cholera. So in order to survive, developing the ability of capturing iron from hemeis one key feature for Vibrio Cholera.Now the mechanism of capturing and transporting heme has been well known. A largeamount of researches demonstrate that a high affinity protein, siderophore, can besynthesized by Vibrio Cholera to capture the heme from host and then thesiderophore-heme complex activates the specific receptor on the surface of Vibrio Choleraouter membrane, and then siderophore-heme complex is brought back to periplasm byTonB system, delivered to BPB and finally be transported into cell through ATP-bindingcasette. However, the heme brought inside the cell can not be used directly and aggregationis toxic to cell membrane. Therefore, degradation of heme before utilization is needed, butwe know little about the destination after heme entering cell.HutZ protein of Vibrio Cholera has been reported to be involved in the process ofdegradating heme in cell recently, and it plays the role of degrading enzyme or storage.Because the HutZ protein shares the same promoter with HutZ protein and translatedsimultaneously, it is reasonable to speculate that HutX protein should be also involved inthe process of utilizing heme in cell. In addition, HutX protein belongs to HutX-ChuXsuperfamily, in which all members have been proved to have a conserved domainDUF1008and have the ability to combine with heme according to the previous study, suchas HemS protein is considered to be heme-degrading enzyme. Also as the homologousprotein of HutX as well as a member of HutX-ChuX superfamily, ChuX has been provedto have the ability to combine with heme and serve as the role of temporary storage in E.coli cells for buffing the possible toxicity from heme molecular, which is believed todamage the membrane structure through promoting non-redox reaction. All the previous studies surpport our hypothesis that HutX protein can combine with heme potentially andgets involved in the process of utiliztion of heme.Objective:To test the heme-binding ability of HutX protein in Vibrio cholerae, exploring theheme-binding corresonding tructure of HutX protein, and on the basis of study to speculatethe role that HutX protein plays in the process of iron taking, transportation and utilizationin the cell of Vibrio cholerae.Methods:In order to verify our hypothesis, the genome of O1biovar EI Tor str. N16961ofVibrio Cholera was used as template to express HutX protein. After nickcl sepharoseaffinity chromatography, anion exchange chromatography (source Q), molecular sievechromatography (Superdex200) and identification by western blotting analysis, thepurified HutX protein was mixed with heme. The mixture was measured by UV-visspectrophotometer. Further, we located the combining points according to the structure ofhomologues. The mutations were mixed with heme before the measurement separately byUV-vis spectrophotometer, and the results were compared with that of wild-typeHutX-heme complex.Results:Through the verification tests, the purified substance we get finally is HutX protein.The results of wild HutX protein-heme complex UV-visible spectroscopy show that thereare two crests visible, one big, another small, which are corresponding the typically patternof six-coordinate bonds according to the well studied spectral results of other substancesand homologues of HutX protein. Six-coordinate bonding pattern means the stability of thecombination, not easy to react with other substances. However, it is noticed that the smallcrest is much smaller than the second crest of six-coordinate bonding spectral result,similar to the spectrum of five-coordinate bonds, which indicated that not onlysix-coordinate bonds exist in the HutX protein-heme complex, but also five-coordinatebonds existing. So basis on the analysis above, we demonstrates that the HutX protein inVibrio cholerae cells has the ability to combine with heme moleculaer with a pattern ofmixing five-coordinate bonds and six-coordinate bonds. In solution, two kinds of bondingpattern reach balance, similar to the previous result of ChuX protein, which has the most similar amino acid residues sequence according to the protein sequence analysis. Furtherthrough analysizing the well-known tructure of homologues basis on the previous studiesusing the way of3D reconstruction of ChuX protein, we speculate that the potential hemecombining pots probabily including H103and F119. Separately mutating the two potentialheme combinding points, we find that the two crests of HutX protein-heme complexabsolutely disappeared, instead of one high crest at about200nm left, which is believed tobe the spectral result of HutX dimer. That is to say, we find that the HutX protein losesthe ability to combine with heme any more. It is indicated that the amino acid residuesH103and F119are the key points for HutX protein combinding with heme molecular.Conclusions:our study declaims that the HutX protein has the ability of combinding with hememolecular. However, due to the instability of the HutX-heme complex, the iron which is atthe center part of the complex is at the state of activitation, that is to say, the iron in thecomplex can easily react with other compounds and take off from the HutX-proteincomplex. Because of the feature of HutX protein, it is thought that the HutX proteinprobabily has the function of storing heme taken up by vibrio cholerae temporarily, for ashort time in order to lower the amount of heme in cell for the protection of cell from hemetoxicity. It is notice that the mixing five-coordinate bonds and six-coordinate bonds patternof HutX protein-heme complex is good for its function, combining with heme to protectthe cell and easy to take the heme away for iron utilization. The result of our study providea priori knowledge for the further studies on the HutX function and the whole process andmechanism of iron utilization in Vibrio Cholera.