Cytochrome B <sub> 5 </ Sub> To The Structural Transformation Of Cytochrome C And The Different Expression System, Its Structure, Nature And Function Of The Impact

Hemoproteins are a class of metalloproteins that contain protoheme IX (heme) as the prosthetic group, which play crucial roles in living life, such as oxygen transport (e.g. hemoglobin, Hb and myoglobin, Mb), electron transfer (e.g. cytochrome b₅, cyt b₅ and cytochrome c, cyt c), biological catalysis (e.g. cytochrome P450, cyt P450 and cytochrome c peroxidase, CcP) and biological sensor (e.g. CooA for CO and sGC for NO). How the same heme prosthetic group used by different hemoproteins to perform a range of functions, is major subject of chemical biology and protein chemistry. People are attempting to answer this question for understanding the precise structure-property- reactivity-function relationships of metalloproteins.Cyt b₅ is a typical electron-transfer hemoprotein that associates a heme prosthetic group non-covalently. The heme-holding ability depends mostly on the strong axial ligation provided by residues His63 and His39, and on the hydrogen and hydrophobic interactions as well. Cyt b₅ is different from cyt c, in which two thioether bonds were formed between heme vinyl groups and the cysteine residues of a classic Cys-Xaa-Xaa-Cys-His (CXXCH) heme-binding peptide motif within the polypeptide chain, resulting in covalent attachment of the heme group to the protein matrix. At the same time, the heme of cyt b₅ is relatively more exposed to solvent in comparison with cyt c or Mb. All these factors lead to a lower heme-binding stability of cyt b₅.From the past research of cyt bs, we can find that alteration studies on the axial ligation of cyt b₅ have been somewhat limited due to the fact that such alteration would lead to the substantial decrease of the heme-binding stability of cyt b₅. As a result, we could not obtain the holo-protein, while obtains the apo-protein of the axial residue mutants that fail to incorporate heme during fermentation. In these cases, the low stability of apo-proteins combined with the absence of typical red color makes the purification procedures more complicated compared with the wild-type cyt b₅. Much time and effort should be invested in order to obtain large quantities of desired proteins with high purity necessary for biochemical and biophysical studies.However, many studies on the heme axial ligands of hemoproteins are related to their structures, properties and functions. Therefore, how to improve the heme-binding stability of cyt b₅, and how to simplify the purification procedures for cyt b₅, especially how to use a simple method to obtain the apo-cyt b₅ axial residue mutants, which are all subjects deserved to pursue.Because there is no classic CXXCH heme-binding peptide motif in cyt b₅ as that found in cyt c, and there is even no cysteine residue within the polypeptide chain of cyt b₅, the formation of thioether bond as formed in cyt c is impossible for cyt b₅. By a closer examination of the X-ray crystallography of cyt b₅, we found that the residue Asn57 and Ser71 are close to heme 4-vinyl and 2-vinyl groups with distances of 3.86 and 4.32 A, respectively, which might be suitable for covalent bond formation if these two residues are replaced with cysteine. It is probable that the introduced cysteine residues would form the thioether bond as in cyt c with the heme vinyl groups during fermentation. Therefore, we constructed the cyt b₅ N57C, cyt b₅ S71C and cyt b₅ N57C/S71C mutants by means of site-directed mutagenesis, and further characterized these variants.Our study demonstrates that by introducing cysteine residue close in space to the heme vinyl groups and expressed in vivo, the heme group could be covalently attached to the protein matrix of cyt b₅, where two different kinds of cysteine-heme covalent linkages were formed: cysteine-57 forms the typical thioether linkage with heme 4-vinyl group, while cysteine-71 forms an unusual [heme-CO-CH2-S-CH₂-C_α] linkage with heme 2-vinyl group. The difference mainly depends on distance between the heme vinyl groups and cysteine residues, the vinyl group spatial positioning and their microenvironments. This study also showed that cyt b₅ could be converted into a cyt c-like protein without constructing the classic CXXCH heme-binding motif of cyt c, which demonstrates that the classic heme-binding peptide motif CXXCH is not essential for heme covalent attachment to protein matrix in cytochromes. In addition, we find that with heme covalently linked, the constructed cyt c-like cyt b₅ displays high peroxidase activity in its unfolded form. All these findings provide us more information for understanding the formation mechanism of a cyt c-like protein and for delineating the structure-property-reactivity-function relationship of hemoproteins.In order to simplify the purification procedures for preparation of cyt b₅, we tested the possibility of using the glutathione S-transferase system to express cyt b₅ in E. coli as a GST-cyt b₅ fusion protein and then purify it. The results showed that cyt b₅ can be successfully expressed as a GST-cyt b₅ fusion protein, and it can be matured in vivo as a holo-protein with heme incorporated into cyt b₅ during the fermentation. The GST-cyt b₅ fusion express system has many advantages over other systems in that the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. At the same time, characterization ofthe fusion protein reveals that the protein-protein interactions between GST and cyt b₅ are apparently very weak, and the fusion of them has not made significant structural and functional alterations of their counterparts. The GST-cyt b₅ fusion protein shares similar properties and functions to that of isolated cyt b₅. On the other hand, it retains the binding capacity of GST to glutathione, which makes it have double functions and applications. However, when expressing the axial residue mutants of GST-cyt b₅ fusion protein, we can only obtain the apo-protein of the mutants. Furthermore, the high affinity of GST to heme has dramatically affected the reconstitution of the apo-protein of GST-cyt b₅ mutants with heme in vitro. Therefore, the GST-cyt b₅ fusion expression system was not suitable to obtain the holo-protein of GST-cyt b₅ mutants that related to the alteration of cyt b₅ axial ligands.Furthermore, we have also used the histidine-tagged expression system by introducing a six-histidine tag to the N-terminus of cyt b₅ to facilitate the purification on a nickel affinity column. The results showed that (His)₆-cyt b₅ has similar spectroscopic and electrochemical properties as well as the chemical stability to that of cyt b₅, which suggests the introduction of (His)6-tag has not altered the structure, property and function of cyt b₅ significantly. At the same time, the (His)₆-tag has not affected the in vitro reconstitution of the apo-protein with heme. Therefore, this method is more suitable to obtain the holo-protein of cyt b₅ axial residue mutants. Using this method, we have obtained the (His)₆-cyt b₅ H39G single-mutant and (His)₆-cyt b₅ H39G/G42H double-site mutant where the positions of the native heme axial ligand His39 and Gly42 were reversed. Characterization of the mutants reveals that after removed from the amino acid position 39 to 42, the original axial histidine still can act as an axial ligand for the heme group, while the heme ligand provided by His42 is considerably weaker than the original axial His39. This study illustrates that the polypeptide chain of cyt b₅ has some flexibility, and it is more difficulty to construct a distal histidine within the heme pocket of cyt b₅ than the case in myoglobin that has a comparatively more rigid protein scaffold. All these findings further extend our understanding of the structure-property-reactivity-function relationships of cyt 65.