Cytochrome C Peroxidase Structure - Function Of The Conversion Of Research

Hemoproteins, a large class of metalloproteins which have iron protohemeâ…¨(heme) as a common prosthetic group, play versatile roles in biological systems, such as oxygen transport (e.g. hemoglobin, Hb and myoglobin, Mb), electron transfer (e.g. cytochrome c, Cyt c and cytochrome b₅, Cyt b₅) , 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). By sharing the same prosthetic group, how various hemoproteins can perform a range of functions by means of different combination of heme and protein scaffolds is a major subject of chemical biology and protein chemistry. People, including chemists and biologists, are attempting to answer such a question so as to understand the precise structure-property-reactivity-function relationship of metalloproteins.Cyt c is a typical electron-transfer hemoprotein. In addition to the two axial ligations provided by His18 and Met80, the hydrogen bond and hydrophobic interactions between the heme and the protein polypeptide, the heme also covalently linked to the protein matrix through two thioether bonds which are formed between vinyl groups of the heme and cysteine residues of a classic Cys-Xaa-Xaa-Cys-His (CXXCH) heme-binding motif of the polypeptide chain. Therefore, Cyt c is a very stable protein.It is the stability of Cyt c that makes it not only the typical representative for studying the structure-function relationship, but also an excellent template for structure-function convention study of hemoproteins. In living systems, the main functions of Cyt c are electron transfer and inducing apoptosis, but Cyt c, under certain conditions, can also catalyze peroxidase-like reactions and presents several advantages. For example, Cyt c is able to perform catalytic reactions at high temperature and high concentration of organic solvents. However, compared with natural peroxidase, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), the peroxidase activity of Cyt c is very low because of the differences of their structures. After careful comparison of the structures of Cyt c and HPR, we could find out that there are structural differences between them. HRP has a 5-cooridination high spin heme iron, and a distal histidine as well as a positively charged arginine in the heme pocket, whereas, Cyt c has a 6-coordination low spin heme iron but it lacks the distal histidine as well as the distal arginine in its heme pocket. The structural characters of HRP not only benefit the formation of compound I but also facilitate the access of substrate in the peroxidase reaction cycle.Even though many researches on site-directed mutation and peroxidase reaction of Cyt c have been reported, no one has specialized on how to convert Cyt c into a peroxidase-like enzyme by molecular design. Therefore, by means of molecular design and protein engineering, converting Cyt c, a stable electron-transfer hemoprotein, into an efficient, peroxidase-like metalloenzyme can not only help us to understand the structure-property-reaction-function relationship of hemoproteins but also probably create a new metalloenzyme with potential application in industry.Based on the crystal structure analysis of Cyt c (PDB ID 2YCC), we found that Pro71 is almost located at the distal position of the heme pocket. If it is replaced by a histidine, the distance between Nεatom of His71 and heme ferric iron is～5．62 ?, which is close to the distance between Nεatom of the distal histidine and heme iron in HRP and CcP (～5．84 and～5．55 ?, respectively). Therefore, we first constructed a single-site mutant (Cyt c M80V, M80V) by replacing the heme axial ligand Met80 with valine that has not coordination capacity, and then we introduced a "distal" histidine at position 71 by constructing a double-site mutant of Cyt c P71H/M80V using site-directed mutagenesis.As expected, the peroxidase activity of the Cyt c M80V mutant was enhanced by the vacancy at the sixth ligand site of the heme. In contrast, the introduction of a "distal" histidine in the double-site variant, Cyt c P71H/M80V, did not increase the peroxidase activity at all, but rather caused it to decrease. Therefore, we characterized the Cyt c P71H/M80V variant protein electrochemically and spectroscopically. All the experimental results showed that the introduced histidine at position 71 did not act as a functional distal-histidine, but most likely coordinated to the heme iron, maybe due to the flexibility of theΩloop segment (resides 70-84) of Cyt c protein.Because of failed construction of a distal histidine at position 71 in Cyt c, we further examined the crystal structure of Cyt c and found out that Tyr67 is also situated above the heme plane. At the same time, molecular simulation suggests that the replacement of Tyr67 with histidine should place the Nεof His67 at～5．18 ? from the heme iron, a distance that approximates the distance between the Ns of the distal histidine and the heme iron in HRP and CcP (5．84 and 5．55 ?, respectively). Thus we constructed Cyt c Y67H and Y67H/M80V mutant genes and expressed them to proteins. Kinetic studies showed that the obtained Cyt c variants, Cyt c Y67H and Y67H/M80V have much higher peroxidase activities than the wild-type cytochrome protein. Moreover, the apparent second-order rate constant (K_cat/K_m) toward guaiacol oxidation of Cyt c Y67H was even higher than that of the native horseradish peroxidase under the same conditions.Besides a distal histidine, typical peroxidases, such as HRP and CcP, usually have a distal arginine in the distal heme pocket. In order to mimic this feature we introduced an arginine in the heme pocket of cytochrome c by constructing Cyt c Y67R and Y67R/P71H/M80V variants. The two mutant proteins were expressed, and their peroxidase activities were examined with guaiacol as substrate. The results showed that the introduced arginine indeed enhances the peroxidase activity of Cyt c. However, the Michaelis-Menten constant (K_m) is also increased probably due to the steric effect brought up by the large R-group of arginine which inhibits the substrate access to the heme pocket.In order to further examine the effects of Pro71His mutation on the protein structure and property in the presence of Met80, we prepared another mutant of Cyt c, Cyt c P71H variant protein. UV-visible, CD and resonance Raman spectroscopies indicate that in the oxidized mutant the histidine (His71) serves as the sixth axial ligand, while the Met80 still coordinate to the heme in the reduced form. This study illustrates that the Pro71, in theΩloop D segment, plays important roles in stabilizing the structure of Cyt c. So, further molecular designs are required in order to construct a functional distal histidine at position 71 in the heme pocket of Cyt c.In summary, the above research results not only enrich our knowledge about Cyt c per se, but also extend our understanding of the structure-property-reactivity-function relationships of other hemoproteins.