| Cytochrome c (Cyt c) is a 13 kDa small hemoprotein which is located between the inner and outer membrane of the mitochondrion. It is highly conserved across the spectrum of species, found in bacteria, yeast, plants and animals. Cyt c is the only soluble protein in the cytochrome family of proteins. The iron atom in the center of its heme group is coordinated by the nitrogen of His18, the sulfur of Met80 and four nitrogen atoms of the porphyrin. Meanwhile, the c-type heme also covalently linked to the protein matrix through two thioether bonds which are formed between vinyl groups of the heme and two cysteine residues, Cys14 and Cys17. The heme is closely surrounded by the protein matrix, which brings Cyt c a globular shape. These structural characteristics make Cyt c a very stable protein.The ancient protein Cyt c is an essential component in the energy metabolism pathway. It transfers electrons between cytochrome c reductase and cytochrome c oxidase. Although thoroughly studied as an electron carrier in the fundamental metabolic process of oxidative phosphorylation, only recently was it found to play central roles in both pro-apoptotic and apoptotic pathways, opening the door to death. In 1996, Wang et al. found that in the beginning of apoptosis, Cyt c would be released from mitochondria and bind apoptotic protease activating factor-1 (Apaf-1), which forms apoptosome and activates caspsase-9 and other caspases and initiates apoptosis. In 2005, Kagan's group further found that Cyt c also takes part in a so-called "pro-apoptotic pathway. When Cyt c binds cardiolipin (CL), mitochondriaspecific phospholipid, it undergoes a conformational change and acquires peroxidase activity. The catalytic Cyt c peroxidizes CL to its oxidized form (oxidized-CL), which is required for the release of pro-apoptotic factors from mitochondria, and for the execution of the subsequent apoptotic steps. These finding revealed that Cyt c dominates not only survival but also the death of the cell, reignited an intense research interest of this ancient protein.Recently, conformational transitions of Cyt c are being realized to be responsible for its multi-functions. Two kinds of conformational transitions are the most noticed. One is called "pro-apoptotic conformational transition", and the other is "alkaline conformational transition". The molecular mechanism for the pro-apoptotic conformational transition is unknown, and the mechanism and the real biological consequence of the alkaline conformational transition are also unrecognized. Thus, in order to explore their mechanisms and functions, we investigated these two conformation transitions systematically.The pro-apoptotic conformational transition is a kind of conformational change when Cyt c binds the membrane of mitochondria. This conformational change is closely associated with the peroxidase activity, and the mechanism of this conformational change is completely unknown. We noticed that in Cyt c, Tyr67 forms a hydrogen bond network with the axial ligand Met80 and conserved Asn52 and Thr78. Interestingly, these two loops which these residues were located (residues 40-57Ωloop and residues 71-85Ωloop) were recently found to be the least stable and fastest unfolding units in Cyt c, and to participate in two initial steps in a stepwise unfolding pathway. Clearly, this hydrogen bond network of Cyt c possesses an important biological consequence. In order to further verify the importance of the hydrogen bond network and to reveal its biological implications, two non-conserved mutants Y67H and Y67R of yeast iso-1 Cyt c were developed. Through property characterization via UV-Vis, CD and fluorescence spectra, we found these two proteins were easily to undergo conformational changes. With increasing temperature, the secondary structures of the proteins changed heavily while their structures kept intact. The Tm of the conformational transition of the Y67H variant is 50℃and the Tm of the conformational transition of the Y67R variant is 30℃. By the kinetics measurement of their peoxidase activities, we found the conformational transitions were highly related to their peroxidase activities. With increasing temperature, the peroxidase activity of the Y67H variant underwent 10-fold increased, and the Y67R variant increased over 50-fold. As the first evidence for the correlation between peroxidase activity and conformational change of Cyt c under the physiological conditions at neutral pH and without denaturant, ours results suggest that the hydrogen bond network associated with Tyr67 is involved in the conformational switch and thus orients the protein to the apoptotic pathway. The interaction of Cyt c with cardiolipin leads to a damage of the hydrogen bond network associated with Tyr67, triggering the conformational transition and inducing peroxidase activity, and thereby initiating apoptosis by releasing Cyt c.Besides, the equilibrium and kinetics of the alkaline transition of human Cyt c have been systematically investigated for the first time in this thesis. Cyt c is highly conserved across the spectrum of species, and it does not undergo much alteration from yeast to human. As human Cyt c is hard to obtain, Cyt c from some other species, such as yeast and horse, are often selected for research instead of human Cyt c. However, as recently found to be involved in apoptotic and pro-apoptotic pathway, Cyt c is being realized to possess great potential applications in biomedical area. The cDNA of human Cyt c is cloned by RT-PCR for the first time and a high-effective expression system for human Cyt c has been developed in this study. Besides, we have also cloned the Cyt c-binding region of Apaf-1, and developed an effective expression system for soluble proteins, which would facilitate further investigation of the mechanism for the interactions between Cyt c and Apaf-1. With the expression and purification system for human Cyt c, we successfully obtained human Cyt c with the yield of 20 mg per liter culture. The equilibrium and kinetics of the alkaline transition of human Cyt c have been systematically investigated for the first time, and compared with those of yeast and horse Cyt c from an evolutionary perspective. We found that although human Cyt c possessed similar spectral property with yeast and horse Cyt c, their alkaline transition showed apparently difference. The pKa value for the alkaline transition of human Cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of Cyt c from yeast, horse and human. Molecular modeling of human Cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human Cyt c than in yeast iso-1 and horse heart Cyt c. Although the driving force governing the rapid transition of the primate Cyt c molecule to its current human form remains elusive, it is evident that the threshold for alkaline transition was elevated along this evolution. Understanding the driving force for this elevation is an interesting issue that needs further investigation. These results clearly demonstrate that the alkaline transition in Cyt c has great biological implications, which invokes our further interested aimed at understanding the alkaline transition in Cyt c.Although considerable effort has been devoted to the study of the alkaline conformational transition in Cyt c, its real biological consequence is still unknown. Now it is widely accepted that the alkaline conformational transition obeys to a two-step mechanism. Firstly, the deprotonation of some group (s) leads to an intermediate state. Then, the intermediate state undergoes a conformational equilibrium and transform to the alkaline conformer. The most significant and controversial issue is concerning the identification of the "trigger group", whose deprotonation is a key requirement for the pathway of alkaline transition, initiating the conformational change in the native Cyt c. Several titratable groups have been suggested, such as Lys72/73/79, Tyr67 or the water molecule connected to Tyr67. We have found that the Y67H and Y67R mutants of Cyt c can be easily switched to a state with high peroxidase activity, indicating that the maintenance of the inner hydrogen bond network associated with Tyr67 is probably the trigger for pro-apoptotic conformational transition in Cyt c. Concerning the alkaline conformational transition in Cyt c, we noticed that Tyr67 is also supposed to be one of the "trigger group" candidates for alkaline transition. Therefore, the Cyt c Tyr67-variants would be a unique example to gain insights into the underlying mechanism for the alkaline transition, and to distinct the two different conformational transitions mentioned above. For this study, the mutant proteins, Y67H/M80V and Y67H/M80D were also developed based on the Y67H variant of yeast iso-1 Cyt c. If Tyr67 could serve as a trigger, the mechanism of the alkaline conformational transition in these mutants would surely be changed. However, by thermodynamics and kinetics characterization of the variants, we found the variants obey the same mechanism with the wild-type Cyt c. Thus, our results demonstrate for the first time that Tyr67 is not the trigger group in the alkaline conformational transition in Cyt c. The alkaline conformational transitions in the Y67H and Y67H/M80D variants are more difficult than the wild-type Cyt c. Tyr67 is the trigger of the pro-apoptotic conformational transition, but it is not the alkaline conformational transition. The Tyr67 variants are easy to undergo pro-apoptotic conformational transition, but hard to undergo alkaline transition. These results demonstrate for the first time that these two conformational transitions undergo distinct mechanism. Besides, we found the peroxidase activities of the variants decreased with increasing pH values. Some interesting biological consequences may emerge from these studies. A large proportion of Cyt c is electrostatically anchored to the anionic cardiolipin molecules in mitochondria, probably by its surface Lys72 and Lys73 residues. Some special biological events could destroy the inner hydrogen bond network in Cyt c, confer the protein high peroxidase activity and make the cell death. The cellular circumstances governing this process has as yet escaped clear definition, however, elaborate molecular machineries must be involved, controlling and regulating the transition between death-promoting and life-supporting functions in Cyt c. Based on the observations in this communication, now we understand that Cyt c has the potential to make another distinct response from its pro-death conformational transition. By deprotonating the inner trigger group, a conformational equilibrium is initiated in which the surface lysine (Lys72, Lys73 or Lys79) could coordinate to the heme prosthetic group. The peroxidase activity as well as the redox potential of Cyt c is prominently reduced during this transition. Thus, we prefer to the proposal that the alkaline conformational transition may serve as a protective mechanism for Cyt c to the abnormal enhancement in peroxidase activity and to avoid to get bogged down in the apoptotic route.The other project in this thesis is to construct a kind of novel hybridized hemoproteins. One of the fundamental purposes in protein engineering is to develop stable and catalytic enzymes for industrial applications. Metalloproteins, which account for nearly half of all proteins in nature, are particularly attractive in engineering because of their efficiency and diversity in function utilizing metal ions or metal-containing cofactors such as heme, and their ubiquitous usage in vivo such as biocatalysis, energy metabolism and signal transduction. Thus, we have been exploring a new route to novel functional metalloproteins which involves constructing hybridized protein scaffolds from different hemoproteins. The hybridized scaffolds contain specific motifs by rational design, and the resulting superb proteins possess merits from distinct metalloproteins. Cyt c, which has remarkable stability attributed to its compact globular structure and covalently bound heme group, could serve as an excellent structural framework in engineering. Besides, we took notice of CYP450, one of the most versatile enzymes in nature, which could efficiently catalyze hundreds of different substrates via a variety of difficult biotransformations such as epoxidations and hydroxylations. Thus, in order to acquire stable and catalytic proteins, the SRSs of CYP450 were constructed into the scaffold of yeast iso-1 Cyt c via overlap extensition PCR method, respectively, replacing its residues 78-85 loop. This loop is one of the least stable and fastest unfolding units in Cyt c and just located at the only opening crevice of its heme pocket. The resulting hybrid proteins were named HY1 (the hybrid protein of CYP450 SRS-1 with Cyt c), HY2, HY3, HY5 and HY6, respectively. By UV-Vis, CD and fluorescence spectral characterization of the hybrid proteins, we found all these hybrids exhibited superb stability. To evaluate whether the hybrid proteins acquire CYP450 activities, styrene, a typical substrate for CYP450 was applied. Through GC-MS characterization we found that while Cyt c, HY2, HY3 and HY5 could not catalyze styrene, HY1 and HY6 could catalyze styrene to styrene oxide. Besides, by the kinetics measurement of their peoxidase activities, we found the peoxidase activity of HY6 was 10-fold increased by Cyt c, and the peroxidase activity of HYl was 40-fold increased by Cyt c. These results indicate that we have developed some novel hybrid metalloproteins by constructing substrate recognition sites of CYP450 into the framework of Cyt c. These hybrids exhibit superb stability and catalytic activity. Structural work is currently underway in our lab to elucidate the enzymatic mechanism and facilitate further construction. |
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