Cytochrome c is a highlyconserved protein across the spectrum ofeukaryotic species, found in plants, animals, fungi, and many unicellular organisms. This, along with its small size (molecular weight about 12,000daltons),[7] makes it useful in studies ofcladistics.[8] Cytochrome c has been studied for the glimpse it gives into evolutionary biology.
Cytochrome c has a primary structure consisting of a chain of about 100amino acids. Many higher-order organisms possess a chain of 104 amino acids.[9] The sequence of cytochrome c in humans is identical to that of chimpanzees (our closest relatives), but differs from that of horses.[10]
Cytochrome c has an amino acid sequence that is highly conserved in eukaryotes, varying by only a few residues. In more than thirty species tested in one study, 34 of the 104 amino acids were conserved (identical at their characteristic position).[11] For example, humancytochrome oxidase reacted with wheat cytochromec,in vitro; which held true for all pairs of species tested.[11] In addition, the redox potential of +0.25 volts is the same in all cytochromec molecules studied.[11]
Tunafish cytochrome c crystals (~5 mm long) grown by liquid–liquid diffusion under microgravity conditions in outer space.[12]
Cytochrome c belongs to class I of thec-type cytochrome family[13] and contains a characteristic CXXCH (cysteine-any-any-cysteine-histidine) amino acid motif that binds heme.[14] This motif is located towards theN-terminus of thepeptide chain and contains a histidine as the 5th ligand of the heme iron. The 6th ligand is provided by amethionine residue found towards theC-terminus. The protein backbone is folded into fiveα-helices that are numbered α1-α5 from N-terminus to C-terminus. Helices α3, α4 and α5 are referred to as 50s, 60s and 70s helices, respectively, when referring to mitochondrial cytochrome c.[15]
While most heme proteins are attached to the prosthetic group through iron ion ligation and tertiary interactions, the heme group of cytochrome c makes thioether bonds with twocysteine side chains of the protein.[16] One of the main properties of heme c, which allows cytochrome c to have variety of functions, is its ability to have different reduction potentials in nature. This property determines the kinetics and thermodynamics of an electron transfer reaction.[17]
The dipole moment has an important role in orienting proteins to the proper directions and enhancing their abilities to bind to other molecules.[18][19] The dipole moment of cytochrome c results from a cluster of negatively charged amino acid side chains at the "back" of the enzyme.[19] Despite variations in the number of bound heme groups and variations in sequence, the dipole moment of vertebrate cytochromes c is remarkably conserved. For example, vertebrate cytochromes c all have a dipole moment of approximately 320debye while cytochromes c of plants and insects have a dipole moment of approximately 340 debye.[19]
Cytochrome c is an essential component of the respiratoryelectron transport chain in mitochondria. Theheme group of cytochrome c accepts electrons from thebc1 Complex III and transports them toComplex IV, while it transfers energy in the opposite direction.[citation needed]
Cytochrome c can also catalyze several redox reactions such ashydroxylation andaromaticoxidation, and showsperoxidase activity by oxidation of various electron donors such as 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2-keto-4-thiomethyl butyric acid and 4-aminoantipyrine.[citation needed]
Cytochrome c was also discovered in 1996 byXiaodong Wang to have an intermediate role inapoptosis, a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage.[21]
Cytochrome c binds tocardiolipin in the inner mitochondrial membrane, thus anchoring its presence and keeping it from releasing out of the mitochondria and initiating apoptosis. While the initial attraction between cardiolipin and cytochrome c is electrostatic due to the extreme positive charge on cytochrome c, the final interaction is hydrophobic, where a hydrophobic tail from cardiolipin inserts itself into the hydrophobic portion of cytochrome c.[citation needed]
During the early phase of apoptosis, mitochondrial ROS production is stimulated, and cardiolipin is oxidized by a peroxidase function of the cardiolipin–cytochrome c complex. The hemoprotein is then detached from the mitochondrial inner membrane and can be extruded into the soluble cytoplasm through pores in the outer membrane.[22]
The sustained elevation incalcium levels precedes cytc release from the mitochondria. The release of small amounts of cytc leads to an interaction with theIP3 receptor (IP3R) on theendoplasmic reticulum (ER), causing ER calcium release. The overall increase in calcium triggers a massive release of cytc, which then acts in the positive feedback loop to maintain ER calcium release through the IP3Rs.[23] This explains how the ER calcium release can reach cytotoxic levels. This release of cytochrome c in turn activatescaspase 9, a cysteineprotease. Caspase 9 can then go on to activatecaspase 3 andcaspase 7, which destroy the cell from within.[citation needed]
One of the ways cell apoptosis is activated is by release of cytochrome c from the mitochondria into cytosol. A study has shown that cells are able to protect themselves from apoptosis by blocking the release of cytochrome c using Bcl-xL.[24] Another way that cells can control apoptosis is by phosphorylation of Tyr48, which turns cytochrome c into an anti-apoptotic switch.[25]
In addition to its well-known roles in the electron transport chain and cell apoptosis, according to a 2008 study cytochrome c can also act as an antioxidative enzyme in the mitochondria; it does so by removingsuperoxide (O−2) andhydrogen peroxide (H2O2) frommitochondria.[26] Therefore, not only is cytochrome c required in the mitochondria for cellular respiration, but it is also needed in the mitochondria to limit the production ofO−2 andH2O2.[26]
Cytochrome c is widely believed to be localised solely in the mitochondrial intermembrane space under normal physiological conditions.[27] The release of cytochrome c from mitochondria to the cytosol, where it activates thecaspase family ofproteases, is believed to be the primary trigger leading to the onset of apoptosis.[28] Measuring the amount of cytochrome c leaking from mitochondria to cytosol, and out of the cell to culture medium, is a sensitive method to monitor the degree of apoptosis.[29][30] However, detailed immuno-electronmicroscopic studies with rat tissues sections employing cytochrome c specific antibodies provide compelling evidence that cytochrome c under normal cellular conditions is also present at extramitochondrial locations.[31] In pancreatic acinar cells and theanterior pituitary, strong and specific presence of cytochrome c was detected inzymogen granules and ingrowth hormone granules, respectively. In the pancreas, cytochrome c was also found in condensingvacuoles and in the acinarlumen. The extramitochondrial localisation of cytochrome c was shown to be specific as it was completely abolished upon adsorption of the primary antibody with purified cytochrome c.[31] Besides cytochrome c, extramitochondrial localisation has also been observed for large numbers of other proteins including those encoded by mitochondrial DNA.[32][33][34] This raises the possibility of the existence of yet-unidentified specific mechanisms for protein translocation from mitochondria to other cellular destinations.[34][35]
Cytochrome c has been used to detect peroxide production in biological systems. As superoxide is produced, the number of oxidised cytochrome c3+ increases, and reduced cytochrome c2+ decreases.[36] However, superoxide is often produced with nitric oxide. In the presence of nitric oxide, the reduction of cytochrome c3+ is inhibited.[37] This leads to the oxidisation of cytochrome c2+ to cytochrome c3+ byperoxynitrous acid, an intermediate made through the reaction of nitric oxide and superoxide.[37] Presence ofperoxynitrite or H2O2 andnitrogen dioxide NO2 in the mitochondria can be lethal since they nitratetyrosine residues of cytochrome c, which leads to disruption of cytochrome c's function as an electron carrier in the electron transport chain.[38]
Cytochrome C has also been widely studied as an enzyme with peroxidase-like activity. Cytochrome C was conjugated to charged polymer to test its peroxidase-like activity.[39][40] Inspired from natural examples of enzyme encapsulation in protein-based cage structures (Example: Carboxysomes, Ferritin, and Encapsulin), Cytochrome C was encapsulated in a 9 nm small self-assembling DNA binding protein from nutrient starved cells (Dps) protein cage using chimeric self-assembly approach. Authors observed unique catalytic activity behavior upon encapsulating enzyme inside a protein-cage, which was different from enzyme in solution. This was attributed to local microenvironment provided by Dps nanocage's interior cavity which is different than bulk.[41]
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^Domazou AS, Gebicka L, Didik J, Gebicki JL, van der Meijden B, Koppenol WH (April 2014). "The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c".Free Radical Biology & Medicine.69:172–80.doi:10.1016/j.freeradbiomed.2014.01.014.PMID24447894.
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