Biology:Cytochrome c peroxidase

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Cytochrome c peroxidase
Ferrous cytochrome c peroxidase.png
Identifiers
EC number1.11.1.5
CAS number9029-53-2
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Cytochrome c peroxidase
Identifiers
OrganismSaccharomyces cerevisiae
SymbolCCP
UniProtP00431

Cytochrome c peroxidase, or CCP, is a water-soluble heme-containing enzyme of the peroxidase family that takes reducing equivalents from cytochrome c and reduces hydrogen peroxide to water:

CCP + H2O2 + 2 ferrocytochrome c + 2H+ → CCP + 2H2O + 2 ferricytochrome c

CCP can be derived from aerobically grown yeast strains and can be isolated in both native and recombinant forms with high yield from Saccharomyces cerevisiae. The enzyme’s primary function is to eliminate toxic radical molecules produced by the cell which are harmful to biological systems. It works to maintain low concentration levels of hydrogen peroxide, which is generated by the organism naturally through incomplete oxygen reduction. When glucose levels in fast growing yeast strains are exhausted, the cells turn to respiration which raises the concentration of mitochondrial H2O2.[1] In addition to its peroxidase activity, it acts as a sensor and a signaling molecule to exogenous H2O2, which activates mitochondrial catalase activity.[2] In eukaryotes, CCP contain a mono-b-type haem cofactor and is targeted to the intermembrane space of the mitochondria. In prokaryotes, CCP contains a c-type diheme cofactor and is localized to the periplasm of the cell. Both enzymes work to resist peroxide-induced cellular stress.[3]

CCP plays an integral role in enabling inter-protein biological electron transfer. The negative charge transfer process is carried out by a complex formed between cytochrome c and cytochrome c peroxidase which occurs in the inter-membrane space of mitochondria. The mechanism involves ferrous cytochrome c (Cc) providing electrons for the Cc-CcP system to reduce hydrogen peroxide to water.[4] The complex is formed by non-covalent interactions.[5]

Cytochrome c peroxidase can react with hydroperoxides other than hydrogen peroxide, but the reaction rate is much slower than with hydrogen peroxide.

It was first isolated from baker's yeast by R. A. Altschul, Abrams, and Hogness in 1940,[6] though not to purity. The first purified preparation of yeast CCP dates to Takashi Yonetani and his preparation by ion exchange chromatography in the early 1960s. The X-ray structure was the work of Thomas Poulos and coworkers in the late 1970s.[7] CCP is the first heme enzyme to have its structure successfully solved through X-ray crystallography.

The yeast enzyme is a monomer of molecular weight 34,000, containing 293 amino acids, and contains as well a single non-covalently bound heme b. It is negatively charged and is a moderately-sized enzyme (34.2 kDa). The apoenzyme, not active and bound to substrates, has an acidic isoelectric point of pH 5.0-5.2.[8] Unusual for proteins, this enzyme crystallizes when dialysed against distilled water. More so, the enzyme purifies as a consequence of crystallization, making cycles of crystallization an effective final purification step.

Much like catalase, the reaction of cytochrome c peroxidase proceeds through a three-step process, forming first a Compound I and then a Compound II intermediate:

CCP + ROOH → Compound I + ROH + H2O
CCP-compound I + e− + H+ → Compound II
Compound II + e− + H+ → CCP
CCP-catalyzed redox cycle

CCP in the resting state has a ferric heme, and, after the addition of two oxidizing equivalents from a hydroperoxide (usually hydrogen peroxide), it becomes oxidised to a formal oxidation state of +5 (FeV, commonly referred to as ferryl heme. However, both low-temperature magnetic susceptibility measurements and Mössbauer spectroscopy show that the iron in Compound I of CCP is a +4 ferryl iron, with the second oxidising equivalent existing as a long-lived free-radical on the side-chain of the tryptophan residue (Trp-191).[9] In its resting state, the Fe atom (Fe (III)) in the CCP heme is paramagnetic with high spin (S= 5/2). Once the catalytic cycle is initiated, the iron atom is oxidized to form an oxyferryl intermediate (Fe(IV)=O) has low spin (S= 1/2).[4] This is different from most peroxidases, which have the second oxidising equivalent on the porphyrin instead. Compound I of CCP is fairly long-lived, decaying to CCP-compound II with a half-life at room temperature of 40 minutes to a couple hours.

CCP has high sequence identity to the closely related ascorbate peroxidase enzyme.

Amino acid composition

Amino acid analyzer studies reveal presence of residues of Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg, Cys, and Trp in crystalline CCP. The enzyme shows an unusual amino acid pattern compared to other peroxidase. Plant peroxidase such as horseradish peroxidase and pineapple peroxidase B have low lysine, tryptophan, and tyrosine contents and high cysteine content. In contrast, CCP has high lysine, tryptophan, and tyrosine content and low cysteine content.[10] The enzyme contains a 68-residue sequence at the N-terminus of its monomeric protein, which targets it to the inter-membrane space of the mitochondria where it can the complex with cytochrome c and where it carries out its sensor, signaling and catalytic roles.[1] Studies indicate the distal arginine (Arg48), a highly conserved amino acid among peroxidase, plays an important role in the catalytic activity of CCP by controlling its active site through stabilization of the reactive oxyferryl intermediate from control of its access.[11]

References

  1. ↑ 1.0 1.1 "Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria". Proceedings of the National Academy of Sciences of the United States of America 111 (49): 17468–73. December 2014. doi:10.1073/pnas.1409692111. PMID 25422453. Bibcode: 2014PNAS..11117468K. 
  2. ↑ "Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense". Free Radical Biology & Medicine 65: 541–51. December 2013. doi:10.1016/j.freeradbiomed.2013.06.037. PMID 23831190. 
  3. ↑ "Structure, mechanism and physiological roles of bacterial cytochrome c peroxidases". Advances in Microbial Physiology 52: 73–106. 2007. doi:10.1016/S0065-2911(06)52002-8. ISBN 9780120277520. PMID 17027371. 
  4. ↑ 4.0 4.1 "The complex of cytochrome c and cytochrome c peroxidase: the end of the road?". Biochimica et Biophysica Acta (BBA) - Bioenergetics 1807 (11): 1482–503. November 2011. doi:10.1016/j.bbabio.2011.07.010. PMID 21820401. 
  5. ↑ "Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link". Proceedings of the National Academy of Sciences of the United States of America 101 (16): 5940–5. April 2004. doi:10.1073/pnas.0306708101. PMID 15071191. Bibcode: 2004PNAS..101.5940G. 
  6. ↑ "Cytochrome c peroxidase". J. Biol. Chem. 136 (3): 777–794. 1941. doi:10.1016/S0021-9258(18)73036-6. http://www.jbc.org/content/142/1/303.full.pdf. 
  7. ↑ "The crystal structure of cytochrome c peroxidase". The Journal of Biological Chemistry 255 (2): 575–80. January 1980. doi:10.1016/S0021-9258(19)86214-2. PMID 6243281. http://www.jbc.org/content/255/2/575.full.pdf. 
  8. ↑ "Cytochromec Peroxidase". Cytochrome c peroxidase. Advances in Enzymology and Related Areas of Molecular Biology. 33. 1970. pp. 309–35. doi:10.1002/9780470122785.ch6. ISBN 9780470122785. 
  9. ↑ "Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES". Science 245 (4919): 738–40. August 1989. doi:10.1126/science.2549632. PMID 2549632. Bibcode: 1989Sci...245..738S. 
  10. ↑ "Cytochrome c peroxidase. 3. The amino acid composition of cytochrome c peroxidase of Baker's yeast". Acta Chemica Scandinavica 21 (10): 2736–42. 1967. doi:10.3891/acta.chem.scand.21-2736. PMID 5585683. 
  11. ↑ "Directed molecular evolution of cytochrome c peroxidase". Biochemistry 39 (35): 10790–8. September 2000. doi:10.1021/bi001121e. PMID 10978164. 

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