Rendiconti Lincei

, Volume 28, Supplement 1, pp 159–167 | Cite as

Heme iron centers in cytochrome P450: structure and catalytic activity

  • Gianfranco GilardiEmail author
  • Giovanna Di Nardo
Concepts in Catalysis


Heme iron centers are found in a wide range of proteins where they play different roles for many crucial biological processes, including catalysis. Among heme-containing enzymes, the cytochromes P450 superfamily comprises members distributed in all domains of life where they participate in the metabolism of endogenous and exogenous compounds. These enzymes can perform a series of oxidative reactions on a broad range of chemically different substrates and for this reason are optimal candidates for biocatalytic purposes and, in general, technological applications. In this review, the general features of these enzymes will be discussed with particular emphasis on the structural insights obtained through X-ray crystallography to understand the key steps of their catalytic mechanism where oxygen is activated. Moreover, one of the finest multi-step reactions catalyzed by the cytochrome P450 aromatase, dealing with the conversion of androgens into estrogens, will be discussed in details. The X-ray structure of this enzyme, together with site-directed mutagenesis experiments has elucidated the role of key residues involved in substrate binding and catalysis. This last example shows how the function and structure in cytochromes P450 are closely inter-correlated to achieve a complex finely tuned catalytic mechanism. It is possible to exploit them for biotechnological applications, but careful attention must be paid in not altering their delicate structure on which their correct function strictly depends.


Cytochromes P450 Heme reactivity Monooxygenases 


  1. Balthazart J, Ball GF (2006) Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci 29:241–249. doi: 10.1016/j.tins.2006.03.004 CrossRefGoogle Scholar
  2. Choi JM, Han SS, Kim HS (2015) Industrial applications of enzyme biocatalysis: current status and future aspects. Biotechnol Adv 33:1443–1454. doi: 10.1016/j.biotechadv.2015.02.014 CrossRefGoogle Scholar
  3. Davis EC, Popper P, Gorski RA (1996) The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain Res 734:10–18. doi: 10.1016/0006-8993(96)00298-3 CrossRefGoogle Scholar
  4. Di Nardo G, Gilardi G (2013) Human aromatase: perspectives in biochemistry and biotechnology. Biotechnol Appl Biochem 60:92–101. doi: 10.1002/bab.1088 CrossRefGoogle Scholar
  5. Di Nardo G, Breitner M, Sadeghi S, Castrignanò S, Mei G, Di Venere A, Nicolai E, Allegra P, Gilardi G (2013) Dynamics and flexibility of human aromatase probed by FTIR and time resolved fluorescence spectroscopy. PLoS One 8:e82118. doi: 10.1371/journal.pone.0082118 CrossRefGoogle Scholar
  6. Di Nardo G, Breitner M, Bandino A, Ghosh D, Jennings GK, Hackett JC, Gilardi G (2015a) Evidence for an elevated aspartate pKa in the active site of human aromatase. J Biol Chem 290:1186–1196. doi: 10.1074/jbc.M114.595108 CrossRefGoogle Scholar
  7. Di Nardo G, Castrignanò S, Sadeghi SJ, Baravalle R, Gilardi G (2015b) Bioelectrochemistry as a tool for the study of aromatization of steroids by human aromatase. Electrochem Comm 52:25–28. doi: 10.1016/j.elecom.2015.01.007 CrossRefGoogle Scholar
  8. Fantuzzi A, Fairhead M, Gilardi G (2004) Direct electrochemistry of immobilised human cytochrome P450 2E1. J Am Chem Soc 126:5040–5041. doi: 10.1021/ja049855s CrossRefGoogle Scholar
  9. Fantuzzi A, Mak LH, Capria E, Dodhia VR, Panicco P, Collins S, Gilardi G (2011) A new standardised electrochemical array for drug metabolic profiling with human cytochromes P450. Anal Chem 83:3831–3839. doi: 10.1021/ac200309q CrossRefGoogle Scholar
  10. Fink G, Sumner BE, McQueen JK, Wilson H, Rosie R (1998) Sex steroid control of mood, mental state and memory. Clin Exp Pharmacol Physiol 25:764–775. doi: 10.1111/j.1440-1681.1998.tb02151.x CrossRefGoogle Scholar
  11. Fisher MT, Scarlata SF, Sligar SG (1985) High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria. Arch Biochem Biophys 240:456–463. doi: 10.1016/0003-9861(85)90050-5 CrossRefGoogle Scholar
  12. Garcia-Segura LM, Azcoitia I, DonCarlos LL (2001) Neuroprotection by estradiol. Prog Neurobiol 63:29–60. doi: 10.1016/S0301-0082(00)00025-3 CrossRefGoogle Scholar
  13. Ghosh D, Griswold J, Erman M, Pangborn W (2009) Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature 457:219–223. doi: 10.1038/nature07614 CrossRefGoogle Scholar
  14. Ghosh D, Lo J, Morton D, Valette D, Xi J, Griswold J, Hubbell S, Egbuta C, Jiang W, An J, Davies HML (2012) Novel aromatase inhibitors by structure-guided design. J Med Chem 55:8464–8476. doi: 10.1021/jm300930n CrossRefGoogle Scholar
  15. Girvan HM, Munro AW (2016) Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology. Curr Opin Chem Biol 31:136–145. doi: 10.1016/j.cbpa.2016.02.018 CrossRefGoogle Scholar
  16. Guengerich FP (2001) Uncommon P450-catalyzed reactions. Curr Drug 2:93–115. doi: 10.1021/tx0002583 CrossRefGoogle Scholar
  17. Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta 1770:330–344. doi: 10.1016/j.bbagen.2006.07.017 CrossRefGoogle Scholar
  18. Hong Y, Yu B, Sherman M, Yuan YC, Zhou D, Chen S (2007) Molecular basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase. Mol Endocrinol 21:401–414. doi: 10.1210/me.2006-0281 CrossRefGoogle Scholar
  19. Horvath TL, Wikler KC (1999) Aromatase in developing sensory systems of the rat brain. J Neuroendocrinol 11:77–84. doi: 10.1046/j.1365-2826.1999.00285.x CrossRefGoogle Scholar
  20. Lamb DC, Lei L, Warrilow AG, Lepesheva GI, Mullins JG, Waterman MR, Kelly SL (2009) The first virally encoded cytochrome P450. J Virol 83:8266–8269. doi: 10.1128/JVI.00289-09 CrossRefGoogle Scholar
  21. Lipscomb JD (1980) Electron paramagnetic resonance detectable states of cytochrome P-450cam. Biochemistry 19:3590–3599. doi: 10.1021/bi00556a027 CrossRefGoogle Scholar
  22. Lo J, Di Nardo G, Griswold J, Egbuta C, Jiang W, Gilardi G, Ghosh D (2013) Structural basis for the functional roles of critical residues in human cytochrome P450 aromatase. Biochemistry 52:5821–5829. doi: 10.1021/bi400669h CrossRefGoogle Scholar
  23. Lombardi P (1995) The irreversible inhibition of aromatase (oestrogen synthetase) by steroidal compounds. Curr Pharm Des 1:23–50. doi: 10.1016/S0925-4439(02)00096-0 Google Scholar
  24. Maurelli S, Chiesa M, Giamello E, Di Nardo G, Ferrero VEV, Gilardi G, Von Doorslaer S (2011) Direct spectroscopy evidence for binding of anastrozole to the iron heme of human aromatase. Peering into mechanism of aromatase inhibition. Chem Comm 47:10737–10739. doi: 10.1039/c1cc13872c CrossRefGoogle Scholar
  25. Mizutani M, Sato F (2011) Unusual P450 reactions in plant secondary metabolism. Arch Biochem Biophys 507:194–203. doi: 10.1016/ CrossRefGoogle Scholar
  26. Naftolin F, Ryan KJ, Davies IJ, Petro Z, Kuhn M (1975) The formation and metabolism of estrogens in brain tissues. Adv Biosci 15:105–121Google Scholar
  27. Nagano S, Poulos TL (2005) Crystallographic study on the dioxygen complex of wild-type and mutant cytochrome P450cam. Implications for the dioxygen activation mechanism. J Biol Chem 280:31659–31663. doi: 10.1074/jbc.M505261200 CrossRefGoogle Scholar
  28. Nahri LO, Fulco AJ (1986) Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem 261:7160–7169Google Scholar
  29. Nelson DR (2009) The cytochrome p450 homepage. Hum Genom 4:59–65. doi: 10.1186/1479-7364-4-1-59 Google Scholar
  30. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. Ii. Solubilization, purification, and properties. J Biol Chem 239:2379–2385Google Scholar
  31. Ortiz de Montellano PR (2015) Cytochrome P450. Structure, Mechanism, and Biochemistry. Springer, New YorkGoogle Scholar
  32. Ortiz de Montellano PR, Nelson SD (2011) Rearrangement reactions catalyzed by cytochrome P450 s. Arch Biochem Biophys 507:95–110. doi: 10.1016/ CrossRefGoogle Scholar
  33. Panicco P, Dodhia VR, Fantuzzi A, Gilardi G (2011) First enzyme-based amperometric platform to determine the polymorphic response in drug metabolism by cytochromes P450. Anal Chem 83:2179–2186. doi: 10.1021/ac200119b CrossRefGoogle Scholar
  34. Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J (1985) The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem. 260(30):16122–16130Google Scholar
  35. Raag R, Poulos TL (1989) Crystal structure of the carbon monoxide-substrate-cytochrome P-450CAM ternary complex. Biochemistry 28(19):7586–7592CrossRefGoogle Scholar
  36. Rittle J, Green MT (2010) Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science 330(6006):933–937. doi: 10.1126/science.1193478 CrossRefGoogle Scholar
  37. Roepke TA, Ronnekleiv OK, Kelly MJ (2011) Physiological consequences of membrane-initiated estrogen signalling in the brain. Front Biosci 16:1560–1573. doi: 10.2741/3805 CrossRefGoogle Scholar
  38. Roselli CE (2007) Brain aromatase: roles in reproduction and neuroprotection. J Steroid Biochem Mol Biol 106:43–150. doi: 10.1016/j.jsbmb.2007.05.014 CrossRefGoogle Scholar
  39. Sadeghi SJ, Fantuzzi A, Gilardi G (2011) Breakthrough in P450 bioelectrochemistry and future perspectives. BBA Protein Proteom 1814:237–248. doi: 10.1016/j.bbapap.2010.07.010 CrossRefGoogle Scholar
  40. Santen RJ, Brodie H, Simpson ER, Siiteri PK, Brodie A (2009) History of aromatase: saga of an important biological mediator and therapeutic target. Endocr Rev 30:343–375. doi: 10.1210/er.2008-0016 CrossRefGoogle Scholar
  41. Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet RM, Ringe D, Petsko GA, Sligar SG (2000) The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287:1615–1622. doi: 10.1126/science.287.5458.1615 CrossRefGoogle Scholar
  42. Schuler MA, Werck-Reichhart D (2003) Functional Genomics of P450s. Ann Rev Plant Biol 54:629–667. doi: 10.1146/annurev.arplant.54.031902.134840 CrossRefGoogle Scholar
  43. Sigel R, Sigel A, Sigel H (2007) The ubiquitous roles of cytochrome p450 proteins: metal ions in life sciences. Wiley, New YorkCrossRefGoogle Scholar
  44. Sohl CD, Guengerich FP (2010) Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1. J Biol Chem 285:17734–17743. doi: 10.1074/jbc.M110.123711 CrossRefGoogle Scholar
  45. Thompson EA Jr, Siiteri PK (1974) The involvement of human placental microsomal cytochrome P-450 in aromatization. J Biol Chem 249:5373–5378Google Scholar
  46. Urlacher VB, Lutz-Wahl S, Schmid RD (2004) Microbial P450 enzymes in biotechnology. Appl Microbiol Biotechnol 64:317–325. doi: 10.1007/s00253-003-1514-1 CrossRefGoogle Scholar
  47. Valetti F, Gilardi G (2004) Directed evolution of enzymes for product chemistry. Nat Prod Rep 21:490–511. doi: 10.1039/b202342n CrossRefGoogle Scholar
  48. Wagner CK, Morrell JI (1997) Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation. J Steroid Biochem Mol Biol 61:307–314. doi: 10.1016/S0960-0760(96)00237-3 CrossRefGoogle Scholar

Copyright information

© Accademia Nazionale dei Lincei 2016

Authors and Affiliations

  1. 1.Department of Life Sciences and Systems BiologyUniversity of TorinoTurinItaly

Personalised recommendations