Tuesday, 12 June 2012

Cytochrome P450: Nature's most versatile catalyst by Coon

MULTIPLICITY OF P450 CYTOCHROMES

  • Catalytic changes in induced microsomes may be caused by:
  1. posttranslational modification by proteolysis or adding other chem grps to cytochrome
  2. alter membrane env
  3. change other components that alter rate-lmiting steps eg NADPH-cytochroms P450 reductase, cyt b5 or phospholipids
MICROSOMAL P450 CYTOCHROMES CATALYZE
NUMEROUS REACTIONS WITH COUNTLESS SUBSTRATES

  •  individual steps in the hydroxylation reaction, which has the following overall stoichiometry:

RH + O2 + NADPH + H
+
→ ROH + H2O + NADP
+
,

  • RH is a drug os substrate
  • ROH is product

Figure 2 Joint function of P450 and reductase in drug metabolism. The schemes
account for the oxygenase, oxidase, and peroxygenase reactions of cytochrome P450
with electron transfer from NADPH via the reductase. (A) The reductase cycle is
modified from that in Reference 94 with the model for rapid interflavin electron transfer
in Reference 95. (B) The P450 cycle is based on that in Reference 96. Fe represents the
heme iron atom, RH a drug or other substrate, and ROH the corresponding product.


  • NADPH →FAD → FMN → P450

  • Flavoprotein cycles between 1 e- and 3 e- reduced states during turnover
  • Electron donation to p450 occurs via reaction FMNH2 →FMNH• the semiquinone. 



In Fig 2A
  1. Flavoprotein in fully oxidised state, Primed for fniction when NADPHreduces FAD
  2. Electron redistribution 
  3. give flavin diradical in equilibrium with FMNH2-FAD
  4. which donates an e to P450 ---> Le-reduced flavoprotein
  5. NADPH reduces FAD
  6. Electron redistriubtion
  7. provides FMNH2 as a potential donor to P450
  8. Alternately, FMNH2-FAD may be reduced by NADPH to give FMNH2-FADH2
  9. as a donor
  • FMNH2 provides reducing equivalents for oxygen activation by P450
  • regardless whether FAD is in fully reduced, semiquinone or oxidised state

.
The scheme in Figure 2B includes the basic reaction cycle for oxygen activation proposed in 1980 by White & Coon (98).

  1. Substrate RH binds ferric P450
  2. First ET from FMNH2
  3. diosygen binding
  4. 2nd ET from FMNH2
  5. uptake 2 protons. heterolytic splitting of O-O bond with generation of putative iron-oxene species
  6. proposed formation substrate radical as a transient intmt 
  7. oxygen inserts into substrate
  8. product dissoc with return of P450 to resting state
  9. H2O2 or an alkyl hydroperoxide donates O atom for substrate hydroxyalation. no requirement for O2 or NADPH as e donor. Homolytic cleavage of O-O bond. 
  10. fg
  11. Release products of O2 reduction not coupled to substrate oxygenation eg H2O2
  12. Superoxide
  13. in 4-electron NADPH oxidase reaction, water when (FeO)3+ species is reduced by e from NADPH
  • Ferrous P450 can donate e in stepwise fashion
  • reduce substrate
  • Lipid hydroperoxise, LCH(OOH)R cleaved to yield a ketone and hydrocarbon forms
  • lipid peroxidation is destructuve in membranes of cells
  • Oxidative deformylation of a aldehyde and loss of aldehyde carbon as formate
  • may be possible route for modifying drugs with a carbonyl function eg aldehyde and ketone


MULTIPLE OXIDANTS AND MULTIPLE MECHANISMS
IN P450 CATALYSIS


Figure 3 Versatility in P450 oxygenating species. The iron-oxygen intermediates
in P450 catalysis and their proposed roles as oxidants.

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