Thursday, 14 June 2012

Strucuture-function relationships in heme proteins by Paoli

  • Heme redox potential is determined by molecular env and axial ligands of iron
  • in cytochromes when axial ligand if samem diff midpoint potentials
  • in multiheme cyts iron is coordinated by His
  • in c-type monoheme cyts his and met ligands are used
  • all c-type hemes in hydroxylamine oxidoreductase have bishistidyl ligation
  • redox midpoint potentials vary form +290 to -410mV
  • Heme redox potentials in these mols range over 1V
  • Heme reactivity varies according to factors:
  1. substituent grps on porphyrin ring
  2. axial ligands to iron
  3. hydrophobic env and electrostatic effects
  4. heme exposure (solvent accessibility)
Ligation of heme iron
  • axial ligands control redox potentials
  • electrostatic effects on metal centre
  • Hbonding to iron's proximal ligand affects distribution of charge and strength of ligand-metal bond
  • In heme proteins, 3 residues are used as proximal ligands
  • his in blogins and peroxidases
  • tyrosine in catalses
  • cysteine in chloroperoxidases and cyt P450 monooxygenases
  • in globins, proximal His donates a H bond to a main chain carbonyl O
  • in peroxidases it interacts with Aspartate
  • Interaction with -ve charged Asp ---> stronger H bond than in globins ---> better e donation to metal centre
  • Contribute to ability of peroxidases to stabilises higher oxidation states of iron during push-pull concept
  • Push comes from e donation by proximal ligand
  • Pull is due to nature of distal grps
  • eg arg in heme-peroxidases
  • polarise peroxide O-O bond
  • In catalase, proximal tyr accepts a H bond from an arg
  • in chloroperoxidase and cyt P450 proximal cys is engaged in H bonding interactions with 2 main chain amide grps
  • CAtalse and chloroperoxidase have electropositive proximal env
  • interactions decrease -ve charge on phenolate and thiolate ligands
  • Contributes to high redox potential of proteins
  • Mutate 1 His of 4 bis-histidyl coordinated hemes  of tetra-heme cyt c3 to Met ---> increase of 200mV in one of 4 redox potentials
  • when methionine-histidyl kligation of mitochondrial cyt c was changed to bis-histidyl ligation ---> decrease of mV in midpoint portential
  • bis-histidyl coordination in c type cyts assoc with lower redox potentials than met-His coordination
Hydrophobic env and electrostatic effects
  • Heme proteins decrease their midpoint potential by increasing polarity of heme env
  • stabilise more highly charged oxidised state.
  • in cyt f from diff species high conservation of most residues in heme pocket except where trp, phe, leu and val are observed
  • trp4 mutated to phe in cyt f from Phormidium lamiosum
  • Phe at sam eposition in cyt f from Chlamdomonas reinhardtii changed to trp
  • wt str shows indole ring or benzyl ring extends over tetrapyrrole perpendicular to heme plane
  • in trp ---> phe mutant, redox potential increases from 297 to 323 mV
  • in phe ---> trp mutant, redoc potential decreases from 370 to 336 mV.
  • decreased midpoint potential induced by trp may be caused by indole ring's pi interactions with porphyrin
  • stabilised oxidised form of heme
  • thru electrostatic repulsion with Fe orbitals
  • Redox properties of heme also governed by electrostatic field from neighbouring hemes
Heme exposure and solvent accessibility
  • level of heme exposure to solvent governs redox potentials
  • as exposure to water increaes (polarity of heme env increases), midpoint potentials decrease
  • In try to phe mutation at position 67 in cyt c, rearranged H bond network and atomic shifts
  • a water mol is bound internally, in addition to water mol already present in wt
  • loss of one hydroxyl grp increases polarity of heme env
  • decrease redox potential

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