Tuesday, 5 June 2012

Structure function relationships in heam proteins by Paoli


CYTOCHROMES  AND OTHER REDOX HEME-PROTEINS: CONTROL OF REDOX PROPERTIES

  • ET reactions take place along potential gradient
          ---extremes determined by reduction potentials of cofactors
  • In catalysis redox properties of heme are exploited by oxygenases and peroxidases
  • Cytochrome c extract O atoms from O2
           --- oxidise organic substrates
  • Redox functions of heme proteins governed by precise protein heme interactions that modeulate chem of iron centre
  • Change in substitutent grps at peripery of porphyrin ring ---> diff types of iron porphyrin dericatives
          --- eg b-heme in globins, catalses, peroxidases
  • This heme is in b-type cytochromes eg cyt b5
  • Most of proteins in ET reactions contain c-hemes covalently bound to protein
  • Linkage via 2 thioether bonds between Cys residues and 19 carbon atoms of vinyl grps in pyrrole rings 2 and 4
  • Covalent assoc involves conserved CxxCH motif
  • In cytochrome c oxidase contains a type hemes
          --- involve in reduction of O2 to water
          --- electrogenic transfer of protons across membrane
  • Heme is bound with same canonical cytochrome c folding topology
  • simple a-helical globular fold
  • In chloroplast cytochrome f,  redox partner of cytochrome b6 and e donor to plastocyanin, c-type heme is covalently linked to unusual fold
  • In cytochrome f, c type heme iron is coordinated at 6th ligand position by N terminal amino grp of protein
  • Protein heme frameworks indicate molecular pathways for controlled transfer of electrons alone precisely positioned prosthetic grps with release of free energy.
  • All c type cytochromes bind thru CxxCH motif
  • Formation of covalent protein-heme thioether bonds requires spec enz eg cytochrome c and c1 heme-lyases
  • Enz involved in import of apo-forms of mitochrondrial cytochromes from cytosol ---> synthesised there
  • Heme lyases contain CPV motifs
  • may be involved in heme binding
  • Heme mol may be more stably anchored
  • mutations in heme pocket better tolerated ---> evo advantage
  • can finetune and optimise redox properties
  • Redox potential of heme determined by nature of its molecular env and axial ligands of iron
  • In multiheme cytochromes the iron is generally coordinated by histidines, 
          ---- unlike most c type mono heme cytochromes where histidine and methionine ligands are used.
  • In cytochrome c3 from sulfur reducing bac all 4 hemes have His ligands to iron atoms
          ---- but midpoint potentials range from 2140 to 2380 mV
  • Reactivity of hemes varies according to following factors:
  1. substituent grps on porphyrin ring
  2. axial ligands to the iron
  3. hydrophobic env and electrostatic effects
  4. heme exposure (solvent accessibility)
LIGATION OF THE HEME IRON
  • Axial important in controlling redox potential
  • electronic effects on metal centre
  • H bonding iron's proximal ligand affects charge distribution and strength of ligand-metal bond
  • In heme proteins, 3 residues are used as proximal ligands
          --- His in globins and perofidases
          --- tyrosine in catalases
          --- cysteine in chloroperoxidases and cytochrome P450 mono-oxygenases
  • In globins, proximal His donates a H bond to a main chain carbonyl O
  • In peroxidases it interacts with an Aspartate
  • Interaction with -vely charged Asp ---> stronger H bond than in globins ---> better e donation to metal centre
  • Peroxidases can stabilise higher oxidation states of iron during catalysis
  • push-pull concept
  • Push is from e donation by proximal ligand
  • Pull is due to nature of distal grps eg Arginine in heme peroxidases
          --- involved in polarising peroxide O-O bond
  • In catalase, proximal tyrosine accepts a H bond from an Arg
  • In chloroperoxidase and cytochrome P450 proximal Cys in H bonding interactions with 2 mainchain amide grps
  • Catalse and chloroperoxidase have electropositive proximal env
          --- interactions decrease -ve charge on phenolate and thiolate ligands
          ---> high redox potential of proteins
  • Mutate 1 his of 4 bis-histidyl coordinate hemes of tetra-heme cytochrome c3 to methionine ---> large increase of 200 mV in 1 of 4 redox potentials
  • Change metionine-histidyl ligation of mitochondrial cytochrome c to bis-His ligation ---> decrease 200 mV in midpoint potential
  • Bis-His coordination in c type cytochromes assoc with lower redox potentials than met-his coordination

HYDROPHOBIC ENVIRONMENT  AND ELECTROSTATIC EFFECTS

  • Kassner proposed heme proteins decrease midpoint potential by increasing polarity of heme env because it stabilises a more highly charged oxidised state
  • In cytrochromes f from diff species high conservation of most residues in heme pocket except at a positoin where typ, phe, leu and val are observed
  • Trp 4 mutated to Phe in cytochrome f from cyanobaterium Phormidium laminosum
  • Phe at same position in cytochrome f from Chlamydomonas reinhardtiiwas changed to trp
  • In wild type str, indole ring extends over tetrapyrrole
  • perpendicular to heme plane
  • In trp --> phe mutant, redox potential increases from 297 to 323 mV
  • In phe ---> trp mutant, redox potential decreases from 370 to 336 mV
  • In both cases changes are 1/2 of 70 mV diff between 2 speices
  • It is proposed decreased midpoint potential induced by trp caused by indole ring's p interactions with porphyrin 
          --- stabilise oxidised form of heme
          --- thru electrostatic repulsion with Fe orbitals
  • In multiheme cytochromes, redox properties governed by polarity of surrounding aa and variable electrostatic field from neighbouring hemes.


HEME EXPOSURE AND  SOLVENT ACCESSIBILITY

  • level of heme exposure important in fgoverning redox potentials
  • As exposure to water increases, polarity of heme env increases
         --- midpoint potentials decrease
  • Study of porphyrin pi-electron polarisation and pH, ionic strength and electrostatic-related factors
  • Tyr-->phe mutation at position 67 in cyt c
  • Str determination of mutant
  • rearrangement of H bonding network and shifts in atomic positions
  • Water mol found bound internally in addition to water mol already present in wild type
  • Loss of one hydroxyl grp increases polarity of local heme env
         ---> decreases redox potential

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