Thursday, 14 June 2012

Flavodoxins: sequence, folding, binding, function and beyong by Sancho

Introduction

  • flavodoxins are small ET proteins that contain 1 mol of noncovalently but tightly bound FMN as redox active component
  • Fouond in cyanobac and clostridia growing in low iron conditions
  • replaced iron-containing ferredoxin in reactions leading to NADP+ and H2 reduction
Folding, stability and 3D str of apoflavodoxin
  • To acquire finctional conform, flavodoxin pp must bind FMN
  • cofactor only binds apoprotein when it is already folded
  • apoflavodoxin folds fast
  • recruit anions in phosphate binding site
  • fill empty space left by missing cofactor
  • bring one of FMN isoalloxazine binding loops in contact with other one
FMN recognition, stability of complex and its influence on flavodoxin stability
  • FMN mol made of a fused, aromantic, triple ring, a phosphate grp and a linking ribityl chain
  • In flavodoxin no positively charged residues interact with phosphate
  • phospate is bound by network of H bonds
  • overall electrostatic field of apoflavodoxin is negative
  • so is appraoching phosphate
  • phosphate site is promiscuous in recruiting anions
  • binding may be driven by hydrophobic/aromatic interactions at isoalloxazine site
  • Once binding takes place, tight complex formed
  • In Anabaena flavdoxin, phosphate grp contributes omst to strength of complex
  • H bonds with thr side chains and main chain NH grps
  • Isoalloxazine contributes much by tight coplanar interaction with conserved tyr 94 and conserved trp 57
  • functional complex is more stable than apoprotein
  • In anabaena flavodoxin bound FMN increases stability and cooperativity of thermal unfolding
  • With FMN, FMN bindings site is stable at moderate temp and unfolds with rest of protein
Tailoring of FMN redox potentials by interaction with apoprotein
  • In photosynthetic reactions flavodoxins must acept e one by one
  • FMN connaot peroform when free in sol
  • low stability of its 1e reduced redox state
  • Flavodoxin can do 
  • apoprotein modieifes redox potentials of cofactor or relative stability of oxisied 1e reduced (semiquinone) or 2e reduced (hydroquinone) forms of FMN
  • form complexes of diff affinities with 3 redox forms
  • conserved tyr is tightly packed onto si face of isolalloxazine ring
  • favourable interactions with 3 redox forms of FMN
  • thosw with fully reduced state are much weaker than those with oxidised and semireduced ones
  • Substantial stabilisation of semiquinone relative to hydroquinone
  • shifts semiquinone/hydroquinone redox potential to much more negative values than that of free FMN
  • negative electrostatic potential of acidic flavodoxin destabilised negatively charged hydroquinone
  • destabilised burial of its uncompensated negatie charge in reduced complex
  • Semiquinone is stabilised relative to oxidised form
  • can form new H bond with main chain grp
  • shift oxidised/semireduced potential to moderately less negative values relative to potential of free FMN

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