Binding Thermodynamics of Ferredoxin:NADP Reductase: Two Different Protein Substrates and One Energetics |
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Authors: | Marta Martí nez-Jú lvez,Milagros Medina |
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Affiliation: | † Departamento de Bioquímica y Biología Molecular y Celular, and Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain ‡ Fundación Aragón I+D (ARAID-BIFI), Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain |
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Abstract: | The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP+ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His299 FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics. |
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Keywords: | apoFld, apoflavodoxin ASA, accessible surface area EPPS, N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid ET, electron transfer Fd, ferredoxin Fld, flavodoxin FMN, flavin mononucleotide FNR, ferredoxin-NADP+ reductase ITC, isothermal titration calorimetry ox, sq, or rd, oxidised, semiquinone, or reduced states of the protein Tricine, N-tris(hydroxymethyl) methylglycine Tris, 2-amino-2-hydroxymethylpropane-1,3 diol |
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