Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

TY - JOUR

T1 - Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

AU - Frago, Susana

AU - Goñi, Guillermina

AU - Herguedas, Beatriz

AU - Peregrina, José Ramón

AU - Serrano, Ana

AU - Perez-Dorado, Inmaculada

AU - Molina, Rafael

AU - Gómez-Moreno, Carlos

AU - Hermoso, Juan A

AU - Martínez-Júlvez, Marta

AU - Mayhew, Stephen G

AU - Medina, Milagros

PY - 2007/11/15

Y1 - 2007/11/15

N2 - Contribution of three regions (phosphate-binding, 50's and 90's loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E(ox/sq) and E(sq/hq) and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E(ox/sq) by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E(sq/hq). Moreover, E(sq/hq) became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E(sq/hq) to less negative values and E(ox/sq) to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.

AB - Contribution of three regions (phosphate-binding, 50's and 90's loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E(ox/sq) and E(sq/hq) and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E(ox/sq) by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E(sq/hq). Moreover, E(sq/hq) became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E(sq/hq) to less negative values and E(ox/sq) to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.

KW - Anabaena/metabolism

KW - Binding Sites

KW - Enzyme Activation

KW - Flavin Mononucleotide/chemistry

KW - Flavodoxin/chemistry

KW - Kinetics

KW - Models, Chemical

KW - Models, Molecular

KW - Oxidation-Reduction

KW - Protein Binding

KW - Protein Structure, Tertiary

U2 - 10.1016/j.abb.2007.08.024

DO - 10.1016/j.abb.2007.08.024

M3 - Journal article

C2 - 17904516

SN - 0003-9861

VL - 467

SP - 206

EP - 217

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

IS - 2

ER -