Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin.
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.
Frago
Susana
S
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009-Zaragoza, Spain.
Goñi
Guillermina
G
Herguedas
Beatriz
B
Peregrina
José Ramón
JR
Serrano
Ana
A
Perez-Dorado
Inmaculada
I
Molina
Rafael
R
Gómez-Moreno
Carlos
C
Hermoso
Juan A
JA
Martínez-Júlvez
Marta
M
Mayhew
Stephen G
SG
Medina
Milagros
M
D017033:Anabaena / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D005418:Flavodoxin; D005486:Flavin Mononucleotide