title: Differential stabilization of the three FMN redox forms by tyrosine 94 and tryptophan 57 in flavodoxin from Anabaena and its influence on the redox potentials.
abstract: Flavodoxins are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule as the redox-active center. The redox potentials of the flavin nucleotide are profoundly altered upon interaction with the protein. In Anabaena flavodoxin, as in many flavodoxins, the flavin is sandwiched between two aromatic residues (Trp57 and Tyr94) thought to be implicated in the alteration of the redox potentials. We have individually replaced these two residues by each of the other aromatic residues, by alanine and by leucine. For each mutant, we have determined the redox potentials and the binding energies of the oxidized FMN--apoflavodoxin complexes. From these data, the binding energies of the semireduced and reduced complexes have been calculated. Comparison of the binding energies of wild-type and mutant flavodoxins at the three redox states suggests that the interaction between Tyr94 and FMN stabilizes the apoflavodoxin--FMN complex in all redox states. The oxidized and semireduced complexes are, however, more strongly stabilized than the reduced complex, making the semiquinone/hydroquinone midpoint potential more negative in flavodoxin than in unbound FMN. Trp57 also stabilizes all redox forms of FMN, thus cooperating with Tyr94 in strong FMN binding. On the other hand, Trp57 seems to slightly destabilize the semireduced complex relative to the oxidized one. Finally, we have observed that reduction of mutants lacking Trp57 is slow relative to that of wild-type or mutants lacking Tyr94, which suggests that Trp57 could play a role in the kinetics of flavodoxin redox reactions.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Lostao
authors.forename: C
authors.initials: C
authors.lastname: Gómez-Moreno
authors.forename: S G
authors.initials: SG
authors.lastname: Mayhew
authors.forename: J
authors.initials: J
authors.lastname: Sancho
mesh_terms: D017033:Anabaena / Q000378:metabolism; D003198:Computer Simulation; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism*; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D014364:Tryptophan; D014443:Tyrosine
chemical_list: D005418:Flavodoxin; D011994:Recombinant Proteins; D014443:Tyrosine; D005486:Flavin Mononucleotide; D014364:Tryptophan