Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.
Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.
Pueyo
J J
JJ
Departamento de Bioquímica y Biología Molecular y Celulare, Facultad e Ciencias, Universidad de Zaragoza, Spain.
Revilla
C
C
Mayhew
S G
SG
Gómez-Moreno
C
C
D017033:Anabaena / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005287:Ferredoxin-NADP Reductase / Q000302:isolation & purification / Q000378:metabolism*; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003574:Cytochrome c Group; D005288:Ferredoxins; D046911:Macromolecular Substances; D005287:Ferredoxin-NADP Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide