Mapping the interaction of the [2Fe-2S] Clostridium pasteurianum ferredoxin with nitrogenase MoFe protein.
The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.
Chatelet
C
C
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble, France.
Meyer
J
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity
C081714:2Fe-2S ferredoxin; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005288:Ferredoxins; D008983:Molybdoferredoxin; D011994:Recombinant Proteins; D009591:Nitrogenase