Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S] heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120.
Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-1 ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH:PetF and PetF:FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP+ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP+ photoreduction.
Schmitz
S
S
Botanisches Institut, Universität Bonn, Germany.
Schrautemeier
B
B
Böhme
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D017033:Anabaena / Q000378:metabolism*; D001483:Base Sequence; D004563:Electrochemistry; D004926:Escherichia coli; D005288:Ferredoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C081714:2Fe-2S ferredoxin; D000596:Amino Acids; D005288:Ferredoxins; D011994:Recombinant Proteins; D009591:Nitrogenase