Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding.
The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.
Curdt
I
I
Botanisches Institut der Universität Bonn, Karlrobert-Kreiten-Strasse 13, D-53115 Bonn, Germany.
Singh
B B
BB
Jakoby
M
M
Hachtel
W
W
Böhme
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis*; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005288:Ferredoxins; D009572:Nitrite Reductases