Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis.
The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.
Schmitz
S
S
Botanisches Institut, Universität Bonn, Germany.
Martínez-Júlvez
M
M
Gómez-Moreno
C
C
Böhme
H
H
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D017033:Anabaena / Q000201:enzymology*; D003574:Cytochrome c Group / Q000378:metabolism; D004563:Electrochemistry; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D000596:Amino Acids; D003574:Cytochrome c Group; D005288:Ferredoxins; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase