INTERACTION OF POSITIVELY CHARGED AMINO-ACID-RESIDUES OF RECOMBINANT,CYANOBACTERIAL FERREDOXIN-NADP(-DIRECTED MUTAGENESIS() REDUCTASE WITHFERREDOXIN PROBED BY SITE)

The petH genes encoding ferredoxin:NADP(+) reductase (FNR) from two An abaena 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, an d crystallographic studies. The following substitutions were introduce d by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K43 0Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K2 75E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the d iaphorase activities, the specific rates of ferredoxin dependent NADP( +)-photoreduction and cytochrome c reduction catalyzed by FNR showed t hat 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 cytochrom e c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP(+) by photosystem I via FNR. A m utation involving the replacement of positive charge by a neutral amid e produced in all cases a smaller inhibitory effect on the activity th an a charge reversal mutation. In addition, it has been found that R21 4 was necessary for stable integration of the non covalently bound FAD -cofactor. (C) 1998 Elsevier Science B.V.