Reduction of the pea ferredoxin-NADP(H) reductase catalytic efficiency by the structuring of a carboxyl-terminal artificial metal binding site

CATALANO DUPUY, D. L.; ORECCHIA, M.; RIAL, D. V.; CECCARELLI, E. A.

BIOCHEMISTRY

American Chemical Society

Lugar: Philadelphia; Año: 2006 vol. 45 p. 13899 - 13909

0006-2960

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliverNADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxylterminalamino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, includingfour histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.c