Reduction of the pea ferredoxin-NADP(H) reductase Catalytic Efficiency by the Structuration of a Carboxyl Terminal Artificial Metal Site.

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

BIOCHEMISTRY

American Chemical Society

Año: 2006 vol. 45 p. 13899 - 13909

0006-2960

Ferredoxin (flavodoxina)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxina, adrenodoxin) to redox-based metabolism 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 reductases to sustain the demands of the photosynthetic process. By the contrary, FNRs from organisms growing on heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20 to 100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight in the FNR structural features that modulates the enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl terminal amino acid (Tyr308) is followed by an artificial metal site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as consequence, strongly reduces the catalytic efficiency of the enzyme. The enzyme affinity for NADP+ and the Km were only slightly affected by the addition of the amino acid extension in either the presence or absence of Zn2+. Our results are the first experimental evidence that mobility of the carboxyl terminal backbone region of the FNR and mainly the Tyr308 are essential for obtaining an FNR enzyme with high catalytic efficiency.