Structural Grounds For The Substrate Specificity Of The Ferredoxin NADP(H) Reductase

PALADINI, S; CARRILLO, N; CECCARELLI, EDUARDO A.

Pinamar, Argentina

Congreso; 10th Congress Panamerican Association for Biochemistry and Molecular Biology; 2005

SAIB - PABMB

Ferredoxin-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that catalyse the reversible electron transfer between NADP(H) and obligatory one-electron carriers as ferredoxin. A highly conserved C‑terminal tyrosine (Y308 in pea) is stacked near to the flavin in plant type FNRs. This amino acid performs a fundamental role in nucleotide discrimination and in increasing catalytic efficiency. Y308 is apparently displaced when the nicotinamide ring of NADP(H) is bound. We have investigated the structural grounds of the FNR–NADP(H) interaction and the involvement of Y308 on it. Differential spectroscopy analysis of the mutant FNR Y308S and analogues of NADP+ shows an autonomous binding of 2’phospho‑AMP or nicotinamide moieties with similar spectroscopy changes. In addition, nicotinamide mononucleotide reduced (NMNH) oxidase activity is detected in Y308S but not in wild‑type FNR indicating that Y308 preventing of NMNH to act as substrate. Moreover, the artificial substrate ferricyanide inhibits FNR increasing NADPH and NADH Michaelis constants and, the specificity for NADPH relative to NADH. Our results indicate that binding of any of the two moieties of the substrate NADP(H) affects the prosthetic environment, reflecting a conformational change on the enzyme which may tailor the enzyme for catalysis