Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin?NADP+ reductases

SÁNCHEZ-AZQUETA, A.; MUSUMECI, M. A.; MARTÍNEZ-JÚLVEZ, M.; CECCARELLI, E. A.; MEDINA, M

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2012 vol. 1817 p. 1063 - 1071

0005-2728

The role of the highly conserved C266 and L268 of pea ferredoxin?NADP+ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constantsfor NADPH oxidation were lower than those for NADP+ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wildtype enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268?270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).