4AF7

PEA FNR C266M MUTANT

Summary for 4AF7

• Entry DOI: 10.2210/pdb4af7/pdb
• Related: 1QFY 1QFZ 1QG0 1QGA 2XNC 4AF6
• Descriptor: FERREDOXIN--NADP REDUCTASE, LEAF ISOZYME, CHLOROPLASTIC, FLAVIN-ADENINE DINUCLEOTIDE (3 entities in total)
• Functional Keywords: oxidoreductase, flavoenzyme, electron transfer, photosynthesi, chloroplast
• Biological source: PISUM SATIVUM (PEA)
• Cellular location: Plastid, chloroplast stroma: P10933
• Total number of polymer chains: 2
• Total formula weight: 71315.33

Authors

Martinez-Julvez, M.,Sanchez-Azqueta, A.,Musumeci, M.A.,Medina, M.,Ceccarelli, E. (deposition date: 2012-01-18, release date: 2012-05-30, Last modification date: 2023-12-20)

Primary citation

Sanchez-Azqueta, A.,Musumeci, M.A.,Martinez-Julvez, M.,Ceccarelli, E.,Medina, M.
Structural Backgrounds for the Formation of a Catalytically Competent Complex with Nadp(H) During Hydride Transfer in Ferredoxin-Nadp(+) Reductases.
Biochim.Biophys.Acta, 1817:1063-, 2012

PubMed Abstract: 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 constants for 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 wild-type 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).

PubMed: 22542899
DOI: 10.1016/J.BBABIO.2012.04.009

Experimental method

X-RAY DIFFRACTION (2.85 Å)