title: Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.
abstract: Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.
authors: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain.
authors: Ana
authors: A
authors: Sánchez-Azqueta
authors: Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
authors: Daniela L
authors: DL
authors: Catalano-Dupuy
authors: Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
authors: Arleth
authors: A
authors: López-Rivero
authors: Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
authors: María Laura
authors: ML
authors: Tondo
authors: Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
authors: Elena G
authors: EG
authors: Orellano
authors: Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina.
authors: Eduardo A
authors: EA
authors: Ceccarelli
authors: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain. Electronic address: mmedina@unizar.es.
authors: Milagros
authors: M
authors: Medina
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010944:Plants / Q000201:enzymology*; D018087:Plastids
chemical_list: D005287:Ferredoxin-NADP Reductase
keywords: Bacterial-type FPR; Charge–transfer complex; Ferredoxin-NADP(+) reductase; Flavoenzyme; Hydride transfer; Kinetic isotope effect; Plastidic-type FNR