Dynamics of the active site architecture in plant-type ferredoxin-NADP reductases catalytic complexes |
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Authors: | Ana Sá nchez-Azqueta,Daniela L. Catalano-Dupuy,Arleth Ló pez-Rivero,Marí a Laura Tondo,Elena G. Orellano,Eduardo A. Ceccarelli,Milagros Medina |
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Affiliation: | 1. Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain;2. Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Unidad Asociada BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain;3. Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina |
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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. |
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Keywords: | FNR, ferredoxin-NADP+ reductase FPR, bacterial-type FNR AnFNR, FNR from the cyanobacterium Anabaena PCC 7119 PsFNR, FNR from Pisum sativum XaFPR, FPR from Xanthomonas axonopodis pv. citri EcFPR, FPR from Escherichia coli FNRox, FNR in the fully oxidised state FNRhq, FNR in the anionic hydroquinone (fully reduced) state HT, hydride transfer DT, deuteride transfer WT, wild-type CTC, charge&ndash transfer complex CTC-1, FNRox:NADPH CTC CTC-2, FNRhq:NADP+ CTC 2&prime -P-AMP, 2&prime -P-AMP moiety of NADP(H) N5-FAD, N5 hydride donor/acceptor of the FADH&minus /FAD isoalloxazine ring of FNR C4-NADP(H), C4 hydride acceptor/donor of the NADP+/NADPH nicotinamide ring NADPD, (4R)-4-2H-NADPH kA &rarr B, kB &rarr C, apparent/observed rate constants obtained by global analysis of spectral kinetic data kHT, kDT, kobsHT, kobsDT, limiting hydride and deuteride transfer first-order rate constants for the reduction of FNR and their corresponding observed values under particular conditions KIE, kinetic isotope effect on rate constants AH, AD, Arrhenius pre-exponential factors for hydride and deuteride, respectively EaH, EaD, activation energies for hydride transfer and deuteride transfer, respectively DAD, donor&ndash acceptor distance |
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