CONICET | Buscador de Institutos y Recursos Humanos

Kinetic isotope effects in reactions involving H-transfer and their temperature dependence are powerful tools to explore flexibility 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 (HT) and deuteride (DT) transfer rates for the 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 isotopic effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) fluctuations in processes for Pisum sativum FNR than for the Anabaena one. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT step and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and stiffer than those for FNRs. These differences must obey 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.