Molecular Insights into the electron transfer mechanism in the Rhodobacter capsulatus FPR

ALMUDENA PONCE; ANA BORTOLOTTI; NESTOR CORTEZ; JUAN. A HERMOSO; CELIA MAYA

Sicilia

Workshop; 45 th Present and Future Methods for Biomolecular Crystallography; 2012

Ferredoxin NADP(H) reductases (FNRs/FPRs, EC 1.18.1.2) carry out the transference of two electrons beween the NADP(H) and two monoelectron carrier molecules, as ferredoxin or flavodoxin, by a reversible mechanism. FPR from Rhodobacter capsulatus is a bacterial ferredoxin-NADP(H) reductase proposed to shuttle electrons from the NADPH pool of the cell to the flavodoxin NifF, a potential electron carrier of the nitrogenase.1 Both, FPR and flavodoxin NifF, are flavoproteins and contain FAD or FMN as a prostetic group. FPR may accept two electrons from a reduced substrate at once, but then, it has two release them one by one, in two consecutive steps. The aim of our study is to explain the mechanism for each of these reactions Three-dimensional structures, at high resolution, have been obtained for three different complexes FPR-NADP+, and for a counterpart compound of NADP+: the AMP-2?P. In none of these complexes the NADP+ was found in a suitable position for the electron transfer between the substrate (NADPH) and the coenzyme (FAD). In fact, until now it has been impossible obtaining a functional complex between wild-type FNR/FPR and NADP(H). The reason for so in FNRs is that there is a tyrosine in the Cterminal end that stabilizes the isoaloxacine ring by a ð-ð interaction. This tyrosine must previously move outside to allow the nicotinamide entrance in the active site. The target now is to obtain a functional crystallographic FPR-NADP+ complex. To this purpose 3 mutant forms of the FPR have been produced: A266Y; A266Ä and A266YÄ. We are now working on the crystallization of these mutants and on their respective complexes with NADP+.