Kinetic and spectroscopic characterization of two flavodoxin-NADP(H) reductases from Acinetobacter sp. Ver3.

BORTOLOTTI A; SARTORIO MG; PALAVECINO NICOTRA MA; CORTEZ N

San Luis

Congreso; XLVIII Reunión Anual SAB 27-29 Noviembre 2019 San Luis, Argentina; 2019

Sociedad Argentina de Biofísica

Bacterial ferredoxin/flavodoxin NADP(H)-reductases (FPR) are monomeric flavoenzymescarrying non covalently bound FAD as cofactor. They are classified in two subclasses thatdiffer in the C-terminus and have been involved in Fe-S clusters reparation afteroxidative damage. Although most bacteria contain one of the two subclasses, somespecies carry both of them. The extremophile UV-resistant Acinetobacter sp. Ver3 hasboth isoenzymes, FPR1ver3 and FPR2ver3. Our previous experiments proved that theseenzymes respond differently to redox stimuli, suggesting distinctive cellular functions.Here we report a biophysical and biochemical characterization of these flavoenzymes.Variances in the FAD environment at the active site were detected using absorption andfluorescence spectra analysis, suggesting particular electronic arrangements for theflavin in each isoenzyme. Kinetic measurements under steady state conditions using amonoelectronic-artificial acceptor, showed a faster electron transfer in FPR1 compared toFPR2 (kcat1=79,3 ± 1.3 s-1 vs. kcat2=12.6 ± 0,1 s-1). A slower transfer was obtainedwith a di-electronic artificial acceptor (kcat1=4.73 ± 0.1 s-1 vs. kcat2=2.2 ± 0,1 s-1). Wecloned, overexpressed and purified a putative redox partner of FPR, Flavodoxinver3.When recombinant flavodoxin was used as a protein acceptor, FPR2 had a faster electrontransfer than the other enzyme (kcat2=1.3 s-1 vs. kcat1=0.5 s-1). Our findins suggeststhat the different catalysis observed in these isoenzymes could be related to theirspecific electronic arrangements in FAD cofactor.