The extremophilic Andean isolate Acinetobacter sp. Ver3 expresses two ferredoxin/flavodoxin-NADPH reductases isoforms with different spectroscopic and catalytic properties.

BORTOLOTTI, ANA; GONZALEZ, JAVIER M.; PALAVECINO-NICOTRA, ALEJANDRO M.

Congreso; Biofísica en tiempos de COVID-19 : Primeras Jornadas Virtuales SAB 2020; 2020

Sociedad Argentina de Biofísica

Ferredoxin/flavodoxin NADPH-reductases are monomeric flavoenzymes, withcovalently bound FAD as cofactor, present in chloroplasts (FNR) and bacteria (FPR).They are grouped into two subclasses that differ in the C-terminus and FADconformation. Although most bacteria have one of the two subclasses, some species likethe extremophile UV-resistant Acinetobacter sp. Ver3 express both, namely, FPR1ver3 and FPR2ver3. Our previous experiments show that these enzymes respond differently to redox stimuli, suggesting distinctive cellular functions. Absorption spectra of purifiedenzymes, revealed typical features of flavoproteins. However, changes in the FADenvironment at the active site were observed as a red-shift in the transition bandmaximum for FPR2ver3, indicating particular electronic arrangements for the flavin in each enzyme. Steady state kinetic measurements using the one-electron carrierpotassium ferricyanide and the two-electron acceptor dichlorophenol indophenol (DCPIP)indicate that flavoenzymes display catalytic effciencies (kcat/KM) in the order of 1-6 uM?1s?1 with both electron acceptors. FPR1ver3 exhibited the highest kcat values (kcat1 = 82 s?1and 4.7 s?1 vs kcat2= 13.1 ± 0.3 s?1 and 2.2 ± 0.1 s?1 for ferricyanide and DCPIP, respectively). We solved both FPR1ver3 and FPR2ver3 crystallographic structures at 1.75 Åand 1.97 Å, respectively. FPR1ver3 exhibits an adenosine 2´,5´-diphosphate molecule near residues R144, R182, and R190, likely indicating the binding site of NADPH. In contrast,even though FPR2ver3 has an equivalent site lined by R149, R182, and R191, no ligands are discernible there. We cloned, overexpressed and purified a putative redox partner ofFPR, flavodoxinver3. When flavodoxin was used as a protein acceptor, the two reductases show dissimilar kcat/KM values, particularly FPR2ver3 (21-fold higher). Our datasuggest the flavodoxinver3 as possible physiological partner of bacterial subclass 2 ofVer3.AcknowldegmentsThis work was supported by the knowledge, experience and love of science of our dear colleague andfriend Dr. Nestor Cortez. To his memory, we will remember him with love and joy.