The ferredoxin (flavodoxin)-NADP(H) oxidoreductase of Rhodobacter capsulatus. Function and Regulation

GABRIEL G LUCCIARINI; ANA BORTOLOTTI; VERÓNICA DUMIT; NÉSTOR CORTEZ

Rosario (Argentina)

Congreso; V CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL; 2008

The purple bacterium <i>Rhodobacter capsulatus</i> has a remarkable capacity of adapting its metabolism to a broad range of environmental conditions. When illuminated in anaerobiosis this microorganism synthesizes ATP through a cyclic electron transport around a single photosyntem, whereas the presence of oxygen promotes the establishment of a respiratory metabolism.We demonstrated before that <i>R. capsulatus</i> contains a ferredoxin(flavodoxin)-NADP(H) oxidoreductase (FPR), capable to reduce the flavodoxin NifF, involved in nitrogen fixation metabolism. The contribution of FPR to the cell antioxidant response was demostrated in enterobacteria, but its physiological role in photosynthetic bacteria is still poorly understood.To characterize FPR metabolic function, we analyzed the interaction between the enzime with the FdVI, which was proposed to participate in assembly of iron-sulfur clusters. Recombinant <i>Rhodobacter</i> FdVI was purified by combination of anionic exchange chromatography and molecular filtration. The oxidase activity of FPR mediated by Fd was monitored by following NADPH consumption under steady-state conditions. Measured rates agree well with the putative function assigned to this enzyme in the biosynthesis of Fe/S clusters. Aconitase and 6-phosphogluconate dehydratase, Fe/S containig enzymes, are sensitive to oxygen exposure. The ability of FPR/FdVI system to repair Fe/S oxidized clusters, was tested on <i>Rhodobacter</i> cells free extracts, but it was found to be unfunctional in reactivation of oxygen damaged Fe/Scluster in an <i>in vitro</i> reconstituted system. Sequence analysis of promoter region of <i>fpr</i> gene showed three RegA binding consensus sequences. RegA/RegB is a two component system and has been involved in regulating the balance between the generation and utilization of reducing power in this bacterium. RegB is a kinase that sense the redox homeostasis cellular and oxygen level, inducing the phosphorilation of RegA.To study the <i>fpr</i> gene expression we constructed translational and transcrptional <i>lacZ</i> fusion and introduced them in <i>Rhodobacter capsulatus</i> cells by biparental conjugation. &beta;-galactosidase activity and FPR activitiy were made on wild type and <i>regA</i>-disrupted strains. Ours results suggest that the RegA/RegB system funct as an aerobic activator of <i>R. capsulatus fpr</i>gene expression.