ABSTRACT
The photosynthetic bacterium Rhodobacter capsulatus contains a ferredoxin (flavodoxin)-NADP(H) oxidoreductase (FPR) that catalyzes electron transfer between NADP(H) and ferredoxin or flavodoxin. The structure of the enzyme, determined by X-ray crystallography, contains two domains harboring the FAD and NADP(H) binding sites, as is typical of the FPR structural family. The FAD molecule is in a hairpin conformation in which stacking interactions can be established between the dimethylisoalloxazine and adenine moieties. The midpoint redox potentials of the various transitions undergone by R. capsulatus FPR were similar to those reported for their counterparts involved in oxygenic photosynthesis, but its catalytic activity is orders of magnitude lower (1-2 s(-)(1) versus 200-500 s(-)(1)) as is true for most of its prokaryotic homologues. To identify the mechanistic basis for the slow turnover in the bacterial enzymes, we dissected the R. capsulatus FPR reaction into hydride transfer and electron transfer steps, and determined their rates using stopped-flow methods. Hydride exchange between the enzyme and NADP(H) occurred at 30-150 s(-)(1), indicating that this half-reaction does not limit FPR activity. In contrast, electron transfer to flavodoxin proceeds at 2.7 s(-)(1), in the range of steady-state catalysis. Flavodoxin semiquinone was a better electron acceptor for FPR than oxidized flavodoxin under both single turnover and steady-state conditions. The results indicate that one-electron reduction of oxidized flavodoxin limits the enzyme activity in vitro, and support the notion that flavodoxin oscillates between the semiquinone and fully reduced states when FPR operates in vivo.
Subject(s)
Ferredoxin-NADP Reductase/chemistry , Ferredoxin-NADP Reductase/metabolism , Rhodobacter capsulatus/enzymology , Anabaena/enzymology , Anaerobiosis , Binding Sites , Catalysis , Crystallography, X-Ray , Flavodoxin/metabolism , Kinetics , Models, Molecular , NADP/metabolism , Oxidation-Reduction , PhotochemistryABSTRACT
Ferredoxin-NADP(H) reductase (272 amino acids) from Rhodobacter capsulatus (FPR) has recently been postulated to be involved in the antioxidant response and to facilitate the provision of reduced flavodoxin for the reduction of nitrogenase. Crystallization trials of recombinant FPR were carried out at 291 K by the hanging-drop vapour-diffusion method. Orthorhombic crystals (unit-cell parameters a = 69.3, b = 93.6, c = 103.5 A) were obtained. However, their diffraction pattern was not satisfactory and an extensive detergent screening was carried out over the initial crystallization conditions. The introduction of n-heptyl-beta-D-thioglucoside produced new trigonal crystals (space group P3(1)21; unit-cell parameters a = b = 120.5, c = 51.1 A) that diffracted to 1.8 A resolution at beamline BM16 at the ESRF. Preliminary structural analysis indicated that detergent molecules could increase the quality of diffraction of FPR crystals by stabilizing the disordered regions of the protein and by increasing the number of contacts in the crystal packing.
Subject(s)
Crystallography, X-Ray/methods , Ferredoxin-NADP Reductase/chemistry , Rhodobacter capsulatus/enzymology , X-Ray Diffraction/methods , Antioxidants/chemistry , Crystallization , Detergents/pharmacology , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry , TemperatureABSTRACT
Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.
Subject(s)
Dihydrolipoamide Dehydrogenase/metabolism , Ferredoxin-NADP Reductase/metabolism , Rhodobacter capsulatus/enzymology , Cloning, Molecular , Dihydrolipoamide Dehydrogenase/genetics , Dihydrolipoamide Dehydrogenase/isolation & purification , Escherichia coli/metabolism , Ferredoxin-NADP Reductase/genetics , Ferredoxin-NADP Reductase/isolation & purification , Flavodoxin/metabolism , Gene Expression Regulation, Bacterial , Genome, Bacterial , NADH Dehydrogenase/metabolism , NADP/metabolism , Oxidative Stress/physiology , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Rhodobacter capsulatus/genetics , Spectrophotometry/methodsABSTRACT
The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.
