Figure 1.
Classification of plant-type FNRs according to structural features.
Plant-type FNRs are classified as plastidic and bacterial FNRs [21]. Structures of representative prototypes of the plastidic and the bacterial groups are shown. Bacterial-type FNRs are subdivided into two subclasses, subclass I and subclass II. FNRs from Azobacter vinelandii and Rhodobacter capsulatus belong to subclass I; however, they differ in length and sequence of the carboxy-terminal region upstream of the alanine that faces the isoalloxazine of FAD. A view of the environments of the different prosthetic groups and the sequences of the carboxy-terminal extensions are shown to the right of each enzyme structure. FNRs from Pisum sativum (1qg0), E. coli (1fdr), A. vinelandii (1a8p) and R. capsulatus (2bgj) were used as model proteins. Figures were generated using PyMol. Available: http://pymol.sourceforge.net/.
Figure 2.
Complementation of the E. coli fpr-null mutant with Xac-FNR.
The E. coli fpr strain (RR6A) was transformed with pEcFNR that contained the endogenous fpr gene, pXacFNR that contained the Xac fpr gene, or pUC119. The susceptibility of E. coli strains to MV toxicity was evaluated using the disk diffusion assay. The diameters of the inhibition zones were measured after 24 h of incubation. Bars indicate mean ± standard deviation of three independent experiments.
Figure 3.
Expression analysis of Xac-FNR in response to oxidative treatments.
(A) Amplified products of the Xac fpr gene by semiquantitative RT-PCR using RNA preparations from Xanthomonas axonopodis pv. citri cultures grown in SB medium to the early exponential phase and exposed to the indicated concentrations of MV for 15 min. 16S rRNA was used as a loading control and for the quantitation of the total RNA in the RT-PCR experiments. (B) Xac-FNR accumulation after MV-dependent induction. Cleared extracts correspond to 25 µg of total soluble protein. Samples were analyzed by SDS-PAGE and immunoblot analysis using specific antisera. (C) Effect of 2,3-dimetoxy-1,4-naphthoquinone (DMNQ) 500 µM, tert-butyl hydroperoxide (tBOOH) 500 µM, menadione 100 µM and dimethyl sulfoxide (DMSO) on Xac-FNR protein expression. Cleared extracts correspond to 30 µg of total soluble protein and were analyzed by Western blot. The graphs below the gels in (A), (B) and (C) show the expression profiles that were obtained by densitometric quantification of the band intensities. Experiments were performed in triplicate with similar results, and the error bars indicate ±1 standard deviation of the mean (IOD, integrated optical density; A.U., arbitrary units).
Figure 4.
Spectroscopic analyses of Xac-FNR and comparison with the pea and E. coli enzymes.
(A) UV-visible spectra displayed by the different plant-type FNRs. (B) Near-UV and visible CD spectra of the enzymes. (C) FAD solvent accessibility studied by quenching with KI. Xac-FNR (○), pea-FNR (▪), Ec-FNR (•), free FAD (▴). (D) Differential UV-visible spectra elicited by the interaction between the enzymes and NADP+. The spectra were obtained by the subtraction of the spectra of FNR in the presence of 0.3 mM NADP+ and the free enzyme. Different colors were employed for each FNR variant: cyan, Xac-FNR; green, pea-FNR; and red, Ec-FNR.
Table 1.
Kinetic parametersa of NADPH and NADH diaphorase reactions that were catalyzed by Xac-FNR, pea-FNR and Ec-FNR, and the dissociation constants for the different complexes with NADP+ b.
Figure 5.
Thermal stability of the different plant-type FNRs according to the folding-unfolding transitions that were observed for the enzymes.
Cyan, Xac-FNR; green, pea-FNR; and red, Ec-FNR.
Table 2.
Kinetic parameters a for cytochrome c reductase with pea ferredoxin, E. coli flavodoxin, and E. coli ferredoxin, and the melting temperatures of thermal unfolding transitions for Xac-FNR, pea-FNR and Ec-FNR b.
Figure 6.
Analysis of Xac-FNR's redox partner by homology sequence.
(A) Alignment of primary structures of subclass I bacterial FNRs from A. vinelandii (Av-FNR, gb: YP_002800963.1), X. axonopodis pv. citri (Xa-FNR, gb: NP_641792.1), Pseudomonas aeruginosa (Pa-FNR, gb: YP_001347117.1), R. capsulatus (Rc-FNR, gb: ADE85336.1), Rhodobacter sphaeroides (Rs-FNR, gb: YP_002524612.1), Paracoccus denitrificans (Pd-FNR, gb: ABL68770.1) and Oceanicaulis alexandrii (Oa-FNR, gb: ZP_00952506.1). Sequence regions from amino acid 200 to the carboxy-terminus are shown. In bold is the amino acid from Av-FNR that is involved in the interaction with ferredoxin I, as was previously reported [42]. (B) Alignment of ferredoxin I from A. vinelandii (Av-FdI, gb: AAA22125.1) and ferredoxins from X. axonopodis pv. citri (XAC1762, gb: NP_642090.1), P. aeruginosa (ferredoxin A, Pa-FdA, gb: AAF89693.1), R. capsulatus (ferredoxin II, Rc-FdII, gb: YP_003578927.1), P. denitrificans (Pd-Fd, gb: ABL69923.1), Rhodobacter sp. (Rs-Fd, gb: ZP_05844833.1) and O. alexandrii (ferredoxin A, Oa-FdA, gb: ZP_00953239.1). In bold is the peptide involved in the interaction with Av-FNR identified by cross-linking experiments, as was previously reported [42]. Potential residues of Av-FdI that interact with Lys258 of Av-FNR are shaded in gray. Numbers over the sequences correspond to the A. vinelandii proteins. The alignments were performed using ClustalX 2.0.11.
Figure 7.
Characterization of ferredoxin XAC1762.
(A) UV-visible spectrum displayed by ferredoxin XAC1762. (B) Kinetics of the cytochrome c reductase reaction of Xac-FNR with ferredoxin XAC1762 as substrate.
Table 3.
Bacterial strains and plasmids used in this study.