Chlorobaculum tepidum ferredoxin: NADP+ oxidoreductase (CtFNR) is a dimeric thioredoxin reductase (TrxR)-type FNR, whose mechanism and redox properties are poorly characterized. In this work, we focused on the reoxidation mechanisms of its flavin adenine dinucleotide (FAD) cofactor using quinones (Q), nitroaromatics (ArNO2), and other nonphysiological oxidants with different single-electron reduction midpoint potentials (E71) and electrostatic charge. Like in other FNRs, the rate-limiting step of the reaction is the reoxidation of FAD semiquinone (FADH•). However, only one FAD per dimer functions in CtFNR due to some nonequivalence of the NADP(H) binding domains in separate subunits. The reactivity of Q increases with increasing E71, while ArNO2 form another analogous series of lower reactivity. The compounds are reduced in a dominant single-electron way. These data are consistent with an "outer sphere" electron transfer mechanism. On the basis of reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of FAD at pH 7.0 is -0.282 V. In CtFNR, 11% FADH• was stabilized at equilibrium. Calculated electron transfer distances in reactions with Q and ArNO2 were in the range of 2.6-3.4 Å. Taken together with previous studies of Rhodopseudomonas palustris and Bacillus subtilis FNRs, this work allows us to generalize the information on the catalytic ant thermodynamic properties of TrxR-type FNRs. In addition, our data may be valuable from an applied perspective, e.g., the use of redox mediators in photobioelectrochemical systems or microbial cells based on anoxygenic phototrophic bacteria.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be −0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

The fully reduced flavin cofactor (FADred) in ferredoxin-NADP+ oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FADred in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FADred and the dynamic properties of the protein environment, we conclude that the excited state of neutral FADred can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts.

In the crystal structure of ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), Tyr50 stacks on the si-face of the isoalloxazine ring portion of the FAD prosthetic group. This configuration is highly conserved among the homodimeric ferredoxin-NAD(P)(+) oxidoreductases (FNR) from Gram-positive bacteria and photosynthetic bacteria. In this report, pre-steady state reactions of Tyr50 variants with NADP(+)/NADPH and ferredoxin from B. subtilis (BsFd) were examined with stopped-flow spectrophotometry to assess the effects of the mutation on the formation of FNR-substrate complexes and following redox equivalent transfer. Mixing oxidized BsFNRs with NADPH resulted in a rapid complex formation followed by a rate-limiting hydride transfer. The substitution substantially modulated the intensity of the charge transfer absorption band and decreased the observed hydride transfer rates compared to the wild type. Reduction of the Y50W mutant by NADPH proceeded in a monophasic manner, while the Y50G and Y50S mutants did in biphasic phases. The reduced Tyr50 mutants hardly promoted the reduction of NADP(+). Mixing oxidized BsFNRs with reduced BsFd resulted in the reduction of the FNRs. The observed FNR reduction rates of the three variants were comparable, but in the Y50G and Y50S mutants, the amount of the reduced FNR at the rapid phase was decreased, and a slow FNR reduction phase was observed. The obtained results suggest that the replacements of Tyr50 with Gly and Ser permitted the conformational change in the reduced form, which induced an asymmetric kinetic behavior between the protomers of the homodimeric BsFNR.

Rhodopseudomonas palustris ferredoxin:NADP+ oxidoreductase (RpFNR) belongs to a novel group of thioredoxin reductase-type FNRs with partly characterized redox properties. Based on the reactions of RpFNR with the 3-acetylpyridine adenine dinucleotide phosphate redox couple, we estimated the two-electron reduction midpoint potential of the FAD cofactor to be −0.285 V. 5-Deaza-FMN-sensitized photoreduction revealed −0.017 V separation of the redox potentials between the first and second electron transfer events. We examined the mechanism of oxidation of RpFNR by several different groups of nonphysiological electron acceptors. The kcat/Km values of quinones and aromatic N-oxides toward RpFNR increase with their single-electron reduction midpoint potential. The lower reactivity, mirroring their lower electron self-exchange rate, is also seen to have a similar trend for nitroaromatic compounds. A mixed single- and two-electron reduction was characteristic of quinones, with single-electron reduction accounting for 54% of the electron flux, whereas nitroaromatics were reduced exclusively via single-electron reduction. It is highly possible that the FADH· to FAD oxidation reaction is the rate-limiting step during the reoxidation of reduced FAD. The calculated electron transfer distances in the reaction with quinones and nitroaromatics were close to those of Anabaena and Plasmodium falciparum FNRs, thus demonstrating their similar “intrinsic” reactivity.

Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron-transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50) is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants, with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that, upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ∼260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan-bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effects of protein environment and relaxations on the spectral properties of those radical intermediates and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counterions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins.

The green sulfur bacterium, Chlorobaculum tepidum, is an anaerobic photoautotroph that performs anoxygenic photosynthesis. Although genes encoding rubredoxin (Rd) and a putative flavodiiron protein (FDP) were reported in the genome, a gene encoding putative NADH-Rd oxidoreductase is not identified. In this work, we expressed and purified the recombinant Rd and FDP and confirmed dioxygen reductase activity in the presence of ferredoxin-NAD(P)+ oxidoreductase (FNR). FNR from C. tepidum and Bacillus subtilis catalyzed the reduction of Rd at rates comparable to those reported for NADH-Rd oxidoreductases. Also, we observed substrate inhibition at high concentrations of NADPH similar to that observed with ferredoxins. In the presence of NADPH, B. subtilis FNR and Rd, FDP promoted dioxygen reduction at rates comparable to those reported for other bacterial FDPs. Taken together, our results suggest that Rd and FDP participate in the reduction of dioxygen in C. tepidum and that FNR can promote the reduction of Rd in this bacterium.

Among the thioredoxin reductase-type ferredoxin-NAD(P)(+) oxidoreductase (FNR) family, FNR from photosynthetic purple non-sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP(+)/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of K-d for NADP(+) and K-M for NADPH in the diaphorase assay; however, the k(ent) values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP(+) resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable ( > 400 s(-1)). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP(+)/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP(+)/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP(+)/NADPH and/or reaction with electron transfer proteins.

Ferredoxin-NAD(P)+ oxidoreductase (FNR) is a soluble flavoprotein catalyzing the redox reaction between NAD(P)+/H and small iron sulfur protein ferredoxin. Among homo-dimer type FNRs which has a significant structural homology with the bacterial NADPH-thioredoxin reductases, FNR from Rhodopseudomonas palustris (RpFNR) is distinctive for the presence of the Tyr residue on the re-face of the isoalloxazine ring portion of FAD prosthetic group. We report the reactivity of the wild type and Tyr mutants with NADP+/H evaluated by a stopped-flow spectrophotometry. Mixing oxidized wild type and mutated RpFNRs with NADPH provided a rapid charge transfer (CT) complex formation followed by the reduction of FAD. Reduction of RpFNR occurred in part even in the presence of 50-fold excess NADPH at equilibrium. Mixing reduced wild type and mutated RpFNRs with NADP+ also resulted in a rapid CT complexes formation followed by the oxidation of FAD. Kinetic analysis indicated that the rate-limiting step was the hydride-transfer process in both directions and the rates of both directions were comparable. Replacement of the Tyr residue did not affect the rate constants of the hydride transfer step significantly, but the observed rates responding to the following release and re-association of NADP+/H decreased. In the reduced form, the absorption band centered at around 450 nm appeared in all RpFNRs, and the wavelength of the absorption maxima shifted toward shorter wavelength in the mutants. Comparison with other homo-dimer type FNRs suggested that the presence of this band relate to the rapid CT complex formation. We will discuss about the relation between the reversibility and the conformation change of the isoalloxazine ring portion of FAD prosthetic group with the data of other homodimer-type FNRs.

Ferredoxin-NAD(P)+ reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP+ and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP+ and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326-Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337-Glu360 and Ser338-Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP+/NADPH.

Ferredoxin-NAD(P)+ oxidoreductases (FNRs) of photoautotroph Chlorobaculum tepidum (CtFNR) and heterotroph Bacillus subtilis (BsFNR) are homo-dimeric flavoprotein exhibiting a significant conservation of structural topology with bacterial NADPH-thioredoxin reductase. Although the redox reaction catalyzed by FNR is theoretically reversible, under the physiological conditions the direction of the reaction is assumed to be optimized toward either NADP+ reduction in photosynthetic process or NADPH oxidation in non-photosynthetic one. In this presentation the directionality of the redox reactions with NADP+/H and the structure-function relationships will be discussed based on the pre-steady-state kinetics. NADPH oxidation and NADP+ reduction reactions by BsFNR occurred with rapid formations of charge transfer complexes (CTCs) followed by the rate-limiting hydride transfer. The rate of the NADPH oxidation direction in the hydride transfer step was much faster than that of NADP+ reduction direction (500 s−1 vs. < 10 s−1), indicating NADPH oxidation was favored in BsFNR-catalyzing reaction. The deletions of the C-terminal amino acid residues almost did not affect the directionality. NADPH oxidation by CtFNR yielded a rapid formation of CTCs followedby the rate-limiting hydride transfer. The amount of reduced CtFNR at the equilibrium was in part even in the presence of ∼50-fold excess NADPH. NADP+ reduction by CtFNR provided no charge transfer band and the observed rate for the NADP+ reduction by CtFNR was rather slow (∼ 5 s−1), suggesting the formation of CTC was the rate-limiting. The hydride transfer rates in both directions were estimated to be comparable, indicating redox reaction with NADP+/H was reversible in the case of CtFNR. The depletion of the C-terminal residues turns out CtFNR to be NADPH oxidizing enzyme, suggesting that the C-terminal residues are required for tuning the redox properties of the FAD prosthetic group.

Halomonas halodenitrificans nitric oxide reductase (NOR) is the membrane-bound heterodimer complex of NorC, which contains a low-spin heme c center, and NorB, which contains a low-spin heme b center, a high-spin heme b3 center, and a non-heme FeB center. The soluble domain of NorC, NorC* (ΔMet1–Val37) was heterologously expressed in Escherichia coli using expression plasmids harboring the truncated norC gene deleted of its 84 5′-terminal nucleotides. Analogous scission of the N-terminal helix as the membrane anchor took place when the whole norC gene was used. NorC* exhibited spectra typical of a low-spin heme c. In addition, NorC* functioned as the acceptor of an electron from a cytochrome c isolated from the periplasm of H. halodenitrificans and small reducing reagents. The redox potential of NorC* shifted ca. 40mV in the negative direction from that of NorC. Unlike NorC, recombinant NorB was not heterologously expressed. However, recombinant NOR (rNOR) could be expressed in E. coli by using a plasmid harboring all genes in the nor operon, norCBQDX, from which the three hairpin loops (mRNA) were deleted, and by using the ccm genes for the maturation of C-type heme. rNOR exhibited the same spectroscopic properties and reactivity to NO and O2 as NOR, although its enzymatic activity toward NO was considerably decreased. These results on the expression of rNOR and NorC* will allow us to develop more profound studies on the properties of the four Fe centers and the reaction mechanism of NOR from this halophilic denitrifying bacterium.

Ferredoxin-NADP+ oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP+, NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP+ and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNRox)-NADPH and reduced FNR (FNRred)-NADP+, both having CT absorption bands centered at approximately 600 nm. After mixing BsFNRox with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNRred was mixed with NADP+, the amount of BsFNRox increased with increasing NADP+ concentration, but BsFNRred remained as the major species at equilibrium even with about 50-fold molar excess NADP+. In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~ 500 s− 1) was much higher than the reverse one (< 10 s− 1). Mixing BsFdred with BsFNRox induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s− 1 at 10 °C, which increased as BsFdred increased from 40 to 120 μM. The reduction rate of BsFNRox by BsFdred was markedly decreased by premixing BsFNRox with BsFdox, indicating that the dissociation of BsFdox from BsFNRsq is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP+/NADPH were compared with those of other types of FNRs.

Ferredoxin-NAD(P) + oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum ( Ct FNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. Ct FNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of Ct FNR with NADP + , NADPH, and (4 S - 2 H)-NADPD by stopped-flow spectrophotometry. Mixing Ct FNR ox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When Ct FNR red was mixed with excess NADP + , the absorbance in the flavin band I increased to about 70 % of that of Ct FNR ox with an apparent rate of ~4 s −1 , whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between Ct FNR and NADP + /NADPH is reversible, in accordance with its physiological function.

Ferredoxin-NAD(P) + oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis ( Bs FNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si - and re -face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si -face Tyr50 residue in Bs FNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis ( Bs Fd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in Bs FNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and Bs Fd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

Ferredoxin-NADP(+) oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to 1(332 (whole C-terminal extension region) and S325 to 1(332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas K-m values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1-10 mu M. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin. (C) 2014 Elsevier Masson SAS. All rights reserved.

Cyanobacteria synthesize a small amount of hydrocarbons accessible as biofuels.This reaction is catalyzed by acyl-(acyl carrier protein) reductase (AAR) and aldehyde decarbonylase (AD).Because both enzymes have low activities, mutational studies for improving their activities are ongoing.It is expected that cyanobacteria expressing highly active mutants of AAR and AD produce high amounts of alkanes.To ensure this, here we carried out transformation of the cyanobacteria Synechocystis sp.PCC6803 with the plasmid containing the wild-type AAR and AD genes.We found that these cyanobacterial mutants produce higher amounts of heptadecane than the control strain, suggesting that expression of highly active mutants of AAR and AD will further improve alkane biosynthesis.