Fd-NADP Reductase Research Articles

Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential.

Recombination can be used in the laboratory to overcome component limitations in synthetic biology by creating enzymes that exhibit distinct activities and stabilities from native proteins. To investigate how recombination affects the properties of an oxidoreductase that transfers electrons in cells, we created ferredoxin (Fd) chimeras by recombining distantly related cyanobacterial and cyanomyophage Fds (53% identity) that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required for similar levels of cellular electron transfer. These results show how Fds can be diversified through recombination and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic metabolic pathway.

Plasmodium-specific basic amino acid residues important for the interaction with ferredoxin on the surface of ferredoxin-NADP+ reductase.

The malaria parasite (Plasmodium falciparum) possesses a plastid-derived, essential organelle called the apicoplast, which contains a redox system comprising plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR). This system supplies reducing power for the crucial metabolic pathways in this organelle. Electron transfer between P. falciparum Fd (PfFd) and FNR (PfFNR) is performed with higher affinity and specificity than that of plant Fd and FNR. To investigate the mechanism for such superior protein-protein interaction, we searched for the Fd interaction sites on the surface of PfFNR. Basic amino acid residues on the FAD binding side of PfFNR were comprehensively substituted to acidic amino acids by site-directed mutagenesis. Kinetic analysis of electron transfer to PfFd and plant Fds, physical binding to immobilized PfFd and thermodynamics of the PfFd binding using these PfFNR mutants revealed that several basic amino acid residues including those in Plasmodium-specific insertion region are important for the interaction with PfFd. Majority of these basic residues are Plasmodium-specific and not conserved among plant and cyanobacteria FNRs. These results suggest that the interaction mode of Fd and FNR is diverged during evolution so that PfFd: PfFNR interaction meets the physiological requirement in the cells of Plasmodium species.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Å resolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

Electron Transfer of Site-Specifically Cross-Linked Complexes between Ferredoxin and Ferredoxin−NADP+ Reductase

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd:FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductase and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.

Cloning and Characterization of Ferredoxin and Ferredoxin-NADP+ Reductase from Human Malaria Parasite

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.

Dynamics of Redox State-Dependent Ferredoxin Structure and Electron-Transfer Complex Formation with Ferredoxin: NADP+ Reductase

We have investigated the conformational and dynamical changes of plant-type ferredoxin (Fd) depending upon the redox state by 2D NMR spectroscopy and the three-dimensional structures of Fd, Fd:NADP+ reductase (FNR) and their complex by X-ray crystallography. These combined data allow us to discuss a molecular mechanism for the redox dependent change in the affinity of Fd with FNR, which is important in modulating redox metabolism in the cells, and also discuss the molecular property of Fd as an electron carrier in living cells.

Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize.

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.

Comparison of the Electrostatic Binding Sites on the Surface of Ferredoxin for Two Ferredoxin-dependent Enzymes, Ferredoxin-NADP+ Reductase and Sulfite Reductase

Plant-type ferredoxin (Fd), a [2Fe-2S] iron-sulfur protein, functions as an one-electron donor to Fd-NADP(+) reductase (FNR) or sulfite reductase (SiR), interacting electrostatically with them. In order to understand the protein-protein interaction between Fd and these two different enzymes, 10 acidic surface residues in maize Fd (isoform III), Asp-27, Glu-30, Asp-58, Asp-61, Asp-66/Asp-67, Glu-71/Glu-72, Asp-85, and Glu-93, were substituted with the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, the redox potential of which was more positive by 67 mV than that of the wild type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interestingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, whereas this relationship was reversed in the reaction with SiR. The static interaction of the mutant Fds with each the two enzymes was analyzed by gel filtration of a mixture of Fd and each enzyme, and by affinity chromatography on Fd-immobilized resins. The contributions of Asp-66/Asp-67 and Glu-93 were found to be most important for the binding to FNR and SiR, respectively, in accordance with the kinetic data. These results allowed us to map the acidic regions of Fd required for electron transfer and for binding to FNR and SiR and demonstrate that the interaction sites for the two enzymes are at least partly distinct.

Ferredoxin–NADP+ reductase is the site of oxygen reduction in pseudocyclic electron transport

The effects of ferredoxin (Fd) and Fd–NADP+ reductase (FNR) on the oxygen photoreduction by photosystem I (PS I) in spinach (Spinacia oleracea L.) thylakoid membranes were investigated using a unique photoelectrochemical cell. This cell was previously shown to monitor the Mehler reaction products of photosynthetic oxygen reduction and represents an excellent tool for studying pseudocyclic electron transport. The magnitude of the photocurrent produced by the thylakoids was increased by as much as 40% in the presence of 60 μM Fd. If thylakoids were supplemented by both Fd and FNR, an additional increase of photocurrent was observed. All these reactions were inhibited by catalase, an enzyme that degrades H2O2, to demonstrate that O2 reduction was involved in all the photoreactions studied. The fact that more O2 was consumed in the presence of FNR was interpreted as evidence that the most effective site of oxygen reduction on the acceptor side of PS I is on FNR and not on Fd. The in vivo implication is that during pseudocyclic electron transport, NADP+ and oxygen directly compete for PS I electrons, with the former having significantly faster reaction kinetics. The advantageous physiological consequences of such a competition are (i) pseudocyclic electron transport would represent a true attenuating mechanism of the redox state of the NADP+–NADPH pool, (ii) oxygen would be a contingent acceptor under high illumination stress, helping to cope with the resultant elevated electron transport rates, and (iii) this mechanism is indisputably a faster response to stress than cyclic electron transport. Key words: Spinacia oleracea, photosystem I, thylakoid membranes, ferredoxin–NADP+ reductase, pseudocyclic electron transport, photoelectrochemistry.

Studies on a soluble thyroid iodotyrosine deiodinase: activation by NADPH and electron carriers.

Unlike the microsomal iodotyrosine deiodinase activity, which was reponsive to both NADPH and dithionite, the enzymatic activity solubilized and purified from bovine thyroid microsomes could no longer utilize NADPH for deiodination. The purified enzyme could, however, be activated by dithionite, as well as by methyl viologen (MV), reduced by NADPH with thyroidal NADPH-cytochrome c reductase, or by ferredoxin (Fd), in presence of NADPH and Fd-NADP reductase. The purified enzyme could not be activated by photoreduced FMN or by reducing agents having more positive oxidation-reduction potentials than the Fd-NADP system (E0′ = −420 mV). N-elhylmaleimide inhibited microsomal deiodination as well as the activation of the purified enzyme by reduced MV or Fd, but had no effect on activation by dithionite, possibly because the latter can release NEM bound to thiol groups. The iron-chelating agents, 2,2′- dipyridyl and o-phenanthroline, inhibited microsomal deiodination but did not affect the dithionite-, MV- or Fd-activation of the microsomes or of the purified enzyme. The data suggest that microsomal deiodination of diiodotyrosine involves reduction of a flavoprotein deiodinase whose catalytic activity requires unblocked sulfur and that the electron flow from NADPH to the substrate is via an intermediate of highly negative redox potential which probably contains iron.