Ferredoxin-NADP+ reductase. Kinetics of electron transfer, transient intermediates, and catalytic activities studied by flash-absorption spectroscopy with isolated photosystem I and ferredoxin

The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (-380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8-10 ms using 1.6 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction.     

Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium.

The unicellular red alga, Cyanidium caldarium, synthesizes phycocyanobilin from protoheme via biliverdin IX alpha. In vitro transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins were previously shown to require NADPH, ferredoxin, and ferredoxin-NADP+ reductase, as well as specific heme oxygenase and phycobilin formation enzymes. The role of NADPH in these reactions was investigated in this study. The C. caldarium enzymatic activities that catalyze biliverdin IX alpha formation from protoheme, and phycobilin formation from biliverdin IX alpha, were partially purified by differential (NH4)2SO4 precipitation. The enzyme fractions, when supplemented with a light-driven ferredoxin-reducing photosystem I fraction derived from spinach leaves, catalyzed light-dependent transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins, with or without the addition of NADPH and ferredoxin-NADP+ reductase. In the dark, neither reaction occurred unless NADPH and ferredoxin-NADP+ reductase were supplied. These results indicate that the only role of NADPH in both reactions of phycobilin biosynthesis, in vitro, is to reduce ferredoxin via ferredoxin-NADP+ reductase and that reduced ferredoxin can directly supply the electrons needed to drive both steps in the transformation of protoheme to phycocyanobilin.     

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.     

Toyopearl HW-65C: ammonium sulfate as a new column chromatographic adsorbent for enzyme purification

We found that Toyopearl HW-65C gel matrix adsorbed ferredoxin and ferredoxin-NADP+ reductase in the presence of concentrated ammonium sulfate. Ferredoxin was strongly adsorbed on the gel in 80% saturated ammonium sulfate, and ferredoxin-NADP+ reductase was adsorbed in 40% saturated ammonium sulfate. The phenomenon was utilized for purification of ferredoxin and the reductase on a Toyopearl HW-65C: ammonium sulfate column. The technique greatly simplified the early stage of purification of ferredoxin and the reductase. The improved purification methods further involved column treatments with DEAE-Toyopearl 650M and Matrex Red A. The effectiveness of the columns is reported. Since a number of other proteins such as cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and glucose oxidase were also adsorbed well in an appropriately concentrated ammonium sulfate solution, the method may be of general use in enzyme purification.     

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.     

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.     

Apicomplexan parasites possess distinct nuclear-encoded, but apicoplast-localized, plant-type ferredoxin-NADP+ reductase and ferredoxin

In searching for nuclear-encoded, apicoplast-localized proteins we have cloned ferredoxin-NADP(+) reductase from Toxoplasma gondii and a [2Fe-2S] ferredoxin from Plasmodium falciparum. This chloroplast-localized redox system has been extensively studied in photosynthetic organisms and is responsible for the electron transfer from photosystem I to NADP+. Besides this light-dependent reaction in nonphotosynthetic plastids (e.g. from roots), electrons can also flow in the reverse direction, from NADPH to ferredoxin, which then serves as an important reductant for various plastid-localized enzymes. These plastids possess related, but distinct, ferredoxin-NADP+ reductase and ferredoxin isoforms for this purpose. We provide phylogenetic evidence that the T. gondii reductase is similar to such nonphotosynthetic isoforms. Both the P. falciparum [2Fe-2S] ferredoxin and the T. gondii ferredoxin-NADP+ reductase possess an N-terminal bipartite transit peptide domain typical for apicoplast-localized proteins. The recombinant proteins were obtained in active form, and antibodies raised against the reductase recognized two bands on Western blots of T. gondii tachyzoite lysates, indicative of the unprocessed and native form, respectively. We propose that the role of this redox system is to provide reduced ferredoxin, which might then be used for fatty acid desaturation or other biosynthetic processes yet to be defined. Thus, the interaction of these two proteins offers an attractive target for drug intervention.     

Studies on photosystem I. II. Involvement of ferredoxin in cyclic electron flow

The effects of ferredoxin (Fd) and ferredoxin-NADP reductase on the light-induced spectral changes of cytochrome f (cyt f) were investigated with specific reference to their possible involvement in the cyclic electron transfort pathway of photosystem I (PS I). The steady-state level of photooxidation of reduced cytochrome f is decreased by ferredoxin but unaffected by either ferredoxin-NADP reductase alone or ferredoxin plus ferredoxin-NADP reductase when present in equimolar concentrations. These data are taken as evidence for a cyclic electron transport pathway of: PS I → “X” → Fd → (cyt f) → PC → PS I. The reduced ferredoxin could either reduce directly plastocyanin (PC) or via cytochrome f; the data do not allow differentiation between these two possibilities. However, neither ferredoxin-NADP reductase nor cytochrome b564 appear to serve as electron carriers in this pathway.     

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.     

Kinetics of some electron-transfer reactions in biological Photosystems II. Study of intramolecular electron-transfer rates between ferredoxin-NADP reductase, NADP radical and oxidized ferredoxin

It has been shown by the pulse radiolysis technique that radiation-generated NADP free radicals (NADP·) first combine with ferredoxin-NADP reductase and then transfer the odd electron by a fast intramolecular process to the enzyme flavin moiety yielding the semiquinone (ferredoxin-NADP reductase, FNR-FADH·). The corresponding first-order rate constant k₁₅ varies with ionic strength from 2.6·10³ s⁻¹ at I = 0.66 M to 2.3·10⁴ s⁻¹ at I = 0.005 M In the presence of ferredoxin-NADP reductase-bound oxidized ferredoxin, the electron cascades, thus further reducing the ferredoxin. The transfer of the electron from the flavin semiquinone (ferredoxin-NADP reductase, FNR-FADH·) to the bound oxidized ferredoxin proceeds at a rate of k₁₈ = 2.36 s⁻¹. This process approaches an equilibrium condition which is in favor of the reverse reaction suggesting that k₋₁₈ > k₁₈.     

Biochemical and crystallographic characterization of ferredoxin-NADP(+) reductase from nonphotosynthetic tissues.

Distinct forms of ferredoxin-NADP(+) reductase are expressed in photosynthetic and nonphotosynthetic plant tissues. Both enzymes catalyze electron transfer between NADP(H) and ferredoxin; whereas in leaves the enzyme transfers reducing equivalents from photoreduced ferredoxin to NADP(+) in photosynthesis, in roots it has the opposite physiological role, reducing ferredoxin at the expense of NADPH mainly for use in nitrate assimilation. Here, structural and kinetic properties of a nonphotosynthetic isoform were analyzed to define characteristics that may be related to tissue-specific function. Compared with spinach leaf ferredoxin-NADP(+) reductase, the recombinant corn root isoform showed a slightly altered absorption spectrum, a higher pI, a >30-fold higher affinity for NADP(+), greater susceptibility to limited proteolysis, and an approximately 20 mV more positive redox potential. The 1.7 A resolution crystal structure is very similar to the structures of ferredoxin-NADP(+) reductases from photosynthetic tissues. Four distinct structural features of this root ferredoxin-NADP(+) reductases are an alternate conformation of the bound FAD molecule, an alternate path for the amino-terminal extension, a disulfide bond in the FAD-binding domain, and changes in the surface that binds ferredoxin.     

Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes

Heparin, an anionic polysaccharide, inhibited the ferredoxin-catalyzed reduction of NADP in spinach chloroplast thylakoid membranes. Under the same conditions of assay, heparin did not interfere markedly with photoreduction of methyl viologen, anthraquinone sulfonate, or ferredoxin. A kinetic analysis of the heparin-induced interference with NADP photoreduction showed partial competitive inhibition. Heparin also interfered with NADPH oxidation by membrane-bound ferredoxin-NADP reductase (with dichlorophenol-indophenol as the acceptor) by a mechanism that involves partial competitive inhibition. This reaction was sensitive to the presence of salts; increasing ionic strength increases the heparin Ki for inhibition of NADPH oxidation. These results show that heparin binds to ferredoxin-NADP reductase, and in doing so interferes with binding to the reductase by both ferredoxin and NADP(H). Since heparin is redox inactive and does not interfere with the photophosphorylation reaction, it is a useful inhibitor of thylakoid membrane reactions which require the catalytic activity of ferredoxin-NADP reductase.     

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.     

Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics

Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin. The catalytic electron-transfer reactivity of the [7Fe-8S] ferredoxin between ferredoxin-NADP+ reductase and cytochrome c was recognized.     

Circular dichroism studies of the complex between ferredoxin and ferredoxin-NADP reductase

Circular dichroism (CD) spectra are presented of ferredoxin, ferredoxin-NADP reductase and their complex. A change in CD occurs on complex formation which is consistent with a decrease in the Cotton effects due to the ferredoxin. This change is interpreted as due to a decrease in interaction in ferredoxin between the iron-sulphur chromophore group and the protein.     

Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.     

Biosynthesis of phycobilins. Ferredoxin-mediated reduction of biliverdin catalyzed by extracts of Cyanidium caldarium

Cell-free extract of the unicellular rhodophyte, Cyanidium caldarium catalyzes enzymatic reduction of biliverdin IX alpha to phycocyanobilin, the chromophore of the light-harvesting phycobiliprotein, phycocyanin. The enzyme activity is soluble, and the required reductant is NADPH. The extract has been separated into three protein fractions, all of which are required to reconstitute biliverdin reduction. One fraction contains ferredoxin, which was identified by its absorption spectrum. This fraction could be replaced with commercial ferredoxin derived from spinach or the red alga, Porphyra umbilicalis. The second protein fraction contains ferredoxin-NADP+ reductase, which was identified by the ability to catalyze ferredoxin-dependent reduction of cytochrome c in the presence of NADPH. This fraction could be replaced with commercial spinach ferredoxin-NADP+ reductase. These two components appear to be identical to previously described components of the algal heme oxygenase system that catalyzes biliverdin IX alpha formation from protoheme in C. caldarium extracts. The third protein fraction, in the presence of the first two (or their commercial counterparts) plus NADPH, catalyzes the reduction of biliverdin IX alpha to phycocyanobilin. The results indicate that the transformation of biliverdin to phycocyanobilin catalyzed by C. caldarium extracts is a ferredoxin-linked reduction process. The results also suggest the possibility that heme oxygenation and biliverdin reduction may occur in C. caldarium on associated enzyme systems.     

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.