Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase

Oxidative biodegradation of aromatic compounds by bacteria usually begins with hydroxylation of the aromatic ring by multi-component dioxygenases like benzene dioxygenase, biphenyl dioxygenase, and others. These enzymes are composed of ferredoxin reductase, ferredoxin, and terminal oxygenase. Reducing equivalents that originate from NADH are transferred from ferredoxin reductase to ferredoxin and, in turn, to the terminal oxygenase, thus resulting in the activation of a dioxygen. BphA4 is the ferredoxin reductase component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102. The amino acid sequence of BphA4 exhibits significant homology with the putidaredoxin reductase of the cytochrome P450cam system in Pseudomonas putida, as well as with various other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) of bacteria. To date, no structural information has been provided for the ferredoxin reductase component of the dioxygenase systems. In order to provide a structural basis for discussing the mechanism of electron transport between ferredoxin reductase and ferredoxin, crystal structures of BphA4 and its NADH complex were solved. The three-dimensional structure of BphA4 is different from those of ferredoxin reductases whose structures have already been determined, but adopts essentially the same fold as the enzymes of the glutathione reductase (GR) family. Also the three-dimensional structure of the first two domains of BphA4 adopts a fold similar to that of adrenodoxin reductase (AdR) in the mitochondrial cytochrome P450 system. Comparing the amino acid sequence with what is known of the three-dimensional structure of BphA4 strongly suggests that the other ONFRs have secondary structural features that are similar to that of BphA4. This analysis of the crystal structures of BphA4 suggests that Lys53 and Glu159 seem to be involved in the hydride transfer from NADH to FAD. Since the amino acid residues around the active site, some of which seem to be important to electron transport, are highly conserved among ONFRs, it is likely that the mechanism of electron transport of BphA4 is quite applicable to other ONFRs.     

NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe-2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).     

Interaction of ferredoxin with ferredoxin:NADP reductase: effects of chemical modification of ferredoxin

Chemical modification studies have been conducted on spinach ferredoxin to determine the nature of the groups on ferredoxin involved in its interaction with its reaction partners. Modification of a limited number (three or four) carboxyl groups or of the single histidine residue resulted in a decreased ability of ferredoxin to participate in NADP photoreduction but not in cytochrome c photoreduction, suggesting that these groups may be involved in interaction with ferredoxin:NADP reductase but are not involved in interaction with the reducing side of Photosystem I. In contrast, modification of amino groups or the single arginine residue on ferredoxin had little effect on the ability of ferredoxin to participate in NADP photoreduction, suggesting these groups are not involved in the interaction of ferredoxin with either ferredoxin:NADP reductase or the reducing side of Photosystem I. Attempts to modify tyrosine residues on ferredoxin resulted in destruction of the iron-sulfur center of the protein.     

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.     

Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.     

Purification and characterization of renal ferredoxin from bovine renal mitochondria

A renal ferredoxin was purified from bovine renal mitochondria to electrophoretic purity. The molecular weight of the renal ferredoxin was estimated by gel filtration and SDS-polyacrylamide gel electrophoresis to be 12,500 and 13,000, respectively. The optical absorption spectrum of renal ferredoxin in the oxidized form showed two peaks at 416 and 457 nm in the visible region, and the EPR absorption spectrum showed peaks at gx = gy =1.94 and gz = 2.02 in the reduced form at 13K. These spectra were typical of the 2S-2Fe type ferredoxins. Dissimilarities were recognized in the amino acid composition and isoelectric point between bovine renal ferredoxin and bovine adrenodoxin, but not in the optical, magnetic, and immunochemical properties. The reconstitution of 25-hydroxyvitamin D3-1 alpha-hydroxylase system was performed with the three components of NADPH-adrenodoxin reductase from bovine adrenal mitochondria, renal ferredoxin, and cytochrome P-450(1) alpha from bovine renal mitochondria. The results showed that the renal ferredoxin was essential for the 1 alpha-hydroxylase activity of 25-hydroxyvitamin D3.     

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.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

Amino acid sequences of ferredoxin isoproteins from radish roots

Three ferredoxin isoproteins (R-Fd A, R-Fd B-1, and R-Fd B-2) were purified from white roots of radish (Raphanus sativus L. var. acantiformis cultivar Miyashige) and two isoproteins (L-Fd A and L-Fd B) from leaves. The amino acid sequences of three of them (L-Fd A, R-Fd B-1, and R-Fd B-2) were determined and compared with one another and with those of other higher plant ferredoxins so far studied. L-Fd A and R-Fd B-1 had heterogeneities at four and two amino acid sites, respectively. Two isoprotein (R-Fd B-1 and R-Fd B-2) were deduced to be expressed only in root tissue on the basis of sequence studies and amino acid compositions of all isoferredoxins isolated from the radish plant. The root ferredoxins sequenced in this study were similar to each other, but quite different from other higher plant ferredoxins, all of which were isolated from leaf tissue. The coupling activities of these ferredoxin isoproteins were measured in the NADP+-photoreduction system of radish chloroplasts and glutamate synthase [EC 1.4.7.1] systems isolated from radish leaf and root tissues. No distinctive physiological characteristics were observed among these isoferredoxins.     

Comparative study of bovine adrenodoxin and renorenodoxin

The ferredoxin from bovine renal mitochondria (renoredoxin) has been obtained in a highly purified state. The A415/A280 ratio of the purified renoredoxin is 0.84. The absorption spectrum of renoredoxin was shown to be identical to that of bovine adrenodoxin. Two forms of renoredoxin (Mr 14200 and 13300) were detected by using polyacrylamide gel electrophoresis. These forms exhibit a very similar immunologic cross-reactivity with polyclonal antibodies to adrenodoxin. The N-terminal amino acid sequence of renal ferredoxin was shown to be identical to that of adrenodoxin; the C-terminal sequences of both ferredoxins undergo a similar post-translational proteolytic modification. The amino acid composition of ferredoxins are also very close. Renal ferredoxin can be replaced by adrenodoxin in reconstituted systems from bovine adrenal cortex mitochondria which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.     

Chemical modification of carboxyl groups on spinach ferredoxin alters its ability to interact with ferredoxin:NADP reductase

Treatment of spinach ferredoxin with glycine ethyl ester in the presence of a water soluble carbodiimide resulted in the modification of 3-4 carboxyl groups and decreased the ability of ferredoxin to participate in NADP photoreduction by chloroplast membranes by about 80%. The ability of the modified ferredoxin to receive electrons from the reducing side of Photosystem I was relatively unaffected. These findings suggest that the modified ferredoxin is unable to interact with ferredoxin:NADP reductase. This has been verified by demonstration that the modified ferredoxin fails to produce difference spectra typical of a ferredoxin-ferredoxin:NADP reductase complex when added to ferredoxin:NADP reductase.     

A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum.

A ferredoxin containing only one [Fe4S4] cluster was purified from Clostridium thermoaceticum. It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25. Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1). The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two [Fe4S4] clusters, and is similar to other ferredoxins with only one [Fe4S4] cluster. The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C. It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum. NADPH is used in the synthesis of acetate from CO2 in this organism.     

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.     

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.     

Chemical modification of spinach ferredoxin with diethylpyrocarbonate: evidence for the essential nature of the histidyl residue

Spinach ferredoxin contains a single ferredoxin which can be chemically modified with diethylpyrocarbonate. By varying the concentration of diethylpyrocarbonate modified ferredoxins could be prepared which had only one or both of the imidazole nitrogens of the histidine modified. A small amount of tyrosine was also modified. Ferredoxin with only one of the imidazole nitrogens modified was fully active in NADP photoreduction by chloroplast membranes. This activity was lost as the second imidazole nitrogen was modified. The results suggest an essential role for the single histidine of ferredoxin.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

Effect of pH, salt, and coupling state on the interaction of ferredoxin with the chloroplast membrane

To determine if the interaction between ferredoxin and ferredoxin:NADP reductase is similar to the interaction between the purified proteins when the ferrodoxin:NADP reductase is membrane bound, the effect of pH, salt, and coupling state on the Km for ferredoxin in NADP reduction by chloroplast membranes has been examined. Increasing pH and salt concentrations as well as uncouplers all resulted in increases in the Km for ferredoxin. The pH and salt effects on the Km are similar to effects observed by others (C. Batie and H. Kamin (1981) J. Biol. Chem. 256, 7756-7763) on the dissociation constant for a complex between the two purified proteins, although the salt effect on the Km appears to be affected by the surface potential of the chloroplast membrane. These results suggest that the interaction between ferredoxin and the membrane-bound ferredoxin:NADP reductase is not greatly different from the interaction which has been characterized between the two purified proteins.     

Tryptophan fluorescence studies of ferredoxin:NADP reductase indicate the presence of tryptophan in or near the ferredoxin binding site

The tryptophan fluorescence properties of the flavoprotein ferredoxin:NADP reductase have been examined. Although not sensitive to changes in pH or salt concentration, the tryptophan fluorescence is affected by the presence of substrates for the flavoprotein. While NADP addition results in a slight quenching of the fluorescence, ferredoxin decreases the fluorescence by nearly 50%, suggesting the presence of tryptophan in or near the ferredoxin binding site. Titration of this effect gives a dissociation constant for the ferredoxin: flavoprotein complex which is similar to that obtained by spectral perturbations. This approach has also been used to demonstrate that a chemically modified ferredoxin which does not produce spectral perturbations when added to flavoprotein is capable of interacting with the flavoprotein although with a higher dissociation constant than for native ferredoxin.     

New perspectives on bacterial ferredoxin evolution

Summary Recent evidence indicates that a gene transposition event occurred during the evolution of the bacterial ferredoxins subsequent to the ancestral intrasequence gene duplication. In light of this new information, the relationships among the bacterial ferredoxins were reexamined and an evolutionary tree consistent with this new understanding was derived. The bacterial ferredoxins can be divided into several groups based on their sequence properties; these include the clostridial-type ferredoxins, theAzotobacter-type ferredoxins, and a group containing the ferredoxins from the anaerobic, green, and purple sulfur bacteria. Based on sequence comparison, it was concluded that the amino-terminal domain of theAzotobacter-type ferredoxins, which contains the novel 3Fe3S cluster binding site, is homologous with the carboxyl-terminal domain of the ferredoxins from the anaerobic photosynthetic bacteria.A number of ferredoxin sequences do not fit into any of the groups described above. Based on sequence properties, these sequences can be separated into three groups: a group containingMethanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II, a group containingDesulfovibrio gigas ferredoxin andClostridium thermoaceticum ferredoxin, and a group containingDesulfovibrio africanus ferredoxin I andBacillus stearothermophilus ferredoxin. The last two groups differ from all of the other bacterial ferredoxins in that they bind only one FeS cluster per polypeptide, whereas the others bind two. Sequence examination indicates that the second binding site has been either partially or completely lost from these ferredoxins.Methanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II are of interest because, of all the ferredoxins whose sequences are presently known, they show the strongest evidence of internal gene duplication. However, the derived evolutionary tree indicates that they diverged from theAzotobacter-type ferredoxins well after the ancestral internal gene duplication. This apparent discrepancy is explained by postulating a duplication of one halfchain sequence and a deletion of the other halfchain. TheClostridium thermoaceticum andBacillus stearothermophilus groups diverged from this line and subsequently lost one of the FeS binding sites.It has recently become apparent that gene duplication is ubiquitous among the ferredoxins. Several organisms are now known to have a variety of ferredoxins with widely divergent properties. Unfortunately, in only one case are the sequences of more than one ferredoxin from the same organism known. Thus, although the major features of the bacterial ferredoxin tree are now understood, a complete bacterial phylogeny cannot be inferred until more sequence information is available.