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PubMed1996

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.

Amino Acid Sequence↗
PubMed1982

Amino acid sequence of Synechocystis 6714 ferredoxin: a unique structural feature of unicellular blue-green algal ferredoxin.

The amino acid sequence of ferredoxin from Synechocystis 6714, a unicellular blue-green alga, was determined by a combination of conventional methods. The ferredoxin was composed of 96 amino acid residues and lacked methionine and tryptophan. The sequence was as follows: Ala-Ser-Tyr-Thr-Val-Lys-Leu-Ile-Thr- Pro-Asp-Gly-Glu-Asn-Ser-Ile-Glu-Cys-Ser-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala- Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys- Ala-Gly-Lys-Ile-Thr-Ala-Gly-Ser-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp- Asp-Gln-Ile-Glu-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Asp-Leu-Tyr. In an alignment of various ferredoxins with high homology from unicellular and filamentous blue-green algae, Synechocystis 6714 ferredoxin showed 4 gaps. Those between residues 9 and 10 and between residues 12 and 13 were unique for the ferredoxins from the unicellular algae Synechocystis 6714 and Aphanothece sacrum (ferredoxin I). Therefore, ferredoxins from unicellular algae were distinguishable from those of filamentous algae in terms of the presence of gaps. This feature appears to coincide with the phylogenetic division between the two types of blue-green algae.

Amino Acid Sequence↗
PubMed2006

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1.

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

Amino Acid Sequence↗
PubMed1990

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.

Binding Sites↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1981

Reduction of methemoglobin by ferredoxin and ferredoxin-NADP reductase system.

The changes in absorption spectra between 450 and 650 nm during the reduction of methemoglobin A, (alpha 2+ beta 3+)2, and (alpha 3+ beta 2+)2 valency hybrids by the system including ferredoxin and ferredoxin-NADP reductase were studied under anaerobic conditions. During the reduction of methemoglobin A, the isosbestic points gradually shifted to different positions. These shifts were clearly observed in the presence of inositol hexaphosphate, i.e. the isosbestic points were initially observed at 525 and 603 nm, and these shifted to 529 and 599 nm, respectively, suggesting that the intermediate hemoglobins are produced during the process of the reaction. This was confirmed by the isoelectric focusing electrophoresis of the partially reduced methemoglobin solutions with ferredoxin-NADP reductase system on Ampholine-polyacrylamide gel plate. On the other hand, the absorption spectra of alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 changed with excellent isosbestic points during th reductive reaction by ferredoxin-NADP reductase system, i.e. (alpha 2+ beta 3+)2 at 526 and 601 nm and (alpha 3+ beta 2+)2 at 529 and 599 nm. From these results, the mechanism of methemoglobin reduction by ferredoxin-NADP reductase system was suggested. 1) There are two pathways for the reduction of methemoglobin including (formula, see text). 2) The beta chains of methemoglobin may be more susceptible to the reduction than the alpha chains in tetrameric methemoglobin, and thereby the (alpha 3+ beta 2+)2 valency hybrid accumulated at the halfway point of the reaction. 3) The shift in isosbestic points of methemoglobin reduction (525 nm leads to 529 nm, 603 nm leads to 599 nm) is due to the accumulation of (alpha 3+ beta 2+)2, whose isosbestic points during the reduction by the ferredoxin-NADP reductase systems were 529 and 599 nm.

Ferredoxin-NADP Reductase↗
PubMed1994

Optical, EPR, and 1H NMR spectroscopy of serine-ligated [2Fe-2S] ferredoxins produced by site-directed mutagenesis of cysteine residues in recombinant Anabaena 7120 vegetative ferredoxin.

Anabaena 7120 vegetative ferredoxin is a plant-type [2Fe-2S] ferredoxin that contains only four cysteine residues. The four cysteines (Cys41, Cys46, Cys49, and Cys79), which ligate the iron-sulfur cluster, were mutated individually to serine. The wild-type and mutant apoprotein genes were overexpressed in Escherichia coli, and the iron-sulfur cluster was assembled in vitro by adding iron and sulfide. UV-vis, EPR, and 1H NMR spectra were recorded on the wild-type ferredoxin and mutants. The optical spectra of all mutant proteins, in the oxidized state, differed from that of wild-type ferredoxin. Three of the mutant proteins (Cys46Ser, Cys49Ser, and Cys79Ser) exhibited a rhombic EPR spectrum in the reduced state, but one (Cys41Ser) showed a near-axial EPR spectrum. The 1H NMR spectra of each of the four oxidized mutants contained a group of broad, hyperfine-shifted peaks between 20 and 30 ppm with anti-Curie temperature dependence. The pattern of these peaks was different for each mutant, and all were distinct from that of the wild-type ferredoxin. Because of problems with protein stability, it was possible to obtain NMR spectra of only two of the mutants when reduced. The downfield hyperfine 1H NMR spectrum of the reduced Cys46Ser mutant resembled that of wild-type ferredoxin, but that of the Cys49Ser mutant did not. The hyperfine-shifted resonances of the 1H NMR spectrum of the reduced Cys46Ser mutant were assigned on the basis of results from temperature dependence studies, measurements of nuclear Overhauser effect, and 1H NMR spectra of the mutant labeled with [beta-2H]cysteine. Four hyperfine-shifted peaks of reduced Cys49Ser at 298 K were observed at 173, 120, 32, and 18 ppm. These peaks exhibited Curie-type temperature dependence and were tentatively assigned to protons from residues coordinated to Fe(III). The reduced Cys49Ser mutant showed an additional 1H NMR peak at -15 ppm (at 298 K) with Curie-type temperature dependence whose origin is unknown at present. [2Fe-2S] clusters can be placed into three different classifications according to their EPR lines shapes, NMR spectra, and reduction potentials: plant type, vertebrate type, and Rieske type. The EPR and NMR results obtained here reveal that mutant Cys46Ser has a "plant-type" cluster but that mutant Cys49Ser has a "vertebrate-type" cluster. Cysteine to serine mutations have been employed in the past to probe whether particular cysteine residues participate as iron-sulfur ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Anabaena↗
PubMed1979

Properties of common wheat ferredoxin, and a comparison with ferredoxins from related species of triticum and aegilops.

Wheat ferredoxin was purified from the leaves of common wheat (Triticum aestivum). The absorption spectrum showed maxima at 465, 425, 332, and 278 nm. The absorbance ratio, A425 nm/A278 nm was 0.49, and the millimolar extinction coefficient at 425 nm was 10.8 mM-1. cm-1. The amino acid composition was determined to be Lys5, His2, Arg1, Asp11, Thr5, Ser7, Glu18, Pro5, Gly6, Ala7, Cys5, Val7, Met1, Ile4, Leu7, Tyr4, Phe1, and Trp1. The total number of amino acid residues was 97. The molecular weight was calculated from the amino acid composition to be 10,829, including iron and sulfur atoms. This value was confirmed by other methods, which were based on the contents of non-heme iron and of terminal amino acid. The N-terminal amino acid was alanine, and the C-terminal amino acid sequence was -Glu-Leu-Thr-AlaCOOH. Comparative studies were performed between T. aestivum ferredoxin and ferredoxins isolated from closely related species; these were T. aegilopoides, T. durum, Ae. squarrosa, and Ae. ovata. No significant differences in the properties of these ferredoxins were detected. It was also shown that these ferredoxins are immunologically homologous. It is, therefore, likely that one molecular species of ferredoxin is distributed through two genera of Triticum and Aegilops.

Amino Acids↗
PubMed1983

Structure of the extracellular ferredoxin from Rhodospirillum rubrum: close similarity to clostridial ferredoxins.

The amino acid sequence of an [8Fe-8S] ferredoxin isolated from the culture medium of Rhodospirillum rubrum, a photosynthetic purple non-sulfur bacterium, was determined by a combination of various conventional procedures. The sequence was A-Y-K-I-E-E-T-C-I-S-C-G-A-C-A-A-E-C-P-V-N-A-I-E-Q-G-D-T-I-F-V-V-N-A-D-T-C-I-D-C - G-N-C-A-N-V-C-P-V-G-A-P-V-A-E (55 amino acid residues). It lacked methionine, leucine, histidine, arginine, and tryptophan. The molecular weight was calculated to be 5,568 excluding iron and sulfur atoms. The distribution of 8 cysteine residues was exactly the same as that of clostridial-type ferredoxin, suggesting retention of the duplication of the bacterial ancestral ferredoxin gene. The extracellular ferredoxin of R. rubrum was compared with other ferredoxins observed in closely related photosynthetic bacteria and the evolutionary significance of this ferredoxin is discussed.

Amino Acid Sequence↗
PubMed1982

Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum.

A second ferredoxin protein was isolated from the thermophilic anaerobic bacterium Clostridium thermoaceticum and termed ferredoxin II. This ferredoxin was found to contain 7.9 +/- 0.3 iron atoms and 7.4 +/- 0.4 acid-labile sulfur atoms per mol of protein. Extrusion studies of the iron-sulfur centers showed the presence of two [Fe4-S4] centers per mol of protein and accounted for all of the iron present. The absorption spectrum was characterized by maxima at 390 nm (epsilon 390 = 30,400 M-1cm-1) and 280 nm (epsilon 280 = 41.400 M-1 cm-1) and by a shoulder at 300 nm. The ration of the absorbance of the pure protein at 390 nm to the absorbance at 280 nm was 0.74. Electron paramagnetic resonance data showed a weak signal in the oxidized state, and the reduced ferredoxin exhibited a spectrum typical of [Fe4-S4] clusters. Double integration of the reduced spectra showed that two electrons were necessary for the complete reduction of ferredoxin II. Amino histidine, and 1 arginine, and a molecular weight of 6,748 for the native protein. The ferredoxin is stable under anaerobic conditions for 60 min at 70 degrees C. The average oxidation-reduction potential for the two [Fe4-S4] centers was measured as -365 mV.

Amino Acids↗
PubMed2001

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.

Amino Acid Sequence↗
PubMed1976

The immune response to oxidized ferredoxin. II. Cross reactivity of cells and antisera to modified ferredoxins and the nature of the cells responding in vitro.

The cross reactivity of sera from rabbits sensitized to performic acid oxidized ferredoxin (O-Fd) and of spleen cells from mice sensitized to O-Fd was analysed using several chemically modified forms of ferredoxin in the complement fixation test and the in vitro lymphocyte stimulation assay. Only O-Fd and native ferredoxin (native-Fd) gave positive responses in both assays. Dinitrophenylated-O-Fd (DNP-O-Fd) and acid precipitated ferredoxin (TCA-Fd) were able to fix complement (C') but did not simulate DNA synthesis in vitro. Ferredoxin alkylated with N-ethylmaleimioxin (CM-Fd) was unable to stimulate DNA synthesis and was marginally able to fix C'. Methylated-O-Fd (meth-O-Fd) was not recognized in either assay. The various ferredoxin preparations were tested for their ability to sensitize mice for use in the in vitro lymphocyte stimulation assay. Only O-Fd, NEM-Fd and native-Fd were capable of sensitizing lymphocytes for a proliferative response in vitro to the test antigens. This correlates with the observation that only these antigens were able to induce DNA synthesis in O-Fd-sensitized lymphocytes. The nature of the cells responding in vitro was examined by treating the cells with rabbit anti-mouse immunoglobulin and C' or rabbit anti-mouse brain associated theta and C'. The 24-hr response was found to be sensitive to both sera while the 120-hr response was sensitive only to the anti-theta sera.

Alkylation↗
PubMed1993

Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin.

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel beta-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.

Amino Acid Sequence↗
PubMed1981

Electrochemical titrations of a ferredoxin-ferredoxin:NADP+ oxidoreductase complex.

Potentiometric titrations employing an electrochemical thin-layer cell indicate that complex formation between ferredoxin and ferredoxin:NADP+ oxidoreductase alters the midpoint oxidation-reduction potentials of both proteins. The midpoint potential of ferredoxin the complex becomes 22 +/- 6 mV more negative compared to ferredoxin alone while the midpoint potential of ferredoxin:NADP+ oxidoreductase becomes 23 +/- 4 mV more positive on complex formation.

Ferredoxin-NADP Reductase↗
PubMed1994

Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin.

A mutant of spinach ferredoxin-NADP+ reductase, in which Lys-88 has been changed to glutamine, has been obtained by site-directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity. By steady-state kinetics, the Km for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the kcat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: Kd values of 110 and 10 nM were determined for the mutant and wild-type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross-linking studies.

Base Sequence↗
PubMed1997

Residue Glu-91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin-thioredoxin reductase.

The [2Fe-2S] soluble ferredoxin from Chlamydomonas reinhardtii was mutated by site directed mutagenesis, using PCR and the expression plasmid pET-Fd as a template. The recombinant mutated proteins were purified to homogeneity and tested in the activation of NADP-malate dehydrogenase, a light dependent reaction in which ferredoxin thioredoxin reductase (FTR) and thioredoxin are involved. The mutation of residue Glu-91 (E92 in spinach, E94 in Anabaena) alone, either to Gln (E91Q) or to Lys (E91K), was found to completely abolish the reaction of the enzyme light activation. On the other hand, the mutants (E92Q) or (E92K) were as efficient as the wild type ferredoxin in this reaction whereas the double mutants (E91Q/E92Q) or (E91K/E92K) had no activity. In addition, a triple mutant (D25A/E28Q/E29Q) was also found to be inactive for this redox dependent light activation. All these mutations had much weaker effects on the ferredoxin/ferredoxin NADP reductase interaction as measured by the cytochrome c reduction assay. These results indicate that there is a recognition site for FTR in the C terminus part of ferredoxin, but also that a core of negatively charged residues in the alpha1 helix of ferredoxin might be important in the general process of light activation.

Animals↗
PubMed2002

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 Sequence↗
PubMed1978

Studies on the ferredoxin-ferredoxin-NADP reductase complex: kinetic and solvent perturbation studies on the location of sulfhydryl and aromatic amino acid residues.

The molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33,100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined. The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1 mol of ferredoxin was found to react with about 8 mol of p-chloromercuribenzoate in 10 min. Under low ionic strength conditions (1 mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250 nm were 3,640 and 3,690 M-1.S-1, respectively; under high ionic strength conditions (100 mM NaCl), these rate constants were 1,360 and 1,270 M-1.S-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength conditions were 54 and 1,040 M-1.S-1, respectively. By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8 mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.

Amino Acids↗