Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).     

Characterization of a covalently linked complex involving ferredoxin and ferredoxin: NADP reductase

Ferredoxin and the flavoprotein, ferredoxin: NADP reductase, have been covalently linked by incubation in the presence of a water soluble carbodiimide. The cross-linking reaction yields an adduct having a 1:1 stoichiometry. The adduct has depressed levels of diaphorase and NADPH oxidase activity and is inactive in reduction of cytochrome c using NADPH as an electron donor. Thus, although similar to an adduct described by Zanetti and coworkers [J Biol Chem 259: 6153–6157 (1984)] in its stoichiometry, the adduct described herein has significantly different enzymatic properties. It is suggested that this may be a reflection of differences in the interaction between the two proteins resulting from differences in experimental conditions in which the two adducts were prepared.Abbreviations Fd ferredoxin - Fp ferredoxin: NADP reductase - Fd Fp covalently linked Fd-Fp adduct - Fd:Fp noncovalently linked complex between Fd and Fp - EDC 1-ethyl-3-(dimethylaminopropyl) carbodiimide - Tris tris-hydroxymethylaminomethane - MOPS 3-(N-morpholino)propane sulfonic acid - DCIP 2,6-dichloropenolindophenol     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.     

The apicomplexan Cryptosporidium parvum possesses a single mitochondrial-type ferredoxin and ferredoxin:NADP+ reductase system

We have successfully expressed recombinant mitochondrial‐type ferredoxin (mtFd) and ferredoxin:NADP⁺ reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time for an apicomplexan. Both C. parvum mtFd (CpmtFd) and FNR (CpmtFNR) were obtained and purified as holo‐proteins, in which the correct assembly of [2Fe–2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/vis and electron paramagnetic resonance. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c‐coupled assay that followed a typical Michaelis‐Menten kinetics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals and could be explored as potential drug targets in Cryptosporidium and other apicomplexans.     

Complex-forming properties of butanedione-modified ferredoxin-NADP+ reductase with NADP+ and ferredoxin.

The plastidic ferredoxin-NADP⁺ reductase from the xanthophycean alga Bumilleriopsis forms a stoichiometric 1:1 complex with ferredoxin and NADP⁺ which is demonstrated by difference spectra of both complexes. Butanedione modification of the flavoprotein results in loss of its enzymatic activities (transhydrogenase and diaphorase) concurrently with its capability to form a complex with NADP⁺, whereas the ferredoxin-binding site is practically not influenced by the modifying reagent and complex formation is still possible. It is assumed, therefore, that butanedione specifically reacts with the arginine residue of the protein involved in binding of pyridine nucleotides at the active site. Further, the data presented strongly support the previous proposal of different binding sites for ferredoxin and pyridine nucleotides at the reductase.     

Reexamination of the interaction between ferredoxin:NADP reductase and NADP

A comparison was made of graphical and subtractive methods for the determination of the dissociation constant of a complex between ferredoxin:NADP reductase and NADP. The subtractive method gave K_d values near 10 μm which are consistent with recently determined values for K_m,NADP in assays of NADP photoreduction by chloroplast membranes. The graphical method gave values which were considerably higher. The difference between the two methods is due to the failure of the graphical method to correct for the amount of each component present in the complex at the low NADP/ flavoprotein ratios necessary for binding studies. A second NADP binding site of much lower affinity (K_d approx 1 mm) was also detected.     

Interactions between spinach ferredoxin and other electron carriers. The involvement of a ferredoxin:cytochrome c complex in the ferredoxin-linked cytochrome c reductase activity of ferredoxin:NADP+ oxidoreductase.

The trinitrophenylation of a single amino group of spinach ferredoxin abolishes its ability to inhibit the diaphorase activity of the flavoprotein, ferredoxin:NADP oxidoreductase (EC 1.6.7.1); in contrast, the ability of ferredoxin to participate in the ferredoxin-linked cytochrome c reductase activity catalyzed by the flavoprotein is unaffected. Comparison with previously published results [Davis, D. J., and San Pietro, A. (1977) Biochem. Biophys. Res. Commun.74, 33–40]indicates that the site of interaction between ferredoxin and the flavoprotein resulting in inhibition if diaphorase activity is responsible for the spectrally observable 1:1 complex between the two proteins and is identical to the site of ferredoxin involvement in NADP photoreduction. The role of ferredoxin in the ferredoxin-linked cytochrome c reductase activity of the flavoprotein has been reexamined under conditions were the entire electron-accepting system (rather than just the ferredoxin component) is rate limiting. The data support a mechanism by which ferredoxin can bind either to the flavoprotein or to cytochrome c, and the ferredoxin:cytochrome c complex serves as the true substrate for reduction by the flavoprotein. Furthermore, Chromatographic evidence is presented for the formation of complexes between ferredoxin and cytochrome c.     

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.     

Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium.

A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233-16241], do not significantly influence the rate of linear electron transport to NADP(+). Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins.     

Inhibition of ferredoxin: NADP+ reductase activity by the hexacyanochromate (III) ion

The small inorganic complex Cr(CN)6(3-) is a clean inhibitor of the ferredoxin: NADP+ reductase-catalysed oxidation of reduced spinach ferredoxin by NADP+. Independent spectrophotometric measurements show that millimolar additions of Cr(CN)6(3-) to mixtures of ferredoxin and ferredoxin NADP+ reductase give a marked attenuation of the difference spectrum characteristic of ferredoxin-ferredoxin: NADP+ reductase complex formation. Since there is no evidence, from NMR studies, for significant binding of Cr(CN)6(3-) to ferredoxin, these results indicate that Cr(CN)6(3-) binds to ferredoxin: NADP+ reductase at a site which is crucial to its interaction with the electron-transfer protein. The effective kinetic binding constant for Cr(CN)6(3-), measured at low ferredoxin concentration, is 445 M-1 (ie Kdiss congruent to 2 mM) at 25 degrees, pH7.5, I = 0.10 M. With assumption of a simple electrostatic interaction, an enzyme domain with an effective charge of 3+/4+ is proposed.     

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.     

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.     

Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)相似文献   

Affinity chromatography of Anacystis nidulans ferredoxin nitrate reductase and NADP reductase on reduced ferredoxin-Sepharose.

The behavior of two ferredoxin-dependent enzymes—nitrate reductase and NADP reductase—fromAnacystis nidulans on a ferredoxin-Sepharose gel was examined. The oxidized gel-bound ferredoxin exhibited very low affinity for these enzymes but effectively bound both nitrate reductase and NADP reductase when reduced by dithionite. Selective procedures are described for the clution of each of these two enzymes from the reduced ferredoxin-Sepharose gel. These simple methods allow substantial purification of both enzymes.     

Synechocystis ferredoxin/ferredoxin-NADP(+)-reductase/NADP+ complex: Structural model obtained by NMR-restrained docking

Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.     

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.     

Arginyl groups involved in the binding of Anabaena ferredoxin--NADP+ reductase to NADP+ and to ferredoxin

Chemical modification of ferredoxin--NADP+ reductase from the cyanobacteria Anabaena has been performed using the alpha-dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome-c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs = 8.3 M-1 min-1) and is responsible for the binding of NADP+, and a second less reactive group (kobs = 0.9 M-1 min-1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin--NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.