Multiple forms of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from spinach.

Crude extracts of ferredoxin-NADP reductase prepared from spinach by three different methods consistently contained two molecular weight forms of the enzyme: P-1, 117,500, and P-2, 50,000. The lower molecular weight form was purified and shown to consist of two different ionic forms. These three forms of the flavoprotein are immunologically identical. A third molecular weight form of the reductase, excluded by Sephadex G-100, generated P-1 and P-2 on rechromatography. Other experiments demonstrated that this enzyme has NADPH-tetrazolium reductase activity and it accounts for essentially all of the tetrazolium reductase activity of isolated chloroplasts.     

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.     

Reduction of methemoglobins M Hyde Park, M Saskatoon, and M Milwaukee by ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase system

The reduction of hemoglobins (Hb) M such as Hb M Iwate, Hb M Boston, Hb M Hyde Park, Hb M Saskatoon, and Hb M Milwaukee by the ferredoxin and ferredoxin-NADP reductase system was studied systematically under anaerobic conditions. The enzyme system could not reduce the abnormal chains in methemoglobin M with an alpha chain anomaly but effectively converted the methemoglobin M with a beta chain anomaly to the fully reduced form. During the reduction of the methemoglobin M with a beta chain anomaly, the spectra showed a shift of the initial isosbestic points, indicating the possible formation of intermediate hemoglobins in the partially reduced state. On the reduction mode of the methemoglobin M, however, it was classified into three types. 1) Only normal chains were reduced (Hb M Iwate and Hb M Boston). 2) Sequential reduction from normal to abnormal chains occurred (Hb M Milwaukee and Hb M Hyde Park). 3) Normal chains were preferentially reduced, but the reduction of abnormal chains also started at the same rate when the reduction of normal ones had proceeded halfway (Hb M Saskatoon). These differences are discussed in relation to the redox potential of each abnormal chain in methemoglobin M.     

C-terminal tyrosine of ferredoxin-NADP+ reductase in hydride transfer processes with NAD(P)+/H

Ferredoxin-NADP+ reductase (FNR) catalyzes the reduction of NADP+ to NADPH in an overall reversible reaction, showing some differences in the mechanisms between cyanobacterial and higher plant FNRs. During hydride transfer it is proposed that the FNR C-terminal Tyr is displaced by the nicotinamide. Thus, this C-terminal Tyr might be involved not only in modulating the flavin redox properties, as already shown, but also in nicotinamide binding and hydride transfer. FNR variants from the cyanobacterium Anabaena in which the C-terminal Tyr has been replaced by Trp, Phe, or Ser have been produced. All FNR variants show enhanced NADP+ and NAD+ binding, especially Tyr303Ser, which correlates with a noticeable improvement of NADH-dependent reactions. Nevertheless, the Tyr303Ser variant shows a decrease in the steady-state kcat value with NADPH. Fast kinetic analysis of the hydride transfer shows that the low efficiency observed for this mutant FNR under steady-state conditions is not due to a lack of catalytic ability but rather to the strong enzyme-coenzyme interaction. Three-dimensional structures for Tyr303Ser and Tyr303Trp variants and its complexes with NADP+ show significant differences between plant and cyanobacterial FNRs. Our results suggest that modulation of coenzyme affinity is highly influenced by the strength of the C-terminus-FAD interaction and that subtle changes between plant and cyanobacterial structures are able to modify the energy of that interaction. Additionally, it is shown that the C-terminal Tyr of FNR lowers the affinity for NADP+/H to levels compatible with steady-state turnover during the catalytic cycle, but it is not involved in the hydride transfer itself.     

Anabaena flavodoxin as an electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of flavodoxin residues in protein-protein interaction and electron transfer

Biochemical and structural studies indicate that electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd). Moreover, it has been shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (Fld), although, so far, no key residue on the Fld surface has been found to be the counterpart of such FNR side chains. In this study, negatively charged side chains on the Fld surface have been individually modified, either by the introduction of positive charges or by their neutralization. Our results indicate that although Glu16, Glu20, Glu61, Asp65, and Asp96 contribute to the orientation and optimization of the Fld interaction, either with FNR or with photosystem I (PSI) (presumably through the formation of salt bridges), for efficient ET, none of these side chains is involved in the formation of crucial salt bridges for optimal interaction with FNR. These data support the idea that the FNR-Fld interaction is less specific than the FNR-Fd interaction. However, analysis of the reactivity of these mutated Flds toward the membrane-anchored PSI complex indicated that all mutants, except Glu16Gln, lack the ability to form a stable complex with PSI. Thr12, Thr56, Asn58, and Asn97 are present in the close environment of the isoalloxazine ring of FMN in Anabaena Fld. Their roles in the interaction with and ET to FNR and PSI have also been studied. Mutants at these Fld positions indicate that residues in the close environment of the isoalloxazine ring modulate the ability of Fld to bind to and to exchange electrons with its physiological counterparts.     

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.     

Ferredoxin-NADP+ reductase uses the same site for the interaction with ferredoxin and flavodoxin

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.     

Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced [4Fe-4S](+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.     

Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119

Hydrophobic interactions play an active role in effective complex formation between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) from Anabaena, where an aromatic amino acid residue on the Fd surface (F65) and three hydrophobic residues (L76, L78, and V136) on the reductase surface have been shown to be essential for the efficient electron transfer (ET) reaction between Fd and FNR (Martínez-Júlvez et al. (2001) J. Biol. Chem. 276, 27498-27510). Since in this system flavodoxin (Fld) can efficiently replace Fd in the overall ET process, we have further investigated if such hydrophobic interactions are also critical in complex stabilization and ET in the FNR/Fld association. Different ET behaviors with Fld are observed for some of the mutations made at L76, L78, and V136 of Anabaena FNR. Thus, the ET interaction with Fld is almost completely lost upon introduction of negatively charged side chains at these positions, while more conservative changes in the hydrophobic patch can influence the rates of ET to and from Fld by altering the binding constants and the midpoint redox potentials of the flavin group. Therefore, our results confirm that nonpolar residues in the region close to the FAD group in FNR participate in the establishment of interactions with Fld, which serve to orient the two flavin groups in a manner such that ET is favored. In an attempt to look for the counterpart region of the Fld surface, the effect produced by the replacement of the only two nonpolar residues on the Fld surface, I59 and I92, by a Lys has also been analyzed. The results obtained suggest that these two hydrophobic residues are not critical in the interaction and ET processes with FNR. The reactivity of these I92 and I59 Fld mutants toward the membrane-anchored photosystem I (PSI) complex was also analyzed by laser flash absorption spectroscopy. From these data, significant effects are evident, especially for the I92 position of Fld, both in the association constant for complex formation and in the electron-transfer rate constant in the PSI/Fld system.     

Principles and patterns in the interaction between mono-heme cytochrome c and its partners in electron transfer processes

Cytochromes c are very widespread proteins that play key roles in the electron transfer events associated to a wide variety of physiological redox processes. The function of cytochromes c is, at the broad level, to interact with different partners in order to allow electrons to flow from one protein to another. Here, we focused our attention on the protein-protein interactions that involve mono-heme cytochrome c domains in order to identify possible general vs. specific patterns of intermolecular interactions at the structural level. We observed that a number of physico-chemical properties are statistically different in transient vs. permanent and fused complexes. These include the extent of the protein interface area, the amino acid composition and the packing density at the interface. The understanding of the features of transient redox complexes is of particular importance because of the difficulty of obtaining co-crystals that preserve the physiologically relevant configuration. In addition, we identified three different structural modes of interaction that cover all the structurally characterized cytochrome c interactions except one. The mode of interaction does not correlate with the nature of the complex (transient, permanent, fused). Regardless of the mode of interaction, the distance between the heme iron and the partner metal center or organic cofactor center of mass is typically around 19-20 ? for complexes permitting direct electron transfer between the two sites.     

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)