Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Pseudomonas putida harbors two ferredoxin-NADP⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k_cat/K_m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.Commonly, Fprs are ubiquitous, monomeric, reversible flavin enzymes. Fprs evidence a profound preference for NADP(H) over NAD(H) (3). They harbor a prosthetic flavin cofactor (FAD) and catalyze the reversible electron exchange between NADPH and either ferredoxin (Fd) or flavodoxin (Fld) (4, 5). In oxygenic photosynthesis, the Fd is reduced by the photosystem and subsequently passes electrons on to NADP⁺ via the Fpr. This reaction provides the cellular NADPH pool required for CO₂ assimilation and other biosynthetic processes (4, 5). In heterotrophic organisms such as bacteria, reduced ferredoxin, owing to the reverse enzymatic activity of the Fpr, can donate an electron to several Fd-dependent enzymes, such as nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase, allowing ferredoxin to function in a variety of systems, including oxidative stress (1, 4, 5).Iron is the fourth most abundant element in the natural environment and exists primarily as an oxidized form, Fe(III), which has very low solubility under neutral pH conditions (9, 34) and thus presents problems in terms of bioavailability. However, ferrous iron, of Fe(II), is soluble and available at neutral pH in bacterial cytosol (34). Most bacteria secrete siderophores, which are natural chelators of ferric iron. After they bind to ferric iron, that complex enters the bacteria and releases ferric iron into the cytosol in ferric or ferrous form (9). In the bacterial cytosol, ferric iron must be reduced to ferrous form, and thus ferric reductase is essential to bacterial iron utilization.Commonly, prokaryotic ferric reductases are divided into two groups—namely, the bacterial and archaeal types (34). The typical bacterial type ferric reductase is Escherichia coli Fre, which also functions as a flavin reductase. In other words, the ferric reductase can reduce free flavin as flavin reductase, rather than having the flavin cofactor as a prosthetic group in E. coli (38). The archaeal ferric reductase harbors a flavin cofactor in the enzyme and thus does not require a flavin carrier for ferric reduction (26, 34). E. coli Fre includes a Rosmann folding structure at the NAD(P) binding region, whereas the archaeal ferric reductase (FeR) of Archaeoglobus fulgidus does not evidence that folding structure (6, 34). Many bacterial ferric reductases utilize free flavins, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and riboflavin, as electron carrier and, NADH (NAD) or NADP as electron donors to ferric reductase (14, 34). However, reduced ferric iron by reduced free flavin gives rise to the Fenton reaction, which generates the hydroxyl radical within the cell (20, 38). The Fenton reaction is known to generate hydroxyl radicals from ferrous iron and hydrogen peroxide (20). The hydroxyl radical is the most reactive radical and can damage DNA, proteins, and membrane lipids (16, 20, 34, 38). Therefore, the fine-tuning of ferric reduction regulation is required for the survival of bacterial cells.Many Pseudomonas strains, including Pseudomonas putida, a gram-negative soil model bacteria, and Pseudomonas aeruginosa, a human pathogen bacteria, do not harbor annotated ferric reductase within their genome sequences. Commonly, the pathogens compete with the host for available iron, whichis crucial for their survival within the host. Thus, studies of P. aeruginosa regarding iron utilization, siderophores, and ferric reduction are considered to be essential for a better understanding of human infections (9, 19). Studying the physiology and ecology of P. putida also provides us with a new framework for elucidating the basis of the metabolic versatility and environmental stress response of soil microorganisms. Thus, the study of ferric reductase in strains of Pseudomonas at the molecular level is certainly required. From the structural perspective, ferric reductases are generally considered to be contained within the structurally diverse ferredoxin-NADP⁺ reductase (Fprs; EC 1.18.1.2) superfamily, which is frequently involved in the transfer of electrons between Fd/Fld and NADP(H) (2, 15, 34). Thus, we tested the role of the Fpr as a ferric reductase using free flavin (FMN or FAD), NADH, or NADPH as electron donors, and ferric-citrate or ferric-EDTA as terminal electron acceptors (37). We determined that FprA could efficiently utilize NADPH in ferric reduction. Rather, FprB could use NADH as an electron donor and may perform a crucial role as a NADH-dependent ferric reductase under iron stress conditions.     

Reassociation of the Small Form of Ferredoxin-NADP+ Reductase to the Large Form

The large form of ferredoxin-NADP⁺ reductase (FNR) was isolatedand partially purified from spinach leaves. It was then convertedinto the small form of FNR at low ionic strength. The smallform was further purified and separated into two fractions,FNR-Sr and FNR-Sir. FNR-Sr could associate to reconstitute thelarge form, but FNRSir could not. Therefore, FNR-Sr is the trueconstructive form of FNR-L and FNR-Sir is the secondary derivativefrom FNR-Sr. ¹ Present address: Central Research Laboratory, Toyo-Soda Mfg.Co. Ltd., Tonda, Shinnanyo, Yamaguchi 746, Japan. (Received February 26, 1983; Accepted July 7, 1983)     

Effect of Light on Chemical Modification of Chloroplast Ferredoxin-NADP Reductase

Chemical modification of spinach chloroplasts by phenylglyoxal and dansyl chloride resulted in inhibition of NADP photoreduction. The rate of inactivation was higher with both reagents when modification was carried out in the light with methylviologen or phenazine methosulfate present. Uncouplers prevent the effect of light. Electron transport from water to methylviologen was not affected by the modifiers.     

Effect of Ferredoxin on the Diaphorase Activity of Cyanobacterial Ferredoxin-NADP Reductase

The interaction of ferredoxin-NADP reductase (FNR) and ferredoxin (Fd) results in an enhanced rate of reaction and a shift of the pH optimum for the FNR-mediated diaphorase reaction. Low concentrations of NaCl (<100 millimolar), favorable for formation of the FNR:Fd complex, further magnify the alteration of the diaphorase reaction; the activity is enhanced 3-fold and pH optimum is shifted from 9.5 to 7.8. The Fd-stimulated diaphorase activity of FNR may result either from a conformational change of the enzyme and/or from a transition from a two electron to a one electron reaction.     

Ferredoxin and Ferredoxin-NADP Reductase from Photosynthetic and Nonphotosynthetic Tissues of Tomato

Ferredoxin and ferredoxin-NADP⁺ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.     

Two Different Forms of Gonadotropin-releasing Hormone in Amphioxus

Since a GnRH was isolated from mammalian hypothalamus and purified in 1971 and 1972, severalvariants have been identified in various forms of lower vertebrates. However, the presence of GnRHin amphioxus is still an open question. Chang et al. (1984) observed the presence of immunopositivegranules for GnRH in Hatschek's pit of amphioxus. In this paper, HPLC was used for the isolationand purification of GnRH-like peptide from amphioxus tissues, while radioimmunoassay was appliedto determine the immunoreactivity of the peptide. Based on the immunological and chromatographiccharacteristics, two kinds of GnRH (mGnRH and sGchH) were identified in amphioxus and theseGnRH-like peptide were found to be present in the "head", "middle" and "tail" regions ofamphioxus.     

Molecular Weight of Two Oncornavirus Genomes: Derivation from Particle Molecular Weights and RNA Content

Sedimentation analysis and intensity fluctuation spectroscopy have been used in conjunction with the Svedberg equation to determine the particle molecular weights of Rous sarcoma virus (Prague strain) and avian myeloblastosis virus (BAI strain). The molecular weights of these two viruses are (294 +/- 20) x 10(6) and (256 +/- 18) x 10(6), respectively. Values for the molecular weight of the RNA contained in each particle have been calculated as (5.58 +/- 0.5) x 10(6) and (5.88 +/- 0.5) x 10(6). Since the proportion of the viral RNA represented by 4 to 7S low-molecular-weight material is known, the molecular weight of the 60 to 70S genomes may be calculated to lie in the range (3.8 +/- 0.3 to 4.8 +/- 0.4) x 10(6) for both particles. These estimates for the molecular weight of the 60 to 70S genome are much lower than previous estimates and fall within the range of current estimates of the size of a single 35S subunit. The implications of this finding are discussed in terms of current theories for the structure of the genome of RNA tumor viruses.     

Fungicidal and Bactericidal Activity of Chitosans with Different Molecular Weights and Copper Complexes Based on Them

Applied Biochemistry and Microbiology - This work examines the antibacterial and antifungal activity of chitosans with a deacetylation degree of 85% and molecular weights (MWs) in a wide range of...     

不同分子量壳聚糖对五种常见茵的抑制作用研究

体外抑菌法研究了六种不同相对分子量的壳聚糖对金黄色葡萄球菌、大肠杆菌、绿脓杆菌、白色念珠菌、变形杆菌的抑菌活性,并对壳聚糖的抑菌机理做了探讨.     

A New Protein Factor, Connectein, as a Constituent of the Large Form of Ferredoxin-NADP Reductase

The large form of ferredoxin-NADP reductase (FNR) was treatedwith 66% iso-propyl alcohol and fractionated. The precipitatecontained the small form of FNR, which was incapable of reassociatingto the large form. The supernatant contained a new protein factorof low molecular weight. When the protein factor was isolatedfrom the supernatant and added to the small form of FNR, thelarge form of FNR was reconstituted under high salt conditions.Experimental findings indicate that the large form of FNR wascomposed of two molecules of the small form of FNR which wereconnected by a protein factor. The protein factor was purifiedby hydrophobic interaction column chromatography using butyl-Toyopearl650M and its molecular weight was determined to be about 10,000by gel filtration. It was a colorless protein with an unusualabsorption spectrum in ultra-violet regions. The protein factorwas very stable against heat but was digested by trypsin. Itwas named "Connectein" after its connective action. ¹ Present address: Biotechnology Research Laboratory, Toyo SodaManufacturing Co., Ltd., Hayakawa, Ayase-shi, Kanagawa 252,Japan. ² Present address: Nagase Biochemicals, Ltd., Osadano-cho, Fukuchiyama620, Japan. (Received November 9, 1984; Accepted February 9, 1985)     

In Situ Association of Calvin Cycle Enzymes,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase,Ferredoxin-NADP+ Reductase,and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy

The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.     

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N₂ and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH₄)₂SO₄ fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO₃. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO₂⁻ per minute per milligram protein). A sequential elution with NADH followed by KNO₃ (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO₃⁻-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.     

Development of pH-sensitive Insulin Nanoparticles using Eudragit L100-55 and Chitosan with Different Molecular Weights

Insulin is a polypeptide hormone and usually administered for treatment of diabetic patients subcutaneously. The aim of this study was to investigate the efficiency of enteric nanoparticles for oral delivery of insulin. Nanoparticles were formed by complex coacervation method using chitosan of various molecular weights. Nanoparticles were characterized by drug loading efficiency determination, particle size analysis, Scanning Electron Microscopy (SEM), Zeta potential and CD spectroscopy (Circular Dichrosim). The in vitro release studies were performed at pH 1.2 and 7.4. The drug loaded nanoparticles showed 3.38% of entrapment, loading efficiency of 30.56% and mean particle size of 199 nm. SEM studies showed that the nanoparticles are non-spherical. Zeta potential increased with increasing molecular weight of chitosan. The CD spectroscopy profiles indicated that the nano-encapsulation process did not significantly disrupt the internal structure of insulin; additionally, pH-sensitivity of nanoparticles was preserved and the insulin release was pH-dependent. These results suggest that the complex coacervation process using chitosan and Eudragit L100-55 polymers may provide a useful approach for entrapment of hydrophilic polypeptides without affecting their conformation.     

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.     

Interaction of Ferredoxin-NADP Oxidoreductase with the Thylakoid Membrane

The binding of ferredoxin-NADP reductase to spinach chloroplast membranes was studied by washing the membranes with different media. Release of the enzyme from the thylakoids was greater in 0.75 millimolar EDTA but was not complete inasmuch as 20% the activity remained membrane-bound after three washes.     

Study on Different Molecular Weights of Chitosan as an Immobilization Matrix for a Glucose Biosensor

Two chitosan samples (medium molecular weight (MMCHI) and low molecular weight (LMCHI)) were investigated as an enzyme immobilization matrix for the fabrication of a glucose biosensor. Chitosan membranes prepared from acetic acid were flexible, transparent, smooth and quick-drying. The FTIR spectra showed the existence of intermolecular interactions between chitosan and glucose oxidase (GOD). Higher catalytic activities were observed on for GOD-MMCHI than GOD-LMCHI and for those crosslinked with glutaraldehyde than using the adsorption technique. Enzyme loading greater than 0.6 mg decreased the activity. Under optimum conditions (pH 6.0, 35°C and applied potential of 0.6 V) response times of 85 s and 65 s were observed for medium molecular weight chitosan glucose biosensor (GOD-MMCHI/PT) and low molecular weight chitosan glucose biosensor (GOD-LMCHI/PT), respectively. The apparent Michaelis-Menten constant () was found to be 12.737 mM for GOD-MMCHI/PT and 17.692 mM for GOD-LMCHI/PT. This indicated that GOD-MMCHI/PT had greater affinity for the enzyme. Moreover, GOD-MMCHI/PT showed higher sensitivity (52.3666 nA/mM glucose) when compared with GOD-LMCHI/PT (9.8579 nA/mM glucose) at S/N>3. Better repeatability and reproducibility were achieved with GOD-MMCHI/PT than GOD-LMCHI/PT regarding glucose measurement. GOD-MMCHI/PT was found to give the highest enzymatic activity among the electrodes under investigation. The extent of interference encountered by GOD-MMCHI/PT and GOD-LMCHI/PT was not significantly different. Although the Nafion coated biosensor significantly reduced the signal due to the interferents under study, it also significantly reduced the response to glucose. The performance of the biosensors in the determination of glucose in rat serum was evaluated. Comparatively better accuracy and recovery results were obtained for GOD-MMCHI/PT. Hence, GOD-MMCHI/PT showed a better performance when compared with GOD-LMCHI/PT. In conclusion, chitosan membranes shave the potential to be a suitable matrix for the development of glucose biosensors.     

Comparative Studies on Ferredoxin-NADP+ Oxidoreductase Isoenzymes Derived from Different Organs by Antibodies Specific for the Radish Root- and Leaf-Enzymes

Determination of the prosthetic group and titration of sulfhydryl group of ferredoxin-NADP+ oxidoreductase (FNR) from roots of radish (Raphanus sativus var acanthiformis cv Miyashige) confirmed its similarity to leaf-FNR. Antisera directed against radish root-FNR and leaf-FNR distinguished the enzyme forms from roots and leaves of radish as well as other flowering plants. The FNR isoenzymes showed organ-specific distributions. In horsetail (Equisetum arvense L.) and cultured liverwort cells (Marchantia polymorpha), at least two FNR isoenzymes were distinguished by the antisera. FNR from Chlorella vulgaris reacted only with the anti-root-FNR antiserum. FNR from a cyanobacterium, Spirulina spp., failed to react with either antiserum.     

Growth Stimulation Activity of Alginate-Derived Oligosaccharides with Different Molecular Weights and Mannuronate/Guluronate Ratio on Hordeum vulgare L

Journal of Plant Growth Regulation - The differentiated components of alginate-derived oligosaccharides (AOS) lead to different activities on plant. Optimizing AOS use efficiency is, therefore, of...     

Binding Thermodynamics of Ferredoxin:NADP Reductase: Two Different Protein Substrates and One Energetics

The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP⁺ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His²⁹⁹ FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics.