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Query: UMLS:C1832526 (PCC)
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Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.
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PMID:Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies. 131 39

Ferredoxin-NADP+ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the alpha-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPH-cytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-14C]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin.
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PMID:Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates. 144 67

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.
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PMID:Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase. 173 24

Chemical cross-linkage of the positively charged viologen N-methyl-N'-(aminopropyl)-4-4'-bipyridinium dibromide (APMV) to the enzyme ferredoxin-NADP+ reductase from the cyanobacterium Anabaena PCC 7119 has been performed using the carbodiimide 1-ethyl[3-(3-dimethylaminopropyl)]carbodiimide. 0.5-1 mol, depending on the preparation, is introduced for each mol enzyme. The residue involved in the covalent linkage with the viologen, Glu139, has been identified using HPLC separation of the modified proteolytic peptides and subsequent sequencing. Modification of the enzyme changes its catalytic specificity since it is able to react directly with oxygen; this is observed by a high NADPH oxidase activity, which is completely absent in the native enzyme. More important, this new enzymic activity is indicative of the intramolecular electron transfer between the natural redox cofactor FAD and the artificially introduced viologen. Electrons can also flow in the reverse direction, from the viologen to the FAD group, then to NADP+, when the reaction is performed using glassy-carbon electrodes to reduce the viologen. Cyclic voltammetry experiments have shown that there is a small catalytic current between the electrode and the enzyme which is not observed in the native enzyme.
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PMID:The covalent linkage of a viologen to a flavoprotein reductase transforms it into an oxidase. 758 6

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 degrees C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 degrees C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP+ photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.
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PMID:Mutational analysis of photosystem I polypeptides in the cyanobacterium Synechocystis sp. PCC 6803. Targeted inactivation of psaI reveals the function of psaI in the structural organization of psaL. 760 90

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.
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PMID:ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803. 776 63

The flavins of ferredoxin-NADP+ reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H2O and 1.48 mT in D2O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP+, was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 7119 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in D2O, but the coupling Aiso attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP+. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D2O. Effects of NADP+ binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Electron spin resonance and electron nuclear double resonance studies of flavoproteins involved in the photosynthetic electron transport in the cyanobacterium Anabaena sp. PCC 7119. 785 33

The subunit requirements for NADP+ reduction by photosystem I were assessed in mutants of Synechocystis sp. PCC 6803 created by targeted inactivation of the psaD, psaE, psaF, and psaL genes. The PsaE-less, PsaF-PsaJ-less, and PsaL-less mutants showed normal photoautotrophic growth, while the growth of PsaD-less mutants was slower without glucose. In isolated wild-type membranes, the rate of flavodoxin reduction and flavodoxin-mediated NADP+ reduction were 800 and 480 mumol/mg of chlorophyll/h, respectively. The rate of ferredoxin-mediated NADP+ photoreduction was 460 mumol/mg of chlorophyll/h. There was no diminution in NADP+ photoreduction in membranes isolated from the PsaF-less and PsaL-less mutants. The rates of ferredoxin-mediated NADP+ photoreduction in membranes of the PsaE-less mutants were 25 mumol/mg of chlorophyll/h. However, the rate of flavodoxin reduction was 380 mumol/mg of chlorophyll/h, and that of flavodoxin-mediated NADP+ photoreduction was 170 mumol/mg of chlorophyll/h. PsaD-less membranes showed < 20% of the wild-type rates of flavodoxin-mediated NADP+ photoreduction, but were completely deficient in ferredoxin-mediated NADP+ photoreduction. Therefore, the roles of PsaE and PsaD are more crucial for "docking" of ferredoxin than of flavodoxin. Proteolysis studies showed that while PsaD was susceptible to rapid in vitro degradation by thermolysin, the number and sizes of protease-resistant fragments were not affected by the absence of PsaE. Protease accessibility studies further indicated that the C-terminal domain of PsaD is surface-exposed on the n-side. These results suggest that PsaE and the C-terminal domain of PsaD generate the docking site for the electron acceptors of photosystem I.
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PMID:Mutational analysis of photosystem I polypeptides in Synechocystis sp. PCC 6803. Subunit requirements for reduction of NADP+ mediated by ferredoxin and flavodoxin. 806 87

The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP(+) reduction, even though charge separation between P700 and FA/FB occurred with high efficiency. Unlike the wild type, FB became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of FA- and FB- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the FA/FB iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys106 of PsaD, which can be crosslinked to Glu93 of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of FA and FB in membranes of the Lys106 mutants were similar to those of the wild type. Membranes of all Lys106 mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP+ reduction, but had 10-54% decrease in the ferredoxin-mediated NADP+ reduction rates. This implies that Lys106 is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys106 of PsaD and Glu93 of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of FA and FB, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP+ photoreduction on the reducing side of photosystem I.
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PMID:Mutational analysis of photosystem I polypeptides. Role of PsaD and the lysyl 106 residue in the reductase activity of the photosystem I. 866 33


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