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Query: UMLS:C1832526 (PCC)
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The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.
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PMID:Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase. 939 38

The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.
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PMID:Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119. 948 22

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635-2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion.
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PMID:Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus. 959 5

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse-phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100, 000 g. Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.
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PMID:Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin-dependent heme oxygenase from Synechocystis sp. PCC 6803. 974 99

Flavodoxin can function as an alternative electron acceptor for photosystem I (PSI) in place of ferredoxin under iron-limiting conditions. The isiB gene, encoding the flavodoxin in Synechococcus sp. PCC 7002, was overexpressed in Escherichia coli. Under the conditions employed, most recombinant flavodoxin (rFlvd) was in soluble form with cofactor correctly inserted. The absorption spectrum of rFlvd was identical to that of the native flavodoxin of the cyanobacteria. Photoreduction of rFlvd by PSI particles and thylakoid membranes was determined directly by monitoring the absorption change at 467 nm. The optimal conditions for rFlvd photoreduction were determined. Compared to other methods currently employed to measure PSI activity such as oxygen uptake in the presence of methyl viologen and NADP+ photoreduction in the presence of ferredoxin and ferredoxin:NADP+ oxidoreductase, measurement of PSI activity with flavodoxin as an electron acceptor has several advantages. It measures the full-chain electron transfer chain of PSI since flavodoxin accepts electrons from FA/FB and it is much simpler than the method with NADP+ photoreduction. With this method, we found that the affinity of wild-type PSI for rFlvd was 35% higher than that of the PsaE-less PSI, showing that this method is sensitive to structural changes of PSI. Our results demonstrate that rFlvd photoreduction is an effective and simple method for PSI activity measurement.
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PMID:Measurement of photosystem I activity with photoreduction of recombinant flavodoxin. 986 92

Expression of the isiA and isiB genes was analysed in the cyanobacterium Synechocystis sp. PCC 6803 grown in high salt or in iron-deficient medium. The detection of a 2.3-knt transcript in Northern blot experiments indicated cotranscription of isiAB in an operon, which was confirmed by reverse transcriptase PCR. The abundance of a monocistronic 1.25-knt isiA-specific mRNA was about 10-fold higher than the dicistronic message. The isiAB-specific transcripts were most abundant in cells adapted to 342 mM NaCl and under iron deficiency. The promoter of the operon was mapped to 211 bp upstream of the translational start. A putative Fur binding site was detected immediately upstream of the GTG start codon. A preliminary transcript of about 0.2 knt was detected in cells grown in conditions in which the isiAB operon was not transcribed. This indicates that a repressor binds to the identified Fur binding site and thus inhibits isiAB transcription under low salt and iron replete conditions.
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PMID:Transcriptional analysis of the isiAB operon in salt-stressed cells of the cyanobacterium Synechocystis sp. PCC 6803. 986 77

Interactions of the primary quinone acceptor QA of photosystem II (PS II) with surrounding amino acid residues were studied by analysis of FTIR difference spectra of QA upon its photoreduction (QA-/QA). Structural coupling with a His side chain was revealed by identifying the imidazole bands in the QA-/QA spectrum using the PS II core complexes from Synechocystis PCC 6803 in which both of the two imodazole nitrogens of His side chains were specifically labeled with 15N. Strong hydrogen bonding of the imidazole NH was shown by (i) the presence of several peaks at 2600-3000 cm-1, which arise from Fermi resonance of harmonics or combinations of imidazole ring modes with the hydrogen bonding NH stretching vibration, and (ii) the 1179 cm-1 band, which can be assigned to the mode including NH deformation, is at a frequency significantly higher than the corresponding 1151 cm-1 band of model compounds 4- and 5-methylimidazole in aqueous solution. Also, the presence of the bands specific to the Npi-protonated state at 1109/1102/1090 and 1359 cm-1 suggests that the QA-coupled His is protonated at the Npi site. These results are in good agreement with the model of QA interaction in which His215 (D2), which coordinates to the non-heme iron at Ntau, is hydrogen bonded to the QA carbonyl through the Npi-H bond. In contrast, no bands of Trp side chains were detected in the QA-/QA spectrum upon labeling of the indole ring of Trp residues with indole-d5. This result indicates that Trp254 (D2), which corresponds to Trp252 (M) of the bacterial reaction center that is located in van der Waals contact with QA, is not strongly coupled with QA in PS II. Probably, the predicted pi-pi interaction is not strong enough to influence the vibrations of the indole ring of Trp upon QA reduction, or Trp254 (D2) is located rather far from QA in PS II.
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PMID:Hydrogen bonding interaction between the primary quinone acceptor QA and a histidine side chain in photosystem II as revealed by Fourier transform infrared spectroscopy. 989 Sep 22

A cytosolic catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803 was purified to homogeneity by a six-step purification procedure. It is a homodimeric enzyme with a subunit molecular mass of 85 kDa. The isoelectric point of the protein is at pH 5.5; Michaelis constant, turnover number, and catalytic efficiency of the catalase activity for H2O2 were measured to be 4.8 mM, 3450 s-1, and 7.2 x 10(5) M-1 s-1, respectively. Preparation and spectroscopy of the pyridine ferrohemochrome identified an iron protoporphyrin IX as the prosthetic group. The enzyme was shown to exhibit both catalase and peroxidase activities, both of which were inhibited by cyanide, leading to a high-spin to low-spin transition of the heme iron center as detected by a shift of the Soret peak from 405 to 421 nm. The catalase-specific inhibitor 3-amino-1,2,4-triazole proved ineffective. o-Dianisidine, pyrogallol and guaiacol functioned as a peroxidatic substrate, but no reaction was detected with NADH, NADPH, glutathione, and ascorbate. Peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry showed the identity between the purified protein and a putative katG gene derived from the genome of Synechocystis PCC 6803. A comparison of amino acid sequences of the catalase-peroxidase from Synechocystis PCC 6803 and those from other bacteria showed a high homology around the assumed distal and proximal histidine residues, suggesting a highly conserved histidine as the fifth ligand of the heme iron.
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PMID:Purification and characterization of a hydroperoxidase from the cyanobacterium Synechocystis PCC 6803: identification of its gene by peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry. 991 46

Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus.
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PMID:Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803. 1020 Oct 99

IdiA (iron-deficiency-induced protein A) is a protein expressed at highly elevated levels in Synechococcus sp. strains PCC 6301 and PCC 7942 under Fe- or Mn-limiting growth conditions. Besides being similar to two bacterial Fe-binding proteins, SfuA and FbpA, IdiA shows similarity to two ORFs (slr0513 and sir1295) of Synechocystis sp. PCC 6803. Northern blot analysis detected one transcript of about 1300 nt in RNA extracted from Synechococcus sp. PCC 6301 and PCC 7942 grown under Fe deficiency. The intensity of this transcript was considerably reduced in Fe-sufficient culture. It could be further shown that the regulation of IdiA expression is at the transcriptional level and that transcription and translation of IdiA are closely linked. Primer extension analysis indicated a single transcriptional start site 193 nt upstream of the first presumed translational start codon. Moreover, molecular characterization of the entire 5.8 kb chromosomal HindIII DNA fragment carrying the idiA gene from Synechococcus sp. PCC 6301 led to the identification of six long ORFs in addition to idiA. The two genes adjacent to idiA, and dpsA located 2018 nt downstream of idiA, were insertionally inactivated in Synechococcus sp. PCC 7942 and the corresponding mutants were partially characterized. These experiments provide evidence that the gene products of idiB, located immediately downstream of idiA, and of dpsA are involved in the activation of IdiA expression, since the absence of each of these two gene products prevents the greatly elevated expression of IdiA under nutrient deficiency.
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PMID:Molecular characterization of idiA and adjacent genes in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942. 1041 Dec 74


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