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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 pyridoxal phosphate-dependent monomeric L-cysteine/cystine C-S-lyase (C-DES), previously isolated from Synechocystis PCC 6714 by its capacity to direct [2Fe-2S] cluster assembly of ferredoxin in vitro (Leibrecht, I., and Kessler, D. (1997) J. Biol. Chem. 272, 10442-10447), has now been cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence of C-DES was found to be nearly identical (92% identity) to the open reading frame slr2143 of Synechocystis PCC 6803 and showed a more distant relationship to the NifS family of proteins (about 27% identity). Recombinant C-DES displayed activities equal to the isolate from Synechocystis in terms of the cyst(e)ine lyase reaction and holoferredoxin formation which recommended its use for functional and mechanistic studies. Investigation of the substrate spectrum for beta-elimination found L-cysteine to be a poor substrate (kcat approximately 0.15 s-1) in contrast to L-cystine (kcat = 36 s-1) and several related compounds. Of these compounds, desaminocystine (S-(carboxyethylthio)-L-cysteine) was used for C-DES-mediated persulfide generation. Stabilization of the linear persulfide 3-(disulfanyl)-propionic acid was achieved by cyclization as a novel intramolecular trapping reaction; this yielded 1,2-dithiolan-3-one which was isolated and identified by chemical analyses.
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PMID:Evidence for cysteine persulfide as reaction product of L-Cyst(e)ine C-S-lyase (C-DES) from Synechocystis. Analyses using cystine analogues and recombinant C-DES. 986 29

The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX.
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PMID:The cysteine-proximal aspartates in the Fx-binding niche of photosystem I. Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties. 1018 75

In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.
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PMID:Role of a NifS-like protein from the cyanobacterium Synechocystis PCC 6803 in the maturation of FeS proteins. 1072 36

Peroxidases are enzymes that utilize hydrogen peroxide to oxidize substrates. A histidine residue on the proximal side of the haem iron ligates most peroxidases. The various oxidation states and ligand complexes have been spectroscopically characterized. HRP-I is two oxidation states above ferric HRP. It contains an oxoferryl (= oxyferryl) iron with a pi-radical cation that resides on the haem. HRP-II is one oxidation state above ferric HRP and contains an oxoferryl iron. HRP-III is equivalent to the oxyferrous state. Only compounds I and II are part of the peroxidase reaction cycle. CCP-ES contains an oxoferryl iron but the radical cation resides on the Trp-191 residue and not on the haem. CPO is the only known peroxidase that is ligated by a cysteine residue rather than a histidine residue, on the proximal side of the haem iron. CPO is a more versatile enzyme, catalysing numerous types of reaction: peroxidase, catalase and halogenation reactions. The various CPO species are less stable than other peroxidase species and more elusive, thus needing further characterization. The roles of the amino acid residues on the proximal and distal sides of the haem need more investigation to further decipher their specific roles. Haem proteins, especially peroxidases, are structure-function-specific.
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PMID:Haem iron-containing peroxidases. 1073 Jan 88

Bacterial metallothioneins bind, sequester and buffer excess intracellular zinc. At present, the vast majority of the available experimental data relate to cyanobacterial metallothionein, SmtA, from Synechococcus PCC 7942. SmtA is required for normal resistance to zinc and smtA-mediated zinc resistance has been used as a selectable marker. The imidazole groups of histidine residues, in addition to the thiol groups of cysteine residues, co-ordinate zinc in bacterial metallothioneins. The structure of bacterial metallothionein must facilitate some discrimination between 'adventitious' and 'adventageous' zinc-binding sites such that under excess zinc conditions metal is predominantly scavenged from the former. It remains unclear whether or not bacterial metallothionein also acts as a zinc store that supplies zinc-requiring proteins or if under some conditions it deactivates a subset of proteins via zinc removal. Expression of smtA is induced in response to elevated concentrations of zinc via the action of SmtB. SmtB has some sequence similarity to the arsenic responsive repressor ArsR and genes encoding related proteins are present in many bacterial genomes. Metal perception by SmtB differs from ArsR. The latter contains a characteristic Cys-Val-Cys motif associated with a DNA-binding helix-turn-helix (the ArsR motif), while the former contains metal-binding motifs associated with a carboxyl-terminal alpha-helix that forms the interface between SmtB dimers (the SmtB motif). Some SmtB-ArsR family proteins, including the zinc sensor ZiaR from the cyanobacterium Synechocystis PCC 6803, have the metal-sensory motifs of both SmtB and ArsR. The mechanisms of action, and the features that allow discrimination between different metal ions by these sensors, are discussed.
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PMID:Microbial metallothioneins. 1140 13

The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed.
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PMID:Cloning and characterisation of chlorophyll synthase from Avena sativa. 1150 54

We have utilized the unicellular cyanobacterium Synechocystis sp. PCC 6803 to incorporate site-directed amino acid substitutions into the photosystem I (PSI) reactioncenter protein PsaB. A cysteine residue (position 565 of PsaB) proposed to serve as a ligand to the [4Fe-4S] center Fx was changed to serine, histidine, and aspartate. These three mutants--C565S, C565H, and C565D--all exhibited greatly reduced accumulation of PSI reaction-center proteins and failed to grow autotrophically, indicating that this cysteine most likely does coordinate Fx, which is crucial for PSI biogenesis. Interestingly, the strain C565S accumulated significantly more PSI than the other two cysteine mutants and displayed photoreduction of the [4Fe-4S] terminal electron acceptors FA and FB. Mutations were also introduced into a leucine zipper motif of PsaB, proposed to participate in reaction-center dimerization. The mutants L522V, L536M, and L522V/L536M all exhibited wild-type characteristics and grew autotrophically, whereas the L522P mutation prevented PSI accumulation. These data do not provide support for a major structural role of the leucine zipper in reaction-center dimerization or in assembly of Fx. However, the amino acid substitutions incorporated were conservative and might not have perturbed the leucine zipper.
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PMID:Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803. 1160 63

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.
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PMID:A reaction-induced FT-IR study of cyanobacterial photosystem I. 1173 22

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.
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PMID:A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition. 1173 76


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