[Show abstract][Hide abstract] ABSTRACT: Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.
PLoS ONE 09/2012; 7(9):e44339. DOI:10.1371/journal.pone.0044339 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of growth irradiance and respiration on ascorbic acid (AA) synthesis and accumulation were studied in the leaves
of wild-type and transformed Arabidopsis thaliana with modified amounts of the mitochondrial alternative oxidase (AOX) protein. Plants were grown under low (LL; 50 μmol photons
m−2 s−1), intermediate (IL; 100 μmol photons m−2 s−1), or high (HL; 250 μmol photons m−2 s−1) light. Increasing growth irradiance progressively elevated leaf AA content and hence the values of dark-induced disappearance
of leaf AA, which were 11, 55, and 89 nmol AA lost g−1 fresh weight h−1, from LL-, IL-, and HL-grown leaves, respectively. When HL leaves were supplied with L-galactone-1,4-lactone (L-GalL; the precursor of AA), they accumulated twice as much AA and had double the maximal L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activities of LL leaves. Growth under HL enhanced dehydroascorbate reductase and monodehydroascorbate reductase activities.
Leaf respiration rates were highest in the HL leaves, which also had higher amounts of cytochrome c and cytochrome c oxidase (CCO) activities, as well as enhanced capacity of the AOX and CCO electron transport pathways. Leaves of the AOX-overexpressing
lines accumulated more AA than wild-type or antisense leaves, particularly at HL. Intact mitochondria from AOX-overexpressing
lines had higher AA synthesis capacities than those from the wild-type or antisense lines even though they had similar L-GalLDH activities. AOX antisense lines had more cytochrome c protein than wild-type or AOX-overexpressing lines. It is concluded that regardless of limitations on L-GalL synthesis by regulation of early steps in the AA synthesis pathway, the regulation of L-GalLDH activity via the interaction of light and respiratory controls is a crucial determinant of the overall ability of
leaves to produce and accumulate AA.
[Show abstract][Hide abstract] ABSTRACT: Cultured cells of tobacco (Nicotiana tabacum L. cv Petit Havana) were used to investigate signals regulating the expression of the "model" nuclear gene encoding the alternative oxidase (AOX) (AOX1), the terminal oxidase of the mitochondrial alternative respiratory pathway. Several conditions shown to induce AOX1 mRNA accumulation also result in an increase in cellular citrate concentrations, suggesting that citrate and/or other tricarboxylic acid (TCA) cycle intermediates may be important signal metabolites. In addition, mitochondrial reactive oxygen species (ROS) production has recently been shown to be a factor mediating mitochondria-to-nucleus signaling for the expression of AOX1. We found that the exogenously supplied TCA cycle organic acids citrate, malate and 2-oxoglutarate caused rapid and dramatic increases in the steady-state level of AOX1 mRNA at low, near physiological concentrations (0.1 mM). Furthermore, an increase in AOX1 induced by the addition of organic acids occurs independently of mitochondrial ROS formation. Our results demonstrate that two separate pathways for mitochondria-to-nucleus signaling of AOX1 may exist, one involving ROS and the other organic acids.
[Show abstract][Hide abstract] ABSTRACT: Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction. One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 22.214.171.124), although the precise role of this enzyme is not yet known. Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX). We have partially purified mtICDH from tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS-PAGE, which was verified by peptide sequencing. In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak. Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity. In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type. When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line. These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX.
[Show abstract][Hide abstract] ABSTRACT: The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.
International Journal for Parasitology 04/2004; 34(3):297-308. DOI:10.1016/j.ijpara.2003.11.002 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The suf operon is composed of four genes (sufB, sufC, sufD, and sufS) and is highly conserved in the genomes of cyanobacteria. Open reading frame sll0088 in Synechocystis sp. strain PCC 6803 is located near the 5' end of the suf operon but is transcribed in the direction opposite that of the suf operon. We previously reported the isolation of two independent suppressor strains of C14S(PsaC) that mapped to sll0088 and restored photoautotrophic growth. The protein encoded by sll0088 has two significant features: (i) a DNA-binding domain near the N terminus and (ii) four highly conserved cysteine residues near the C terminus. The protein has high sequence similarity to transcription regulatory proteins with a conserved DNA-binding domain and can be classified in the DeoR family of helix-loop-helix proteins. The protein falls into a further subclass that contains a C-X(12)-C-X(13)-C-X(14)-C motif near the C terminus, which may represent a metal-binding site. The expressed Sll0088 protein harbored an iron-sulfur cluster as shown by optical and electron paramagnetic resonance spectroscopy. Compared to the wild type, expression levels of the sufBCDS genes were elevated when cells were grown under conditions of oxidative and iron stress and were even higher in a null mutant of Synechococcus sp. strain PCC 7002 in which the sll0088 homolog was insertionally inactivated. In agreement with the proposed role of the sufBCDS genes in iron metabolism, the growth rate of the null mutant was significantly higher than that of the wild type under iron-limiting conditions. We propose that the protein encoded by sll0088 is a transcriptional repressor of the suf operon, and we name the gene sufR.
Journal of Bacteriology 03/2004; 186(4):956-67. DOI:10.1128/JB.186.4.956-967.2004 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In previous work, some members of our group isolated mutant strains of Synechocystis sp. strain PCC 6803 in which point mutations had been inserted into the psaC gene to alter the cysteine residues to the F(A) and F(B) iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem. 272:8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll a and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 micromol m(-2) s(-1)). Two separate suppressor strains of C14S(PsaC), termed C14S(PsaC)-R62 and C14S(PsaC)-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp. strain PCC 6803 genome named sll0088. C14S(PsaC)-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482-->C change in sll0088, and C14S(PsaC)-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in sll0088. These suppressor strains showed near-wild-type levels of chlorophyll a and photosystem I, yet the serine oxygen ligand to F(B) was retained as shown by the retention of the S > or = 3/2 spin state of the [4Fe-4S] cluster. The inactivation of sll0088 by insertion of a kanamycin resistance cartridge in the primary C14S(PsaC) mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in psaC gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an sll0088 deletion mutant. The sll0088 gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including Arabidopsis thaliana. The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis. This suggests that the product of sll0088 has a role in regulating the biogenesis of photosystem I.
Journal of Bacteriology 07/2003; 185(13):3878-87. DOI:10.1128/JB.185.13.3878-3887.2003 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Physiological evidence indicates that flower formation is hormonally controlled. The floral stimulus, or florigen, is formed in the leaves as a response to an inductive photoperiod and translocated through the phloem to the apical meristem. However, because of difficulties in obtaining and analyzing phloem sap and the lack of a bioassay, the chemical nature of this stimulus is one of the major unsolved problems in plant biology. A combination of microbore high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to compare the contents of the phloem sap from flowering and non-flowering plants. Instead of using one- or two-dimensional gel electrophoresis, microbore HPLC separations allowed us to detect proteins/peptides that were very small and present at very low levels. We detected more than 100 components in the phloem sap of Perilla ocymoides L. and Lupinus albusL. Sequences for 16 peptides in a mass range from 1 to 9 kDa were obtained. Two of these could be identified, 11 showed similarity to known or deduced protein sequences, and three showed no similarity to any known protein or translated gene sequence. Four of these peptides were specific to, modified, or increased in plants that were flowering, indicating their possible role in flower induction. The sequences of these peptides showed similarities to two purine permeases, a protein with similarity to protein kinases, and a protein with no similarities to any known protein.
[Show abstract][Hide abstract] ABSTRACT: Using the mRNA differential display technique, seven cDNAs have been isolated that are rapidly induced when cultured tobacco (Nicotiana tabacum) cells are treated with the mitochondrial electron transport inhibitor antimycin A (AA). Interestingly, six of the cDNAs show distinct similarity to genes known to be induced by processes that involve programmed cell death (PCD), such as senescence and pathogen attack. All of the cDNAs as well as Aox1, a gene encoding the alternative oxidase, were found to also be strongly induced by H2O2 and salicylic acid (SA). AA, H2O2 and SA treatment of tobacco cells caused a rapid rise in intracellular ROS accumulation that, when prevented by antioxidant treatment, resulted in inhibition of gene induction. Besides AA, both H2O2 and SA were found to disrupt normal mitochondrial function resulting in decreased rates of electron transport and a lowering of cellular ATP levels. Furthermore, the pre-treatment of tobacco cells with bongkrekic acid, a known inhibitor of the mitochondrial permeability transition pore in animal cells, was found to completely block gene induction when AA, H2O2 or SA were subsequently added. These findings suggest that the mitochondrion may serve an important role in conveying intracellular stress signals to the nucleus, leading to alterations in gene expression.
The Plant Journal 03/2002; 29(3):269-79. DOI:10.1046/j.1365-313X.2002.01216.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The short-day plant Pharbitis nil is a model plant for the study of photoperiodic control of floral initiation. Flower formation can be induced at the cotyledon stage by a single long night of at least 14 h in duration. Using differential display of mRNA we identified a P. nil ortholog of the Arabidopsis CONSTANS (CO) gene, which will be referred to as PnCO. Expression of PnCO was high after a 14-h night, but low when the dark period was 12 h or less. Our results indicate that the level of the PnCO transcript is photoperiodically regulated. After transfer from continuous light to darkness, PnCO showed a circadian pattern of expression. Expression of the CAB gene, which is a molecular marker for the circadian clock, exhibited a different pattern of expression than did PnCO and was not subject to the same photoperiodic control. A major portion of the PnCO transcripts contained an unspliced intron. Only the intron-free PnCO was able to complement the co mutant of Arabidopsis by shortening the time to flower.
[Show abstract][Hide abstract] ABSTRACT: Mitochondria depend on the nuclear genome to encode the vast majority of their proteins; in turn they control the expression of certain nuclear genes to maintain proper functioning. In this work, Arabidopsis leaves were employed as a model to study nuclear gene expression in response to inhibition of the mitochondrial electron transport by antimycin A. Microarrays containing 11 514 Arabidopsis expressed sequence tags supplied through the Arabidopsis Functional Genomics Consortium (AFGC) were used. Transcript levels of 579 nuclear genes were increased ≥ 2-fold, and the levels of 584 nuclear genes were decreased ≥ 2-fold after antimycin A treatment. While functions of a large number of the gene products are unknown, others are involved in diverse metabolic activities such as phosphorylation, transcription, and energy metabolism. Data from microarray experiments were repeatable and were confirmed by northern hybridization for specific test genes. It was found through cluster analysis that plant cells show significant common response to chemical inhibition of mitochondrial function, aluminum stress, cadmium stress, hydrogen peroxide and virus infection. The results imply that these stresses may act on mitochondria and the responses are in part mediated by mitochondrial-nuclear communication. Most nuclear-encoded respiratory genes involved in the TCA cycle, electron transport and ATP synthesis did not respond to signals from the inhibited mitochondria, while genes for cytochrome c and alternative oxidase were induced. The result indicates that these two genes may be targets in the transcriptional regulation of the two respiratory pathways.
[Show abstract][Hide abstract] ABSTRACT: Besides the cytochrome c pathway, plant mitochondria have an alternative respiratory pathway that is comprised of a single homodimeric protein, alternative oxidase (AOX). Transgenic cultured tobacco cells with altered levels of AOX were used to test the hypothesis that the alternative pathway in plant mitochondria functions as a mechanism to decrease the formation of reactive oxygen species (ROS) produced during respiratory electron transport. Using the ROS-sensitive probe 2',7'-dichlorofluorescein diacetate, we found that antisense suppression of AOX resulted in cells with a significantly higher level of ROS compared with wild-type cells, whereas the overexpression of AOX resulted in cells with lower ROS abundance. Laser-scanning confocal microscopy showed that the difference in ROS abundance among wild-type and AOX transgenic cells was caused by changes in mitochondrial-specific ROS formation. Mitochondrial ROS production was exacerbated by the use of antimycin A, which inhibited normal cytochrome electron transport. In addition, cells overexpressing AOX were found to have consistently lower expression of genes encoding ROS-scavenging enzymes, including the superoxide dismutase genes SodA and SodB, as well as glutathione peroxidase. Also, the abundance of mRNAs encoding salicylic acid-binding catalase and a pathogenesis-related protein were significantly higher in cells deficient in AOX. These results are evidence that AOX plays a role in lowering mitochondrial ROS formation in plant cells.
Proceedings of the National Academy of Sciences 08/1999; 96(14):8271-6. DOI:10.1073/pnas.96.14.8271 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Synechocystis 6803 is a model organism for the study of oxygenic photosynthesis. Its genome has recently been sequenced, and it is naturally competent and can be easily transformed. It also has an active homologous recombination mechanism. This chapter describes a procedure for isolating spontaneous pseudorevertants from PSI mutants of Synechocystis 6803, localization and identification of the suppressor mutations, and some of the properties of the genetic transformation system in this cyanobacterium. Photoautotrophic and mixotrophic growth under white light was used to isolate and characterize pseudorevertants. Isolates obtained from one selective condition were tested for their ability to grow in other conditions by streaking on plates. Three classes of pseudorevertants have been found: those capable of growth under (1) photoautotrophic conditions, but not mixotrophic conditions; (2) mixotrophic conditions, but not photoautotrophic conditions; and (3) both conditions. Pseudorevertants generally grow more slowly than wild-type under these conditions.
Methods in enzymology 12/1998; 297C:18-26. DOI:10.1016/S0076-6879(98)97004-5 · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.
The Plant Cell 10/1998; 10(9):1551-60. · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Higher plant mitochondria contain two terminal oxidases, cytochrome c oxidase and a cyanide-resitant ‘alternative’ oxidase. Electron flux through these two respiratory pathways is controlled by environmental conditions, stimuli received by mitochondria. In general, stresses such as cold, wounding, pathogen attack and others favor electron flow through the alternative oxidase. One of the proposed functions of the alternative pathway is to relieve the tricarboxylic acid (TCA) cycle of inhibition from cytochrome pathway products and allow the cycle to furnish carbon skeletons for anabolic requirements. We are currently investigating, with an NADP-linked isocitrate dehydrogenase in plant mitochondria, a possible link between respiratory control and carbon flux from the TCA cycle. Regulation of the nuclear gene encoding the alternative oxidase, Aox1, is also being employed as a model for perception of the many stresses by the mitochondria and transfer of these signals to the nucleus. Our initial results indicate that hydrogen peroxide is an intermediate in this signalling process.
[Show abstract][Hide abstract] ABSTRACT: Plants, some fungi, and protists contain a cyanide-resistant, alternative mitochondrial respiratory pathway. This pathway branches at the ubiquinone pool and consists of an alternative oxidase encoded by the nuclear gene Aox1. Alternative pathway respiration is only linked to proton translocation at Complex 1 (NADH dehydrogenase). Alternative oxidase expression is influenced by stress stimuli-cold, oxidative stress, pathogen attack-and by factors constricting electron flow through the cytochrome pathway of respiration. Control is exerted at the levels of gene expression and in response to the availability of carbon and reducing potential. Posttranslational control involves reversible covalent modification of the alternative oxidase and activation by specific carbon metabolites. This dynamic system of coarse and fine control may function to balance upstream respiratory carbon metabolism and downstream electron transport when these coupled processes become imbalanced as a result of changes in the supply of, or demand for, carbon, reducing power, and ATP.
[Show abstract][Hide abstract] ABSTRACT: A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.
[Show abstract][Hide abstract] ABSTRACT: Two [4Fe-4S] clusters, FA and FB, function as terminal electron carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in photosynthetic electron transport, site-directed mutants were created in the transformable cyanobacterium Synechocystis sp. PCC 6803. Cysteine ligands in positions 14 or 51 to FB and FA, respectively, were replaced with aspartate, serine, or alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied. All mutant strains were unable to grow photoautotrophically, and compared with wild type, mixotrophic growth was inhibited under normal light intensity. The mutant cells supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PS I subunits PsaC, PsaD, and PsaE and lower rates of PS I-mediated substrate photoreduction compared with the wild type. The alanine and double aspartate mutants have no detectable levels PsaC, PsaD, and PsaE. Electron transfer rates, measured by cytochrome c6-mediated NADP+ photoreduction, were lower in purified PS I complexes from the aspartate and serine mutants. By measuring the P700(+) kinetics after a single turnover flash, a large percentage of the backreaction in the aspartate and serine mutants was found to be derived from A1 and FX, indicating an inefficiency at the FX --> FA/FB electron transfer step. The alanine and double aspartate mutants failed to show any backreaction from [FA/FB]-. These results indicate that the various mutations of the cysteine 14 and 51 ligands to FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and function can be biochemically separated and analyzed.