Influence of inorganic and organic selenium on number of living mycelial cells and their ultrastructure in culture of Hericium erinaceum (Bull.: Fr. Pers.).

Mycelium of the white-rot fungus (Hericium erinaceum (Bull.: Fr. Pers.) produces polysaccharides showing anticancer and immunostimulating activity. In our previous works, we have shown that organic selenitetriglycerides (Selol) contribute to the increase of biosynthesis of exopolysaccharides (EPS) having antioxidative properties and containing large amounts of selenium. The present work is a study of influence of inorganic and organic form of selenium on viability of H. erinaceum mycelium and on ultrastructural changes taking place during its development in submerged culture. The mycelium was grown on media containing sodium selenite (Na2SeO3), a mixture of Na2SeO3 + Selol2% and on control medium (no selenium added). It was shown that mycelium cultured for 3 days in control conditions on standard media contained almost 100% of living cells, with over 80% after 24 days. Treatment with 100 ppm of Na2SeO3 lowered the number of viable cells to 11.8% and 9.1% after 3 and 24 days, respectively. The addition of 2% Selol caused the amounts of living cells to remain at ca 90%. Apparently, Selol helped the cells to cope with the toxic activity of inorganic selenium ions. The addition of sodium selenite induced degradative changes in cell organelles. Such changes were not observed in the case of Na2SeO3 + Selol mixture, in which case cells contained numerous ribosomes and small lipid bodies.

Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

Proton-coupled electron transfer at the Q(o) site: what type of mechanism can account for the high activation barrier?

In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc(1) complex shows kinetic features (a slow rate (approx. 1.5 x 10(3)/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, lambda (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 A of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E(m,7) and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Q(o) site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b(L). The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.

Ultrastructure of Gentiana tibetica proembryogenic cells before and after cooling treatments.

The influence of increased concentrations of sucrose, 0.4 M sorbitol, DMSO and vitrification solution (PVS2) on the ultrastructure of non-frozen and frozen suspensions of Gentiana tibetica King ex Hook. F.tissue cells was investigated. Embryogenic aggregates were composed of three groups of cells of different size with various types of plastids. The ultrastructural changes resulting from increasing the sucrose concentration in the medium from 3 to 6 percent for 4 weeks and from treatment with 0.4 M sorbitol for 48 h were similar. Observations showed replacement of large vacuoles by numerous small ones, condensation of cytoplasm, accumulation of starch, and fragmentation of endoplasmic reticulum. Treatment with PVS2 led to degradation of starch, coalescence of amyloplasts and to shrinking of nucleoli from the third group of cells when originating from 6 percent sucrose medium. The mitochondria initially had various shapes, but after PVS2 treatment showed only spherical shapes with sparse cristae. After programmed freezing of tissue protected by sorbitol and DMSO, lethal damage was observed: membrane and nuclei degradation, and cell destruction. Reversible changes after freezing were observed in tissue pretreated with vitrification solution: dilation of cell membranes, mitochondria with electron-lucent vessels, aggregation of numerous vesicles, and degradation of starch in amyloplasts. In cells cooled by a vitrification method, cell organelles appeared normal as early as 5 h after thawing, and anomalies were not observed after 48 h of post-thawing culture.

Nosema portugal, N. SP., isolated from gypsy moths (Lymantria dispar L.) collected in portugal

A microsporidium Nosema portugal n. sp. was isolated from gypsy moths, Lymantria dispar L, collected near Lisbon, Portugal, in 1985. The life cycle includes two sequential developmental cycles, a primary and a secondary cycle. The primary cycle occurs in midgut epithelial cells, where primary spores are produced within 48 h. The primary spores immediately extrude their polar filaments, presumably to infect other cells. In the target tissues (salivary glands and fat body) the secondary development cycle is followed by the formation of environmental spores. Primary spores were also sometimes present in target tissues. Fresh unfixed and unstained primary spores have a large posterior vacuole and measured 4.8 x 2.7 &mgr;m. Ultrastructurally, they have 5-8 polar filament coils, a large posterior vacuole, abundant endoplasmic reticulum, and were binucleate. Mature unfixed and unstained environmental spores were highly refractive and the posterior vacuole and nuclei could not be seen through the spore coat. Fresh environmental spores measured 4.5 x 1.9 &mgr;m. Ultrastructurally, environmental spores were binucleate, with a typical polaroplast, 10-11 isofilar polar filament coils, and a series of 4-6 thin polar filament-like tubules situated at the posterior end of the row of typical polar filament coils. The ssu rRNA sequences strongly suggest that this species is more closely related to the Vairimorpha subgroup within the Nosema/Vairimorpha clade than to the Nosema subgroup. Copyright 1999 Academic Press.

Intimacies of the impersonal.

This paper is focused on the kinds of awareness that are exposed by clinical work conducted in the depth psychological tradition. The paper begins with an examination of Freud's clinical methodology, which is a description of a particular kind of attentiveness that reveals material normally obscured from conscious awareness. This type of awareness is thought to reveal the contents and the specific organizational features of the unconscious. In this paper, the organizational processes of the unconscious are interpreted to actually be the referents of a privileged type of perceptual awareness leading to a unique relationship to the immediate world. This is taken to be an assumption that is implicit in the models of psychotherapy authored by Jung and Reich. Both developmental and clinical researches are used to support this position. The differentiation between this awareness and the more traditional modes of awareness is further highlighted by an examination of the phenomenon of transference. Finally, a clinical example is presented to support the theoretical claims in the paper.

The effect of humic acids on the reverse osmosis treatment of hazardous landfill leachate.

This study deals with the treatment of hazardous waste landfill leachate with the help of reverse osmosis. The landfill is located in an abandoned brown coal pit in northern Bohemia. The leachate contained 7.2 g/L of dissolved inorganic salts. Among other contaminants were heavy metals, arsenic, ammonia nitrogen and associated organic pollutants, especially chlorinated compounds. A mobile membrane unit (LAB M30) equipped with a spiral wound element (FILMTEC SW30-4040), with a membrane area equaling 7.4 m2 was used for the pilot plant experiments. All experiments were carried out in batch mode. 94% conversion of the input stream into the permeate was achieved by use of a two-stage arrangement. Removal efficiencies of the monitored contaminants in the feed ranged from 94% for ammonia nitrogen to 99% for the two-valent ions. Removal efficiency for total dissolved solids was 99.3% on average. Due to varying levels of humic acids in the leachate throughout the year, fouling experiments were performed to investigate the separation process under different conditions than those used in the pilot plant. Leachates containing different concentrations of added humic acids were separated using a thin film composite on a propylene membrane. The added humic acids were obtained from samples of contaminated oxihumolite.

Changes in the organization of the tubulin cytoskeleton during the early stages of Solanum lycopersicoides Dun. protoplast culture.

Changes in the tubulin cytoskeleton during protoplast culture and plant regeneration of Solanum lycopersicoides Dun. were analyzed using an immunodetection method. Directly after isolation, four groups of protoplasts were distinguished: (1) mononuclear, (2) polynuclear, (3) homogeneous, (4) anuclear. The tubulin cytoskeleton of the protoplasts underwent rearrangements, correlating to the number and structure of cell nuclei in the protoplast. All protoplast groups with the exception of mononuclear were characterized by perturbations in the organization of the tubulin cytoskeleton. Anuclear and homogeneous protoplasts did not have a tubulin cytoskeleton. Polynuclear protoplasts had cortical microtubules, but were not capable of re-forming their original arrangement and did not possess a radial or perinuclear cytoskeleton. Irregularities in microtubule arrangement of these three groups of protoplasts caused their inability to regenerate a cell wall and to divide. Anuclear, polynuclear and homogeneous protoplasts were eliminated from the culture. Mononuclear protoplasts rearranged their cortical microtubules and reestablished the radial and perinuclear tubulin cytoskeleton. Re-formation of the cell suspension and subsequent regeneration of plants occurred exclusively from mononuclear protoplasts, which were able to regenerate cell walls and to divide.

Ultrastructural determination of the early stage of conversion from root primordia to shoot primordia in vitro cultures of Solanum lycopersicoides Dun.

The object of the research were in vitro cultures of root primordia and the early stage of conversion from root to shoot primordia cultures (ESCRS) of Solanum lycopersicoides Dun. The cell ultrastructure of both culture types was studied in order to determine processes underlying the root and shoot morphogenesis type. It was found that a change of a morphogenesis type occurs together with reorganization of aggregate structure and the cell ultrastructural organization. There were 4 zones detected in root primordia culture aggregates (the surface, the starch, the division and the differentiation zone) and 3 zones in ESCRS culture aggregates (the surface, the inner and the differentiation zone). The differences were observed also in storage substances accumulated in a culture (starch in root primordia cultures; proteins, lipids and starch in ESCRS ones).

Perturbation in linker histone content has no effect on the cell cycle but affects the cell size of suspension grown tobacco BY-2 cells.

Histone H1, a key structural element of eukaryotic chromosomes can be perturbed in plants in vivo by overexpression or by a change in the proportion of native H1 variants (Prymakowska-Bosak M., Przewloka M., Iwkiewicz J., Egierszdorff S., Kuras M., Chaubert N., Gigot C., Spiker S., Jerzmanowski A., Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 10250-10255; Prymakowska-Bosak M., Slusarczyk J., Przewloka M., Kuras M., Lichota J., Kilianczyk B., Jerzmanowski A., Linker Histones Play a Role in Male Meiosis and the Development of Pollen Grains in Tobacco, Plant Cell 11 (1999) 2317-2330). In order to analyze the possible causes of the specific phenotypic changes observed in whole plants we employed a simpler system of tobacco BY-2 cell line. We show that the BY-2 cells engineered to overexpress a major variant of Arabidopsis H1 or with the level of native major variants of H1 decreased by antisense strategy maintain the normal ability to grow and the normal length of the cell cycle. In the cells with perturbed H1 histones no change was observed in the organization of mitotic spindle or actin filaments of the cytoskeleton. The only visible phenotypic change occurred in cells overexpressing H1 that showed an increased frequency of cells with unusually large size. This phenotype was correlated with subtle but reproducible changes in the organization of cortical microtubules.

Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Linker histones play a role in male meiosis and the development of pollen grains in tobacco.

To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at approximately 25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.

Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.

Structure and function of cytochrome bc complexes.

The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc1 complexes and the chloroplast and cyanobacterial b6f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b6f complex.

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex.

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].

Structural and ultrastructural analysis of root primordia in vitro cultures (RPC) of Solanum lycopersicoides Dun.

The structure and ultrastructure of cell aggregates and derived root primordia were analysed in established suspension cultures of Solanum lycopersicoides Dun. A total of four modified Murashige and Skoog (1962) (MS) media were used, two containing alpha-naphthaleneacetic acid (NAA) and two containing 2,4-dichlorophenoxyacetic acid (2,4-D). Considerable differences were observed in the size and structure of the aggregates regardless of the medium. The largest aggregates had a four-zone structure (cover, starch, dividing and differentiation zones) with distinct ultrastructural organization. The degree of histological and ultrastructural differentiation in the aggregates indicated rhizogenesis initiation. It begins with the protrusion of mounds of root primordia, which are the result of radial growth of cells in defined zones of the aggregate. Further growth from the primordium forms a root meristem with three tiers initial centre and wholly distinguished histogens (dermatogen, periblem, plerome). Rhizogenesis was more regular on media with NAA than on media with 2,4-D. Primordia and fully organized roots were liberated due to fragmentation of the aggregate and underwent changes in the permanently active developmental cycle of the culture which gave rise to successive generations of aggregates. Growth regulators had different influences on the organellar composition and phenolic compounds presence in the different zones of aggregates.

The energy landscape for ubihydroquinone oxidation at the Q(o) site of the bc(1) complex in Rhodobacter sphaeroides.

Activation energies for partial reactions involved in oxidation of quinol by the bc(1) complex were independent of pH in the range 5. 5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH(2). Separation to products after the first electron transfer and movement of semiquinone formed in the Q(o) site would allow rapid electron transfer to heme b(L). This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation.

Evidence for two active branches for electron transfer in photosystem I.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

Pathways for proton release during ubihydroquinone oxidation by the bc(1) complex.

Quinol oxidation by the bc(1) complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q(o) site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c(1). Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or beta-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H(+) transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.