A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.

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.

Two Menkes-type atpases supply copper for photosynthesis in Synechocystis PCC 6803.

Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of CPx-type ATPases, two of which are now designated pacS and ctaA. We show that CtaA and PacS (but not the related transporters, ZiaA or CoaT) facilitate switching to the use of copper (in plastocyanin) as an alternative to iron (in cytochrome c(6)) for the carriage of electrons within the thylakoid lumen. Disruption of pacS reduced copper tolerance but enhanced silver tolerance, and pacS-mediated restoration of copper tolerance was used to select transformants. Disruption of ctaA caused no change in copper tolerance but reduced the amount of copper cell(-1). In cultures supplemented with 0.2 microm copper, photooxidation of cytochrome c(6) (PetJ) was depressed in wild-type cells but remained elevated in both Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS). Conversely, plastocyanin transcripts (petE) were less abundant in both mutants at this [copper]. Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS) showed increased iron dependence with impaired growth in deferoxamine mesylate (iron chelator)-containing media. Double mutants also deficient in cytochrome c(6), Synechocystis PCC 6803(petJ,ctaA) and Synechocystis PCC 6803(petJ,pacS), were viable, but the former had increased copper dependence with severely impaired growth in the presence of bathocuproinedisulfonic acid (copper chelator). Analogous transporters are likely to supply copper to plastocyanin in chloroplasts.

Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts.

Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.

Co-association of cytochrome f catabolites and plastid-lipid-associated protein with chloroplast lipid particles.

Distinguishable populations of lipid particles isolated from chloroplasts of yellow wax bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves have been found to contain plastid-lipid-associated protein (J. Pozueta-Romero, F. Rafia, G. Houlné, C. Cheniclet, J.P. Carde, M.-L. Schantz, R. Schantz [1997] Plant Physiol 115: 1185-1194). One population is comprised of plastoglobuli obtained from sonicated chloroplasts by flotation centrifugation. Higher density lipid-protein particles isolated from chloroplast stroma by ultrafiltration constitute a second population. Inasmuch as the stromal lipid-protein particles contain plastid-lipid-associated protein, but are distinguishable from plastoglobuli in terms of their lipid and protein composition, they appear to be plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and the higher density lipid-protein particles both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of protein catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover.

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.

[The cardioprotective action of preparations of biotechnological cytochrome c in acute myocardial ischemia]. / Kardioprotektornoe deistvie preparatov biotekhnologicheskogo tsitokhroma c pri ostroi ishemii miokarda.

The cardioprotective effect of cytochrome c preparations was evaluated according to the test for restriction of the size of the myocardial infarct and the effect on the course of acute myocardial ischemia in acute experiments on dogs. Cytochrome c of biotechnological and animal origin and hemtetradecapeptide caused a marked decrease in the size of the myocardial necrosis in experiments on rats: from 68 +/- 4.3% in the control to 32 +/- 3.4, 46 +/- 8.3 and 44 +/- 4.7%, respectively. In dog experiments the cytochrome c agents reduced the intensity of dp/dt decline and decreased the collateral coronary blood flow in acute myocardial ischemic. They produced a beneficial effect on heart bioenergetics, namely, reduced the lactate level in blood flowing from the zone of the ischemia and glucose consumption by the ischemic myocardium. The cardioprotective effect of biotechnological cytochrome c hemtetradecapeptide was practically identical to the effect of the enzyme of animal origin.

Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, are identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu).

Electron-transfer reactions of cytochrome f with flavin semiquinones and with plastocyanin. Importance of protein-protein electrostatic interactions and of donor-acceptor coupling.

Reduction of turnip ferricytochrome f by flavin semiquinones and oxidation of this ferrocytochrome f by French bean cupriplastocyanin are studied by laser flash photolysis over a wide range of ionic strengths. Second-order rate constants (+/- 15%) at extreme values of ionic strength, all at pH 7.0 and 22 degrees C, are as follows: with FMN semiquinone at 1.00 and 0.0040 M, 5.0 x 10(7) and 3.9 x 10(8) M-1 s-1; with riboflavin semiquinone at 1.00 and 0.0040 m, 1.7 x 10(8) and 1.9 x 10(8) M-1 s-1; with lumiflavin semiquinone at 1.00 and 0.0045 M, 1.8 x 10(8) and 4.5 x 10(8) M-1 s-1; with cupriplastocyanin at 1.00 and 0.100 M, 1.4 x 10(6) and 2.0 x 10(8) M-1 s-1. These reactions of cytochrome f are governed by the local positive charge of the interaction domain (the exposed heme edge), not by the overall negative charge of the protein. Lumiflavin semiquinone behaves as if it carried a small negative charge, probably because partial localization of the odd electron gives this electroneutral molecule some polarity; local charge seems to be more important than overall charge even for relatively small redox agents. The dependence of the rate constants on ionic strength was fitted to the equation of Watkins; this model recognizes the importance of local charges of the domains through which redox partners interact. There is kinetic evidence that a noncovalent complex between cytochrome f and plastocyanin exists at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.

Site-directed mutants of the pea plastocyanin gene in which the codon for the surface-exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped-flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8-fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40-fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.

Cleavage of the precursor of pea chloroplast cytochrome f by leader peptidase from Escherichia coli.

Leader peptidase from Escherichia coli was able to process the precursor of pea cytochrome f synthesised in vitro. N-Terminal sequencing established that cleavage by leader peptidase generated the same mature sequence as in pea chloroplasts. Processing by leader peptidase was much more efficient co-translationally rather than post-translationally, and the extent of post-translational processing declined with time suggesting that the cytochrome f precursor folded to an uncleavable conformation. Detergent extracts of pea thylakoid membranes were unable to process the cytochrome f precursor co- or post-translationally.

An open reading frame encoding a putative haem-binding polypeptide is cotranscribed with the pea chloroplast gene for apocytochrome f.

The nucleotide sequence of a 1 kbp region of pea chloroplast DNA upstream from the gene petA encoding apocytochrome f has been determined. An open reading frame of 231 codons (ORF231) encoding a putative membrane-spanning polypeptide is separated by 205 bp from the coding region of petA. The open reading frame is homologous to open reading frames located in a similar position with respect to petA in chloroplast DNA from Marchantia polymorpha, tobacco, rice, wheat and Vicia faba. The sequence around a conserved histidine residue in a putative membrane-spanning region of the polypeptide resembles sequences present in cytochrome b from chromaffin granules and neutrophil membranes, suggesting that the open reading frame may encode a haem-binding polypeptide, possibly a b-type cytochrome. Northern hybridisation analysis indicates the presence in pea chloroplasts of a complex pattern of transcripts containing ORF231. Large transcripts of 5.5 kb, 4.3 kb, 3.4 kb and 2.7 kb encode both ORF231 and apocytochrome f, indicating that ORF231 and petA are co-transcribed.

Measurement of cytochrome f in thylakoids of higher plants using quantitative gel electrophoresis.

A quantitative method was developed to estimate the concentration of cytochrome (cyt) f in isolated thylakoids, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with a heme-specific reagent containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide. This densitometric technique was at least as sensitive as difference spectroscopy. Analysis of thylakoid preparations by densitometry of stained bands using cyt c as standard gave molar ratios of cyt/chlorophyll which were identical to ratios obtained by difference spectroscopy. Densitometric assays demonstrated that the molar ratio of cyt f/chlorophyll decreased during leaf aging in seven higher plants; however, there was a marked difference in the rate at which cyt f was lost from the leaves of different species.

Synthesis of components of the cytochrome b-f complex by isolated pea chloroplasts.

Three components of the cytochrome b-f complex, cytochrome f, cytochrome b-563 and a 15.2-kDa polypeptide, were labelled with radioactive amino acids in isolated pea chloroplasts incubated in the light with added Mg-ATP. The assembly of cytochrome b-563 (19.5 kDa) into the cytochrome complex required the presence of added Mg-ATP whereas cytochrome f (37.3 kDa) and the 15.2-kDa polypeptide were assembled in its absence. Incorporation of [35S]methionine into the polypeptide chain of cytochrome b-563 and the 15.2-kDa component was confirmed by peptide mapping of the products of partial digestion with papain.

Isolation and characterization of a cytochrome b-f complex from pea chloroplasts.

A cytochrome b-f complex has been extracted from pea chloroplast membranes with octyl glucoside and cholate and has been purified by ammonium sulphate fractionation and polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium deoxycholate. The complex contains cytochromes f and b-563 in a ratio of 1:2, but shows very low levels of the Rieske iron sulphur centre and plastoquinol-plastocyanin oxidoreductase activity. The complex consists of four polypeptides of molecular weights 37300, 34000, 19500 and 15200. The complex is dissociated by electrophoresis in the presence of 2M urea to give pure preparations of cytochrome f and cytochrome b-563, which on sodium dodecylsulphate-polyacrylamide gel electrophoresis give single bands of apparent molecular weights 37300 and 19500 respectively.