The specificity in the interaction between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 is mainly determined by the copper protein

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

A comparative structural and functional analysis of cytochrome cM cytochrome c6 and plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803

Cytochrome cM is a new c-class photosynthetic haem protein whose physiological role is still unknown. It has been proposed previously that cytochrome cM can replace cytochrome c6 and plastocyanin in transferring electrons between the two membrane complexes cytochrome b6-f and photosystem I in organisms growing under stress conditions. The experimental evidence herein provided allows us to discard such a hypothesis. We report a procedure to overexpress cytochrome cM from the cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli cells in mg quantities. This has allowed us to perform a comparative laser flash-induced kinetic analysis of photosystem I reduction by the three metalloproteins from Synechocystis. The bimolecular rate constant for the overall reaction is up to 100 times lower with cytochrome cM than with cytochrome c6 or plastocyanin. In addition, the redox potential value and surface electrostatic potential distribution of cytochrome cM are quite different from those of cytochrome c6 and plastocyanin. These findings strongly indicate that cytochrome cM cannot be recognised by and interact with the same redox partners as the other two metalloproteins.     

Structure of the complex between plastocyanin and cytochrome f from the cyanobacterium Nostoc sp. PCC 7119 as determined by paramagnetic NMR. The balance between electrostatic and hydrophobic interactions within the transient complex determines the relative orientation of the two proteins

The complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mM(-1), and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of plastocyanin relative to cytochrome f with a precision of 1.3 angstroms. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.     

A laser flash-induced kinetic analysis of in vivo photosystem I reduction by site-directed mutants of plastocyanin and cytochrome c6 in Synechocystis sp. PCC 6803

In cyanobacteria, plastocyanin and cytochrome c6 are two soluble metalloproteins which can alternately serve as electron donors to photosystem I. From site-directed mutagenesis studies in vitro, it is well-established that both hydrophobic and electrostatic forces are involved in the interaction between the donor proteins and photosystem I. Hence, two isofunctional areas, a hydrophobic one in the north and an acidic one in the east, have been described on the surface of both electron donors. In this work, we have tested the relevance of such kinds of interactions in the photosystem I reduction inside the cell. Several plastocyanin and cytochrome c6 site-directed mutant strains affecting both the acidic and hydrophobic regions of the two metalloproteins, which were previously characterized in vitro, have been constructed. The photosystem I reduction kinetics of the different mutants have been analyzed by laser flash absorption spectroscopy. Relevant differences have been found between the in vitro and in vivo results, mainly regarding the role played by the electrostatic interactions. Adding positive electrostatic charges to the acidic patch of plastocyanin and cytochrome c6 promotes an enhanced interaction with photosystem I in vitro but yields the opposite effect in vivo. These discrepancies are discussed in view of the different environmental conditions, in vitro and in vivo, for the reaction mechanism of photosystem I reduction, namely, differential interaction of the electron donors with the thylakoidal membrane and kinetics of donor exchange.     

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.     

The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903

Plastocyanin (Pc) and cytochrome c₆ (Cyt) have been purified to homogeneity from the cyanobacterium Pseudanabaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k_obs) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119

Most organisms performing oxygenic photosynthesis contain either cytochrome c ₆ or plastocyanin, or both, to transfer electrons from cytochrome b ₆-f to photosystem I. Even though plastocyanin has superseded cytochrome c ₆ along evolution, plants contain a modified cytochrome c ₆, the so called cytochrome c _6A, whose function still remains unknown. In this article, we describe a second cytochrome c ₆ (the so called cytochrome c ₆-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c _6A than to cyanobacterial cytochrome c ₆. In this article, we conclude that the cytochrome c ₆-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c ₆ and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c ₆ and plastocyanin. In any way, the Cyt c ₆-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.     

Isolation and characterization of a calmodulin-like protein from the cyanobacterium Nostoc sp. PCC 6720

A 21-kDa novel polypeptide which possesses characteristics normally considered to be diagnostic of the calmodulin present in eukaryotic cells was isolated from the cyanobacterium Nostoc sp. PCC 6720. The major technique employed in the isolation of the polypeptide was ion-exchange chromatography on a Mono Q column. The 21-kDa polypeptide was shown: to activate pea NAD kinase in vitro, in a Ca²⁺ requiring reaction; to react with polyclonal antibodies raised against spinach calmodulin, but not with those raised against bovine brain calmodulin; and to exhibit a Ca²⁺ dependent shift in migration during SDS-PAGE.Abbreviations ATCC American Type Culture Collection - DCPIP 2,6-dichlorophenylindophenol - PBS Phosphate buffered saline     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.     

Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c from Nostoc sp. PCC 7119

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions.     

Immunological characterization of hydrogenases in the nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 73102

N₂-fixing Nostoc sp. strain PCC 73102 was examined for the presence of hydrogenases. Native-PAGE/immunoblots demonstrated that two proteins with molecular masses of approximately 200 kDa and 215 kDa are immunologically related to hydrogenases purified from Bradyrhizobium japonicum, Azotobacter vinelandii, Methanosarcina barkeri, and Thiocapsa roseopersicina. SDS-PAGE/immunoblots showed that one polypeptide, with a molecular mass of about 58 kDa, is immunologically related to the hydrogenases purified from all the microorganisms mentioned above. In addition, two polypeptides, with molecular masses of approximately 34 and 70 kDa, are immunologically related to the hydrogenases purified from T. roseopersicina and M. barkeri respectively. Immunogold/transmission electron microscopy showed that the hydrogenase proteins are present in both the heterocysts and the vegetative cells.     

A restriction map of plasmid pDC1 from the filamentous cyanobacterium Nostoc sp. MAC PCC 8009

A plasmid designated pDC1 from the cyanobacterium Nostoc sp. MAC PCC 8009 was incubated with 16 different restriction enzymes, of which 8 cleaved pDC1. Plasmid pDC1 has a single site for ClaI, two sites for each of BglI, EcoRI, EcoRV, and MluI, three sites for HpaI, and four for HindIII. A restriction map of pDC1 for these 7 enzymes was constructed.     

Cloning and expression of a putative cyclodextrin glucosyltransferase from the symbiotically competent cyanobacterium Nostoc sp. PCC 9229

A polymerase chain reaction-based method was used to isolate a Nostoc sp. PCC 9229 cDNA from infected glands of Gunnera chilensis. The complete gene sequence was isolated from a genomic Nostoc sp. PCC 9229 library. Sequence analysis showed 84% amino acid similarity to a putative cyclodextrin glycosyltransferase from Nostoc sp. PCC 7120 and the gene was therefore termed cgt. Southern blot revealed that the cgt gene was present in symbiotically competent cyanobacteria. The cgt gene was expressed in free-living nitrogen-fixing cultures in light or in darkness when supplemented with fructose. This is the first expression analysis of a cgt gene from a cyanobacterium.     

Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.     

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.