Search

The thermal folding of plastocyanin from the mesophilic cyanobacterium Synechocystis is described and the results are compared with those obtained for the homologous thermophilic protein from Phormidium laminosum. Molecular dynamics simulations suggest that Phormidium and Synechocystis pastocyanins follow different unfolding pathways that affect different protein areas and which could be responsible for the observed dissimilar thermal resistance.

The electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp PCC 6803 was investigated using site-directed mutagenesis of wild-type copper protein to extend studies of structure and functions. Results showed that a PsaF polypeptide has an essential role in the reaction mechanism of most evolved systems. Wild-type copper protein reacts with photosystem I following a simples collisional kinetic model.

The reaction mechanisms exhibited by the cyanobacteria Anabaena PCC 7119 and Synechocystis PCC 6803 during complex formation and intracomplex electron transfer were investigated by thermodynamic analysis of Pc and Cyt oxidation by photosystem I (PSI) in the two bacterial lines as well as in spinach. Long-range electrostatic interactions were observed to be attractive in Anabaena but repulsive in Synechocystis and spinach. Ionic strength was also observed to determined the net interaction between PSI and its two donor proteins in Anabaena. A compensation effect between entropy and enthalpy at varying ionic strength was observed in all Pc/PSI and Cyt/PSI systems, except with Cyt and PSI from Anabaena.

An extensive analysis of the effect of tyrosine on the physicochemical properties and reactivity of human respiratory cytochrome c is presented. The results revealed that the change in reactivity of nitrated wild-type cytochrome c is attributed to nitration of tyrosine residue 67 and 74.

The significant contribution of the side chain of Trp57, Ile59, and Ile92 and flavodoxin hydrophobic residues towards the productive orientation of the Fld complexes with ferredoxin-[NADP.sup.+] reductase (FNR) and photosystem I(PSI) in Anabaena is reported.

A laser flash-induced kinetic analysis of the oxidation of site-directed mutants of Nostoc sp. PCC 7119 Cf is provided thereby providing relevent information on the interaction about the role played by the modified residues on the interaction between the two redox partners. The findings are discussed in light of the recently determined structure of the Nostoc Pc-Cf complex.

A study shows that in cyanobacteria, plastocyanin and cytochrome c6 are two soluble metalloproteins, which can serve as electron donors to photosystem I. The photosystem I show the reduction kinetics of the different mutants analyzed by laser flash absorption spectroscopy.

Oxidation of the soluble, truncated form of cyctochrome f by wild-type and mutant species of plastocyanin is analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc sp. PCC 7119. The experimental data match well with the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I.

The complexes of plastocyanin and cytochrome f (Pc:Cf) from Nostoc are analyzed by heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The studies of the crossed complexes show that these different interactions are due to the surface properties of the plastocyanins.

The electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP(super +) reductase (FNR) and ferredoxin (Fd) is indicated by the biochemical and structural studies. The results have shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (FId), even though no key residue on the FId surface has found to be the counter part of such FNR side chains.

Results show that hydrophobic residues in the region close to the FAD group in ferredoxin-NADP(sup)+ reductase facilitate interactions with flavodoxin ensuring electron transport. Substitution of nonpolar residues at positions 159 and 192 by lysine reveals that they are not essential for the interaction between electron transport and the reductase enzyme system.

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein., Competing Interests: The authors declare no competing financial interest.

Electron-transfer kinetics of the thermophilic protein Plastocyanin from Phormidium laminosum adsorbed on 1,ω-alkanedithiol self-assembled monolayers (SAMs) deposited on gold have been investigated. The standard electron-transfer rate constant has been determined as a function of electrode-protein distance and solution viscosity over a broad temperature range (0-90 °C). For either thin or thick SAMs, the electron-transfer regime remains invariant with temperature, whereas for the 1,11-undecanethiol SAM of intermediate chain length, a kinetic regime changeover from a gated or friction-controlled mechanism at low temperature (0-30 °C) to a nonadiabatic mechanism above 40 °C is observed. To the best of our knowledge, this is the first time a thermal-induced transition between these two kinetic regimes is reported for a metalloprotein.

Post-translational nitration of tyrosine is considered to be an important step in controlling the multiple functions of cytochrome c (Cyt-c). However, the underlying structural basis and mechanism are not yet understood. In this work, human Cyt-c variants in which all but one tyrosine has been substituted by phenylalanine have been studied by resonance Raman and electrochemical methods to probe the consequences of tyrosine nitration on the heme pocket structure and the redox potential. The mutagenic modifications of the protein cause only subtle conformational changes of the protein and small negative shifts of the redox potentials which can be rationalized in terms of long-range electrostatic effects on the heme. The data indicate that the contributions of the individual tyrosines for maintaining the relatively high redox potential of Cyt-c are additive. Nitration of individual tyrosines leads to a destabilization of the axial Fe-Met80 bond which causes the substitution of the native Met ligand by a water molecule or a lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of the heme pocket of Cyt-c due to tyrosine nitration and to local electric fields are independent of each other and occur via different molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the peroxidase function.

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.

Biochemical and structural studies indicate that electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd). Moreover, it has been shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (Fld), although, so far, no key residue on the Fld surface has been found to be the counterpart of such FNR side chains. In this study, negatively charged side chains on the Fld surface have been individually modified, either by the introduction of positive charges or by their neutralization. Our results indicate that although Glu16, Glu20, Glu61, Asp65, and Asp96 contribute to the orientation and optimization of the Fld interaction, either with FNR or with photosystem I (PSI) (presumably through the formation of salt bridges), for efficient ET, none of these side chains is involved in the formation of crucial salt bridges for optimal interaction with FNR. These data support the idea that the FNR-Fld interaction is less specific than the FNR-Fd interaction. However, analysis of the reactivity of these mutated Flds toward the membrane-anchored PSI complex indicated that all mutants, except Glu16Gln, lack the ability to form a stable complex with PSI. Thr12, Thr56, Asn58, and Asn97 are present in the close environment of the isoalloxazine ring of FMN in Anabaena Fld. Their roles in the interaction with and ET to FNR and PSI have also been studied. Mutants at these Fld positions indicate that residues in the close environment of the isoalloxazine ring modulate the ability of Fld to bind to and to exchange electrons with its physiological counterparts.

Photosynthesis consists of a series of endergonic redox reactions, with light as the source of energy, chlorophyll as the energy converter, and electrons flowing through membrane and soluble proteins. Here, we give an account of the most recent results on the structure-function relationships of the membrane-embedded complexes cytochrome b(6)-f and photosystem I and of the two soluble proteins (cytochrome c(6) and plastocyanin) that serve as alternative electron carriers between them. Particular attention is paid to the evolutionary aspects of the reaction mechanism and transient protein-protein interactions between the membrane complexes and their partners in cyanobacteria, eukaryotic algae, and plants.