Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.     

The electron-transfer site of spinach plastocyanin

Two sites for electron transfer have been proposed for plastocyanin: one near the copper ion and the other close to the acid patch formed by residues 42-45. Calculations of electrostatic properties of spinach plastocyanin and ionic strength dependences of electron-transfer reactions of this protein have been used to distinguish between these two sites. Calculations show that the electric potential field of spinach plastocyanin is highly asymmetric and that the protein has a dipole moment of 360 D. The negative end of the dipole axis emerges between the negative patches formed by residues 42-45, which is though to be the cation binding site, and residues 59-61. The angles between the dipole vector and vectors from the center of mass to the copper ion and to the acid patch are 90 degrees and 30 degrees, respectively. The angle between the dipole vector and a line from the center of mass to the site of electron transfer is evaluated from the ionic strength dependence of electron-transfer rates at pH 7.8 with the help of equations developed by Van Leeuwen et al. [van Leeuwen, J.W., Mofers, F.J.M., & Veerman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434] and Van Leeuwen [van Leeuwen, J.W. (1983) Biochim. Biophys. Acta 743, 408]. The angles found are 85 degrees, 110 degrees, and 75 +/- 15 degrees for reactions with tris(1,10-phenanthroline)cobalt(III), hexacyanoferrate(III), and ferrocytochrome c, respectively. The electric potential field calculations suggest that the hexacyanoferrate(III) interaction angle corresponds to a unique site of minimum repulsion at the hydrophobic region of the protein surface, close to the copper ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A molecular dynamics simulation study of the solvent isotope effect on copper plastocyanin

The effect of heavy water on the structure and dynamics of copper plastocyanin as well as on some aspects of the solvent dynamics at the protein-solvent interfacial region have been investigated by molecular dynamics simulation. The simulated system has been analyzed in terms of the atomic root mean square deviation and fluctuations, intraprotein H-bond pattern, dynamical cross-correlation map and the results have been compared with those previously obtained for plastocyanin in H2O (Ciocchetti et al. Biophys. Chem. 69 (1997), 185-198). The simulated plastocyanin structure in the two solvents, averaging 1 ns, is very similar along the beta-structure regions, while the most significant differences are registered, analogous to the turns and the regions likely involved in the electron transfer pathway. Moreover, plastocyanin in D2O shows an increase in the number of both the intraprotein H-bonds and the residues involved in correlated motions. An analysis of the protein-solvent coupling evidenced that D2O makes the H-bond formation more difficult with the solvent molecules for positively charged and polar residues, while an opposite trend is observed for negatively charged residues. On the other hand, the frequency of exchange of the solvent molecules involved in the protein-solvent H-bond formation is significantly depressed in D2O. The results are discussed also in connection with protein functionality and briefly with some experimental results connected with the thermostability of proteins in D2O.     

Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

The kinetics and mechanism of oxidation of reduced spinach ferredoxin by molecular oxygen and its reduced products

Reduced spinach ferredoxin reacts with molecular oxygen in an autocatalytic reaction characterized by a hyperbolic dependence on oxygen concentration. The kinetics of the reaction indicate formation of a reduced ferredoxin-oxygen intermediate complex and production of superoxide anion which may also react with reduced ferredoxin. Hydrogen peroxide, which is formed from superoxide, in turn reoxidizes reduced ferredoxin at a rate nearly 10-times faster than that of the comparable reaction with oxygen. The kinetics of reaction of hydrogen peroxide with reduced ferredoxin are biphasic. The substrate dependence of the first phase of the reaction is consistent with a simple one-step equilibrium reaction. The second phase of the reaction could be eliminated by addition of the radical trapper, sodium formate.     

1H-NMR sequential assignments and cation-binding studies of spinach plastocyanin

The essentially complete assignment of the 1H-NMR spectrum of the Cu(i) form of spinach plastocyanin has been achieved using two-dimensional NMR techniques and sequence-specific resonance assignment procedures. A variety of pH and temperature conditions was utilised to overcome the problems of resonance overlap in the spectrum, degeneracy of C alpha H and solvent H2O chemical shifts, and cross-saturation of labile NH resonances. A qualitative analysis of the long-range nuclear Overhauser effects observed indicates that the backbone fold of spinach plastocyanin is very similar to that of poplar plastocyanin, whose structure has been solved by X-ray crystallography and differs in 22 of its 99 amino acid residues. The assignments provide a basis for further investigations into the structural and ion- and protein-binding properties of plastocyanin in solution.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.     

Thermal denaturation of whey proteins and its effect in dairy technology

The irreversible denaturation of the most important whey protein fractions, namely beta-lactoglobulin A and B and alpha-lactalbumin were studied. The orders of the reactions, the rate constants and the activation energies were determined. The experiments were extended to include whey protein solutions of different concentrations and mixtures of whey proteins and caseins in different proportions. The kinetic data found by experiment make it possible to calculate in advance the precise degree of irreversible denaturation. It was found that the denaturation of beta-lactoglobulin was a good test parameter in technological studies and that there was a close correlation between the degree of denaturation and the results of important dairy processes.     

Structure of the intermolecular complex between plastocyanin and cytochrome f from spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.     

H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin

Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.     

A Brownian dynamics computational study of the interaction of spinach plastocyanin with turnip cytochrome f: the importance of plastocyanin conformational changes

Brownian Dynamics (BD) computer simulations were used to study electrostatic interactions between turnip cytochrome f (cyt f) and spinach plastocyanin (PC). Three different spinach PC structures were studied: The X-ray crystal structure of Xue and coworkers [(1998) Protein Sci 7:2099–2105] and the NMR structure of Musiani et al. [(2005) J Biol Chem 280:18833–18841] and Ubbink and co-workers [(1998) Structure 6:323–335]. Significant differences exist in the backbone conformation between the PC taken from Ubbink and coworkers and the other two PC structures particularly the regions surrounding G10, E59–E60, and D51. Complexes formed in BD simulations using the PC of Ubbink and colleagues had a smaller Cu–Fe distance than the other two. These results suggest that different PC conformations may exist in solution with different capabilities of forming electron-transfer-active docks. All three types of complexes show electrostatic contacts between D42, E43, and D44 on PC and K187 on cyt f as well as between E59 on PC and K58 on cyt f. However, the PC of Ubbink and coworkers reveals additional contacts between D51 and cyt f as a result of the difference in backbone configuration. A second minor complex component was observed for the PC of Ubbink and co-workers and Xue and co-workers which had contacts between K187 on cyt f and E59 and E60 on PC rather than between K187 on cyt f and D42-D44 on PC as observed for the major components. This second type of complex may represent an earlier complex which rearranges to form a final complex capable of electron transfer. Professor Elizabeth L. Gross, Professor Emeritus of Biochemistry, The Ohio State University, passed away on June 27, 2007.