A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.     

63/65Cu and 1/2H2O isotope shifts in the low-temperature resonance Raman spectrum of fungal laccase

Resonance Raman spectra are reported for the type 1 Cu site of fungal laccase at 295 and 77 K. The low-temperature spectra show enhanced resolution and reveal several weak bands not previously observed, as well as overtone and combination bands associated with the strong approximately equal to 400 cm-1 fundamentals. A novel low-temperature Raman difference technique has been used to obtain 63/65Cu and 1/2H2O isotope shifts. The strong band at 428 cm-1, and the moderate intensity bands at 408 and 387 cm-1 show small (under 0.6 cm-1 63/65Cu isotope shifts. The aggregate shift is substantially less than that expected for an isolated Cu-S(cys) stretch, implying a high degree of mixing of this coordinate with internal modes of the ligands. 1/2H2O shifts of 1.1 and approximately equal to 0.3 cm-1 are observed for the 387 and 428 cm-1 bands. The isotope shift patterns are quite similar for fungal and tree laccase, as are the frequencies of the dominant bands, indicating that the large differences in relative intensity are primarily associated with differences in the excited state potential. The frequency and isotope shift patterns are appreciably different, however, from those observed for azurin and stellacyanin. In contrast to the other 'blue' Cu proteins, fungal laccase shows no moderate intensity band near 270 cm-1 which can be associated with Cu-imidazole stretching; weak features are seen in this region, but the intensities are too low to determine their 1/2H2O sensitivity. The C-S stretching mode of fungal laccase is identified at 737 cm-1, shifting to 741 cm-1 at 77 K. It is about 10 cm-1 lower than for most 'blue' Cu proteins, and the difference is suggested to reflect smaller kinematic coupling between the C-S and Cu-S coordinates, associated with a smaller Cu-S-C angle. Combination modes of the approx. 400 cm-1 fundamentals are substantially stronger, relative to the overtones, than is predicted by first-order scattering theory, implying changes in the excited-state normal modes (Dushinsky effect) associated with force constant alterations.     

Ternary Cu(II) complexes, Cu(H(-1)L)(ACys-) and Cu(H(-2)L)(ACys-); L = peptides, ACys- = N-acetyl-cysteinate. Analogous complexes to the intermediates in the transport of Cu(II) from Cu(H(-2)L) to cysteine

The Cu(II) in Cu(H(-2)L) has been postulated to be successively transported to cysteine (Cys) as follows; Cu(H(-2)L) <==> Cu(H(-2)L)(Cys*-) <==> Cu(H(-1)L)(Cys*-) --> Cu(H(-1)L)(Cys-), where Cys*- denotes the monodentate Cys-. N-acetyl-cysteinate (ACys-) complexes Cu(H(-2)L)(ACys-) and Cu(H(-1)L)(ACys-), having similar coordination modes to Cu(H(-2)L)(Cys*-) and Cu(H(-1)L)(Cys*-), respectively, exhibited the S --> Cu(II) charge transfer absorption at 325-355 nm and the d-d absorption at 530-610 nm. A linear interrelation existed between the energies of the CD and d-d absorptions. Cu(H(-2)L)(ACys-) were in rapid equilibrium with Cu(H(-1)L)(ACys-). Upon forming the ternary complex, pK(c2) of the parent Cu(H(-1)L) was raised to more than 1.0. The formation constants (K) of the Cu(H(-1)L)(ACys-) species from Cu(H(-1)L) were bigger than those of Cu(H(-2)L)(ACys-) from Cu(H(-2)L). The linear free-energy relationship existed between the free-energy change (deltaG) and the entropy change (deltaS) for the ternary complex formation. The rate constants (k1+) for the Cu(H(-1)L)(Cys-) formation closely correlated with the K values for Cu(H(-2)L)(ACys-). The ternary complexes containing ACys are considered to be analogous complexes to the intermediates in the transport of Cu(II) from peptides to cysteine.     

Spectroscopic characterization and O2 reactivity of the trinuclear Cu cluster of mutants of the multicopper oxidase Fet3p

Fet3p is a multicopper oxidase that uses four copper ions (one type 1, one type 2, and one type 3 binuclear site) to couple substrate oxidation to the reduction of O(2) to H(2)O. The type 1 Cu site shuttles electrons between the substrate and the type 2/type 3 Cu sites which form a trinuclear Cu cluster that is the active site for O(2) reduction. This study extends the spectroscopic and reactivity studies that have been conducted with type 1-substituted Hg (T1Hg) laccase to Fet3p and a mutant of Fet3p in which the trinuclear Cu cluster is perturbed. To examine the reaction between the trinuclear Cu cluster and O(2), the type 1 Cu Cys(484) was mutated to Ser, resulting in a type 1-depleted (T1D) form of the enzyme. Additional His to Gln mutations were made at the trinuclear cluster to further probe specific contributions to reactivity. One of these mutants (His(126)Gln) produces the first stable but perturbed trinuclear Cu cluster (T1DT3' Fet3p). Spectroscopic characterization (absorption, circular dichroism, magnetic circular dichroism, and electron paramagnetic resonance) of the resting trinuclear sites in T1D and T1DT3' Fet3p reveal that the His(126)Gln mutation changes the electronic structure of both the type 3 and type 2 Cu sites. The trinuclear clusters in T1D and T1DT3' Fet3p react with O(2) to produce peroxide intermediates analogous to that observed in T1Hg laccase. Spectroscopic data on the peroxide intermediates in the three forms provide further insight into the structure of this intermediate. In T1D Fet3p, the decay of this peroxide intermediate is pH-dependent, and the rate of decay is 10-fold higher at low pH. In T1DT3' Fet3p, the decay of the peroxide intermediate is pH-independent and is slow at all pH's. This change in the pH dependence provides new insight into the mechanism of intermediate decay involving reductive cleavage of the O-O bond.     

Mechanism of hydrogen peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations

We have reinvestigated the biochemistry of H2O2-induced Cu,Zn-superoxide dismutase (SOD1)-centered radicals, detecting them by immuno-spin trapping. These radicals are involved in H2O2-induced structural and functional damage to SOD1, and their mechanism of generation depends on copper and/or (bi)carbonate (i.e., CO2, CO3(-2), or HCO3-). First, in the absence of DTPA and (bi)carbonate, Cu(II) was partially released and rebound at His, Cys, and Tyr residues in SOD1 with the generation of protein-copper-bound oxidants outside the SOD1 active site by reaction with excess H2O2. These species produced immuno-spin trapping-detectable SOD1-centered radicals associated with H2O2-induced active site ( approximately 5 and approximately 10 kDa fragments) and non-active site (smearing between 3 and 16 kDa) copper-dependent backbone oxidations and subsequent fragmentation of SOD1. Second, in the presence of DTPA, which inhibits H2O2-induced SOD1 non-active site fragmentation, (bi)carbonate scavenged the enzyme-bound oxidant at the SOD1 active site to produce the carbonate radical anion, CO3*-, thus protecting against active site SOD1 fragmentation. CO3*- diffuses and produces side chain oxidations forming DMPO-trappable radical sites outside the enzyme active site. Both mechanisms for generating immuno-spin trapping-detectable SOD1-centered radicals were susceptible to inhibition by cyanide and enhanced at high pH values. In addition, (bi)carbonate enhanced H2O2-induced SOD1 turnover as demonstrated by an enhancement in oxygen evolution and SOD1 inactivation. These results help clarify the free radical chemistry involved in the functional and structural oxidative damage to SOD1 by H2O2 with the intermediacy of copper- and CO3*--mediated oxidations.     

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins)

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.     

Solution structure of the Cu(I) and apo forms of the yeast metallochaperone, Atx1

The (1)H NMR solution structure of the Cu(I)-bound form of Atx1, a 73-amino acid metallochaperone protein from the yeast Saccharomyces cerevisiae, has been determined. Ninety percent of the (1)H and 95% of the (15)N resonances were assigned, and 1184 meaningful NOEs and 42 (3)J(HNH)(alpha) and 60 (1)J(HN) residual dipolar couplings provided a family of structures with rmsd values to the mean structure of 0.37 +/- 0.07 A for the backbone and 0.83 +/- 0.08 A for all heavy atoms. The structure is constituted by four antiparallel beta strands and two alpha helices in a betaalphabetabetaalphabeta fold. Following EXAFS data [Pufahl, R., Singer, C. P., Peariso, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E., and O'Halloran, T. V. (1997) Science 278, 853-856], a copper ion can be placed between two sulfur atoms of Cys15 and Cys18. The structure of the reduced apo form has also been determined with similar resolution using 1252 meaningful NOEs (rmsd values for the family to the mean structure are 0.67 +/- 0.12 A for the backbone and 1.00 +/- 0.12 A for all heavy atoms). Comparison of the Cu(I) and apo conformations of the protein reveals that the Cu(I) binding cysteines move from a buried site in the bound metal form to a solvent-exposed conformation on the surface of the protein after copper release. Furthermore, copper release leads to a less helical character in the metal binding site. Comparison with the Hg(II)-Atx1 solid-state structure [Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Structure 7, 605-617] provides insights into the copper transfer mechanism, and a pivotal role for Lys65 in the metal capture and release process is proposed.     

Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ～60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.     

Computational studies of Cu(II)[peptide] binding motifs: Cu[HGGG] and Cu[HG] as models for Cu(II) binding to the prion protein octarepeat region

The binding of Cu(II) to the prion protein is investigated by computations at the B3LYP level of theory on models of the octarepeat domain of the prion protein. The models incorporate the functionality of the glycine (G) and histidine (H) residues which occur in the octarepeat domain, PHGGGWGQ. The copper complexes are designated Cu[HG] and Cu[HGGG]. Coordination to the metal via the imidazole ring of the histidine, the amide carbonyl groups, and the backbone nitrogen atom of the amide groups were examined, as well as several protonation/deprotonation states of each structure. EPR and CD titration experiments suggest that the octarepeat segments of the unstructured N-terminal domain of prion protein can bind Cu(II) in a 1:1 Cu-to-octarepeat ratio. The results identify the extent to which the Cu(II) facilitates peptide backbone deprotonation, and the propensity of binding in the forward (toward the C-terminus) direction from the anchoring histidine residue. A plausible mechanism is suggested for changing from amide O-atom to deprotonated amide N-atom coordination, and for assembly of the observed species in solutions of Cu[PrP] and truncated models of it. A structure is proposed which has the N2O2 coordination pattern for the minor component observed experimentally by EPR spectroscopy for the Cu[HGGG] model. The most stable neutral Cu[HGGG] structure found, with coordination environment N3O1, corresponds to that observed for Cu[HGGGW] and Cu[HGGG] both in the solid state and as the major component in solution at neutral pH.     

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.     

Structure of the two transmembrane Cu+ transport sites of the Cu+ -ATPases

Cu(+)-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu(+) within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu(+) transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu(+). However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu(+)-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu(+) (Site I K(a)=1.3 fM(-1), Site II K(a)=1.1 fM(-1)), in conjunction with reversible direct Cu(+) transfer from chaperones, points to a transport mechanism where backward release of free Cu(+) to the cytoplasm is largely prevented.     

Concerted involvement of cooperative proton-electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu(A) and heme a3/Cu(B) are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e- cooperative linkage at Fe(a3)/Cu(B) is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e- linkage at heme a (and Cu(A)). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu(A) and heme a3/Cu(B) in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R-->O transition), reductive (O-->R transition) and a full catalytic cycle (R-->O-->R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e- linkage at heme a/Cu(A) and heme a3/Cu(B) with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping.     

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

Quantum chemical calculations of the reorganization energy of blue-copper proteins.

The inner-sphere reorganization energy for several copper complexes related to the active site in blue-copper protein has been calculated with the density functional B3LYP method. The best model of the blue-copper proteins, Cu(Im)2(SCH3)(S(CH3)2)(0/+), has a self-exchange inner-sphere reorganization energy of 62 kJ/mol, which is at least 120 kJ/mol lower than for Cu(H2O)4(+/2+). This lowering of the reorganization energy is caused by the soft ligands in the blue-copper site, especially the cysteine thiolate and the methionine thioether groups. Soft ligands both make the potential surfaces of the complexes flatter and give rise to oxidized structures that are quite close to a tetrahedron (rather than tetragonal). Approximately half of the reorganization energy originates from changes in the copper-ligand bond lengths and half of this contribution comes from the Cu-S(Cys) bond. A tetragonal site, which is present in the rhombic type 1 blue-copper proteins, has a slightly higher (16 kJ/mol) inner-sphere reorganization energy than a trigonal site, present in the axial type 1 copper proteins. A site with the methionine ligand replaced by an amide group, as in stellacyanin, has an even higher reorganization energy, about 90 kJ/mol.     

Solution structure of an M-1 conotoxin with a novel disulfide linkage

The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.     

Copper(I)-alkyl sulfide and -cysteine tri-nuclear clusters as models for metallo proteins: a structural density functional analysis

After having set up the computational methodology for Cu(I)-sulfur systems as models for copper proteins, namely using the simple ligands H(2)S, HS(-), CH(3)SH, and CH(3)S(-), the Cu(I)-Cysteine systems have been investigated: [Cu(I)( S -H(2)Cys) (n) ](+) (H(2)Cys, cysteine, NH(2),SH,COOH) [Cu(I)( S -HCys) (n) ](1-) (n) (NH(2),S(-),COOH). Finally, the structures for bi-nuclear [Formula: see text] (Et, CH(2)CH(3)), [Formula: see text] and tri-nuclear [Cu(I)( S -SH)](3), [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text] (NH(2),SH,COOH), [Formula: see text] (NH(2),S(-),COOH, and NH(2),SH,COO(-)), as well as [Formula: see text] (NH(2),S(-),COO(-)), were also optimized to mimic the active center for a metallo-chaperone copper transport protein (CopZ). The X-ray structures for the biomolecules were matched fairly well as regards the Cu-S bond distances and Cu…Cu contact distances in the case the model cysteine S atom is deprotonated. Upon protonation of ligand S atoms, the conformation of clusters is altered and might bring about the di- and tri-nuclear core breakage. These findings suggest that subtle protonation/deprotonation steps, i.e. small and/or local pH changes play a significant role for copper transport processes.     

Constitutive induction of p-Erk1/2 accompanied by reduced activities of protein phosphatases 1 and 2A and MKP3 due to reactive oxygen species during cellular senescence

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-alpha and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-alpha activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-alpha and metal ion(s).     

Molecular mechanism of oxidative stress perception by the Orp1 protein

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.