Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray crystallographic characterisation of type-2-depleted ascorbate oxidase from zucchini.

The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.     

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1?Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.     

Pseudo 2-fold symmetry in the copper-binding domain of arthropodan haemocyanins. Possible implications for the evolution of oxygen transport proteins

Investigation of the copper-binding centre of Panulirus interruptus haemocyanin led to the discovery of a pseudo 2-fold axis relating two helical pairs surrounding and co-ordinating the two copper ions. The pseudo 2-fold symmetry relating one helical pair, co-ordinating Cu-A, to the second helical pair co-ordinating Cu-B is quite precise with 31 equivalent C alpha atoms having a root-mean-square deviation of only 1.47 A. The 2-fold consists of a rotation of 174.6 degrees and a translation parallel to the rotation axis of 0.7 A. After superposition of the helical pairs, the two copper ions are within 1.1 A and the three C alpha atoms of the histidine ligands of Cu-A are within a root-mean-square deviation of 1.0 A from the C alpha atoms of the histidine residues co-ordinating Cu-B. Of the superimposed residues, 26% are identical in sequence. These data suggest that the current oxygen-binding centre of arthropodan haemocyanins is the result of dimerization, gene duplication and gene fusion of an ancestral mono-copper-binding helical pair. This suggestion is supported by the recent discovery that in the sequence of functional domains of molluscan haemocyanins only amino acid sequence homology with the arthropodan Cu-B helical pair has been found and no evidence for similarity with a Cu-A binding helical pair was observed. This provides strong evidence that a mono-copper-binding helical pair has been the ancestor of both the arthropodan and molluscan haemocyanins. Turning to the Fe-binding helical pairs in haemerythrins, it appears that they are less similar to each other than the two Cu-binding helical pairs in arthropodan haemocyanins. Nevertheless, the Fe-B haemerythrin helical pair superimposes well onto the Cu-A helical pair of Panulirus haemocyanin. A root-mean-square deviation of 1.9 A for 24 equivalent C alpha carbon atoms is obtained, while Fe-B deviates 1.4 A from Cu-A after superposition of the helices. Moreover, the three histidine ligands of the Cu-A helical pair are equivalent with three histidine ligands of the Fe-B pair. The structural similarity and correspondence in metal-binding ligands suggests that both haemocyanins and haemerythrins have originated from an ancestral mono-metal-binding helical pair having two ligands provided by the first helix and one ligand by the second helix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus.

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90 degrees C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70 degrees C and above 70 degrees C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.     

The blue oxidases, ascorbate oxidase, laccase and ceruloplasmin. Modelling and structural relationships

On the basis of the spatial structure of ascorbate oxidase [Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 206, 513-529], an alignment of the amino acid sequence of the related blue oxidases, laccase and ceruloplasmin is proposed. This strongly suggests a three-domain structure for laccase closely related to ascorbate oxidase and a six-domain structure of ceruloplasmin. These domains demonstrate homology with the small blue copper proteins. The relationships suggest that laccase, like ascorbate oxidase, has a mononuclear blue copper in domain 3 and a trinuclear copper between domain 1 and 3 and ceruloplasmin has mononuclear copper ions in domains 2, 4 and 6 and a trinuclear copper between domains 1 and 6.     

Homology modeling of the multicopper oxidase Fet3 gives new insights in the mechanism of iron transport in yeast

Fet3, the multicopper oxidase of yeast, oxidizes extracellular ferrous iron which is then transported into the cell through the permease Ftr1. A three-dimensional model structure of Fet3 has been derived by homology modeling. Fet3 consists of three cupredoxin domains joined by a trinuclear copper cluster which is connected to the blue copper site located in the third domain. Close to this site, which is the primary electron acceptor from the substrate, residues for a potential iron binding site could be identified. The surface disposition of negatively charged residues suggests that Fet3 can translocate Fe(3+) to the permease Ftr1 through a pathway under electrostatic guidance.     

A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO

CueO, a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli, is expressed under conditions of copper stress and shows enhanced oxidase activity when additional copper is present. The 1.7-A resolution structure of a crystal soaked in CuCl2 reveals a Cu(II) ion bound to the protein 7.5 A from the T1 copper site in a region rich in methionine residues. The trigonal bipyramidal coordination sphere is unusual, containing two methionine sulfur atoms, two aspartate carboxylate oxygen atoms, and a water molecule. Asp-439 both ligates the labile copper and hydrogen-bonds to His-443, which ligates the T1 copper. This arrangement may mediate electron transfer from substrates to the T1 copper. Mutation of residues bound to the labile copper results in loss of oxidase activity and of copper tolerance, confirming a regulatory role for this site. The methionine-rich portion of the protein, which is similar to that of other proteins involved in copper homeostasis, does not display additional copper binding. The type 3 copper atoms of the trinuclear cluster in the structure are bridged by a chloride ion that completes a square planar coordination sphere for the T2 copper atom but does not affect oxidase activity.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

A peptide model of the copper-binding region of lysyl oxidase

The copper-binding site of lysyl oxidase remains extremely poorly characterized and although models have been suggested for copper(II) coordination by three histidine ligands, as has been found for other copper-containing amine oxidases, there has been no experimental confirmation of these suggestions. In this work, two synthetic peptides with 24 and 34-amino acid residues, respectively, were chosen from the highly conserved histidine-rich sequence previously suggested as the copper-binding region of lysyl oxidase. These peptides each bind one equivalent of Cu(II), at the same site in the two peptides. Spectroscopic (NMR, electron paramagnetic resonance (EPR), CD, visible absorption and fluorescence) techniques were employed to investigate the nature of the resulting complexes. The results indicate that at neutral pH three histidine ring nitrogen atoms and one carboxylate oxygen atom coordinate as the in-plane ligands of the copper, which is in an approximately tetragonally-distorted octahedral geometry. Modeling of the copper-peptides using the consistent force field (CFF91) produces a minimum energy configuration with three histidines and one water molecule as the copper ligands. CD, EPR and fluorescence results are reported for lysyl oxidase and compared with results for the peptides.     

Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.     

The Fox1 ferroxidase of Chlamydomonas reinhardtii: a new multicopper oxidase structural paradigm

Multicopper oxidases (MCO) contain at least four copper atoms arrayed in three distinct ligand fields supported by two canonical structural features: (1) multiples of the cupredoxin fold and (2) four unique sequence elements that include the ten histidine and one cysteine ligands to the four copper atoms. Ferroxidases are a subfamily of MCO proteins that contain residues supporting a specific reactivity towards ferrous iron; these MCOs play a vital role in iron metabolism in bacteria, algae, fungi, and mammals. In contrast to the fungal ferroxidases, e.g., Fet3p from Saccharomyces cerevisiae, the mammalian ceruloplasmin (Cp) is twice as large (six vs. three cupredoxin domains) and contains three type 1, or “blue,” copper sites. Chlamydomonas reinhardtii expresses a putative ferroxidase, Fox1, which has sequence similarity to human Cp (hCp). Eschewing the standard sequence-based modeling paradigm, we have constructed a function-based model of the Fox1 protein which replicates hCp’s six copper-site ligand arrays with an overall root mean square deviation of 1.4 Å. Analysis of this model has led also to assignment of motifs in Fox1 that are unique to ferroxidases, the strongest evidence to date that the well-characterized fungal high-affinity iron uptake system is essential to iron homeostasis in green algae. The model of Fox1 also establishes a subfamily of MCO proteins with a noncanonical copper-ligand organization. These diverse structures suggest alternative mechanisms for intramolecular electron transfer and require a new trajectory for the evolution of the MCO superfamily.     

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.     

Role of copper in thermal stability of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six domains with one type-1 copper in each of domains 2, 4, and 6; the remaining coppers form a catalytic trinuclear cluster at the interface between domains 1 and 6. To assess the role of the coppers in CP thermal stability, we have probed the thermal unfolding process as a function of scan rate of holo- and apo-forms using several detection methods (circular dichroism, aromatic and 8-anilino-naphthalene-1-sulfonic acid fluorescence, visible absorption, activity, and differential scanning calorimetry). Both species of CP undergo irreversible thermal reactions to denatured states with significant residual structure. For identical scan rates, the thermal midpoint appears at temperatures 15-20° higher for the holo- as compared with the apo- form. The thermal data for both forms were fit by a mechanistic model involving two consecutive, irreversible steps (N → I → D). The holo-intermediate, I, has lost one oxidized type-1 copper and secondary structure in at least one domain; however, the trinuclear copper cluster remains intact as it is functional in oxidase activity. The activation parameters obtained from the fits to the thermal transitions were used to assess the kinetic stability of apo- and holo-CP at physiological temperatures (i.e., at 37°C). It emerges that native CP (i.e., with six coppers) is rather unstable and converts to I in <1 day at 37°C. Nonetheless, this form remains intact for more than 2 weeks and may thus be a biologically relevant state of CP in vivo. In contrast, apo-CP unfolds rapidly: the denatured state is reached in <2 days at 37°C.     

Spectroscopic analysis of the trinuclear cluster in the Fet3 protein from yeast, a multinuclear copper oxidase

The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.     

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.     

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.     

Discrete roles of copper ions in chemical unfolding of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six beta-barrel domains with one type 1 copper in each of domains 2, 4, and 6; the remaining copper ions form a catalytic trinuclear cluster, one type 2 and two type 3 coppers, at the interface between domains 1 and 6. We have characterized urea-induced unfolding of holo- and apo-forms of CP by far-UV circular dichroism, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid binding, visible absorption, copper content, and oxidase activity probes (pH 7, 23 degrees C). We find that holo-CP unfolds in a complex reaction with at least one intermediate. The formation of the intermediate correlates with decreased secondary structure, exposure of aromatics, loss of two coppers, and reduced oxidase activity; this step is reversible, indicating that the trinuclear cluster remains intact. Further additions of urea trigger complete protein unfolding and loss of all coppers. Attempts to refold this species result in an inactive apoprotein with molten-globule characteristics. The apo-form of CP also unfolds in a multistep reaction, albeit the intermediate appears at a slightly lower urea concentration. Again, correct refolding is possible from the intermediate but not the unfolded state. Our study demonstrates that in vitro equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages. When the catalytic site is finally destroyed, refolding is not possible at neutral pH. This implies a mechanistic role for the trinuclear metal cluster as a nucleation point, aligning domains 1 and 6, during CP folding in vivo.     

Structural models of the redox centres in cytochrome oxidase.

Evolutionary conservation, predicted membrane topography of the subunits, and known chemical and physical properties of the catalytic metals in cytochrome oxidase provided the basis for plausible structural models of the enzyme's redox centres. Subunit II probably binds one of the copper ions (CuA) whilst subunit I is likely to bind the two haems (a and a3) and the other redox-active copper (CuB). Two cysteine and two histidine residues of subunit II are the likely ligands of CuA, forming a centre that may be structurally similar to that in azurin. The two haems may be sandwiched between two transmembranous segments of subunit I, one of which also provides a histidine ligand to CuB. A third segment may provide two more histidine ligands to the latter. The model was constructed with a 4 A Fe-Cu distance in the binuclear haem a3-CuB centre, and a 14 A distance between the haem irons. The subunit I model involves only three transmembranous helices which bind three catalytic metal groups. The fit of this model to several known physicochemical properties of the redox centres is analysed.