CuB Site Research Articles

Conformation-dependent Post-translational Glycosylation of Tyrosinase: REQUIREMENT OF A SPECIFIC INTERACTION INVOLVING THE CuB METAL BINDING SITE

Tyrosinase, the rate-limiting enzyme in mammalian melanogenesis, is a copper-containing transmembrane glycoprotein. Tyrosinase undergoes a complex post-translational processing before reaching the melanosomal membrane. This processing involves N-glycosylation in several sites, including one located in the CuB copper binding site, movement from the endoplasmic reticulum (ER) to the Golgi, copper binding, and sorting to the melanosome. Aberrant processing is causally related to the depigmented phenotype of human melanomas. Moreover, some forms of albinism and several other pigmentary syndromes are considered ER retention diseases or trafficking defects. A critical step in tyrosinase maturation is the acquisition of an ER export-competent conformation recognized positively by the ER quality control system. However, the minimal structural requirements allowing exit from the ER to the Golgi have not yet been identified for tyrosinase or other melanosomal proteins. We addressed this question by analyzing the enzymatic activity and glycosylation pattern of mouse tyrosinase point mutants and chimeric constructs, where selected portions of tyrosinase were replaced by the homologous fragments of the highly similar tyrosinase-related protein 1. We show that a completely inactive tyrosinase point mutant lacking a critical histidine residue involved in copper binding is nevertheless able to exit from the ER and undergo further processing. Moreover, we demonstrate that tyrosinase displays at least two sites whose glycosylation is post-translational and most likely conformation-dependent and that a highly specific interaction involving the CuB site is essential not only for correct glycosylation but also for exit from the ER and enzymatic activity.

Activation volumes for intramolecular electron transfer in Escherichia coli cytochrome bo3

In this report we describe the activation volumes associated with the heme–heme electron transfer (ET) and CO rebinding to the binuclear center subsequent to photolysis of the CO-mixed-valence derivative of Escherichia coli cytochrome bo3 (Cbo). The activation volumes associated with the heme–heme ET (k=1.2×105 s−1), and CO rebinding (k=57 s−1) are found to be +27.4 ml/mol and −2.6 ml/mol, respectively. The activation volume associated with the rebinding of CO is consistent with previous Cu X-ray absorption studies of Cbo where a structural change was observed at the CuB site (loss of a histidine ligand) due to a change in the redox state of the binuclear center. In addition, the volume of activation for the heme–heme ET was found to be quite distinct from the activation volumes obtained for heme–heme ET in bovine heart Cytochrome c oxidase. Differences in mechanisms/pathways for heme b/heme o3 and heme a/heme a3 ET are suggested based on the associated activation volumes and previously obtained Marcus parameters.

Structural model for the Cu(B) site of dopamine beta-hydroxylase: crystal structure of a copper(II) complex showing N(3)OS coordination with an axial sulfur ligation.

A copper(II) complex containing a NSO-donor Schiff base and NN-donor 2,2‘-bipyridine has been prepared and structurally characterized. The square pyramidal complex with an axial sulfur ligation is a structural model for the CuB site of dopamine β-hydroxylase in its oxidized form. The copper(II) complex is catalytically active in the oxidation of ascorbic acid by dioxygen mediated by a copper(I) species which is proposed to have a four-coordinate structure with a N3S coordination geometry.

Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.

Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.

Structural model for the CuB site of dopamine β-hydroxylase and peptidylglycine α-hydroxylating monooxygenase: crystal structure of a copper(ii) complex showing N3OS coordination and axial sulfur ligation

A copper(II) complex [Cu(L)(phen)](ClO4) (HL, NSO-donor Schiff base ligand) with a CuIIN3OS geometry showing axial sulfur ligation is a structural model for the CuB site of DβH and PHM, and the complex is catalytically active in the oxidation of ascorbic acid by dioxygen mediated by a copper(I) species.

New imidazole-based tripodal ligands as Cu(B) site mimics of cytochrome C oxidase.

New imidazole-based tripodal ligands as Cu(B) site mimics of cytochrome C oxidase.

Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.

Volume Changes Associated with CO Photolysis from Fully Reduced Bovine Heart Cytochrome aa3

Volume changes associated with CO photodissociation from fully reduced cytochrome aa3 have been determined using photoacoustic calorimetry (PAC). Analysis of PAC data reveals two intermediate steps in the photodissociation reaction with volume changes of +6.8 and +6.9 mL/mol occurring on time scales of <50 ns and ∼1.5 μs, respectively (T = 22 °C). The value of ΔV for the fast process can be attributed to conformational dynamics associated with photoinduced transfer of CO from the high-potential heme a3 to the high-potential CuB site, while the slower phase reaction occurs on a time scale consistent with the thermal dissociation of CO from CuB. It is speculated that these volume changes may be linked to movement of Glu-242 as part of a proton shuttle mechanism in heme/copper oxidases.

Roles of Copper Ligands in the Activation and Secretion ofStreptomyces Tyrosinase

The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as CuA and CuB) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1.MelC2 binary complex function, six single substitution mutations were introduced into the CuA and CuB sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1. MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion.

Copperdioxygen complexes: Functional models for proteins

Abstract

Mutational Analysis of Copper Binding by Human Tyrosinase

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes several reactions in the biosynthesis of melanin pigments and is deficient in patients with type I oculocutaneous albinism (OCA1). Tyrosinase is thought to bind two copper ions, one at each of two conserved sequence motifs, termed CuA and CuB, but to date this has been directly proved only for the Neurospora and mushroom enzyme. Here, we demonstrate that mammalian tyrosinase directly binds copper, and that the CuA and CuB sites are both required for copper binding and for catalytic activity. We show that in human tyrosinase, copper binding by the CuB site is most likely coordinated by residues His363, His367, and His389, and that copper binding may be cooperative, with copper binding at one site facilitating copper binding by the other site. Furthermore, correct folding of the tyrosinase polypeptide appears to be necessary for copper binding, and a number of human OCA1 mutations disrupt copper binding and thus catalytic function of tyrosinase.

The Archaeal SoxABCD Complex Is a Proton Pump in Sulfolobus acidocaldarius

The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lübben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a587 and aa3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme CuB site. Standard midpoint potentials of the cytochrome a587 heme centers were determined as +210 and +270 mV, respectively. The maximum turnover of the complex (1300 s-1 at 65 degrees C) was found to be about three times greater than that of the previously studied isolated cytochrome aa3 subunit alone (Gleissner, M., Elferink, M. G., Driessen, A. J., Konings, W. N., Anemüller, S., and Schäfer, G. (1994) Eur. J. Biochem. 224, 983-990). With N,N,N',N'-tetramethyl-1,4-phenylenediamine as a reductant, the SoxABCD complex reconstituted into liposomes generates a proton motive force. A new method is described by co-reconstitution of SoxABCD with a Sulfolobus Rieske FeS-protein (SoxL), allowing energization by cytochrome c. It is based on the finding that this Rieske protein can equilibrate electrons between cytochrome c and quinones reversibly (Schmidt, C. L., Anemüller, S., Teixeira, M., and Schäfer, G. (1995) FEBS Lett. 359, 239-243). With this system, generating no scalar protons, the stoichiometry of proton translocation could be determined. A net H+/e- ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa3 moiety of the complex does not contain the signature of a H+ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H+ translocation, a mechanism different from that in typical heme-copper oxidases of the aa3 or bo3 type is discussed.

A new EPR signal from cytochrome oxidase--evidence for conformational flexibility at the CuB site?

A new EPR signal from cytochrome oxidase--evidence for conformational flexibility at the CuB site?

Structure of CuB in the Binuclear Heme-Copper Center of the Cytochrome aa3-Type Quinol Oxidase from Bacillus subtilis: An ENDOR and EXAFS Study

We have studied the structure of the CuB site in the binuclear heme-copper center of the fully oxidized form of the quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy. This enzyme is member of the large superfamily of heme-copper respiratory oxidases, which catalyze the reduction of dioxygen to water and link it to translocation of protons across the bacterial or mitochondrial membrane. The EXAFS of the CuB site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands. There are some indications that a Cl- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatters, indicative of a Cl- (or S) bridge between the heme iron and CuB [cf. Powers, L., et al. (1994) Biochim. Biophys. Acta 1183, 504-512]. Proton ENDOR spectroscopy of the CuB site in 1H2O and 2H2O media showed evidence of an oxygenous copper ligand with an exchangeable proton. 14N ENDOR revealed three inequivalent nitrogenous ligands with hyperfine coupling constants consistent with histidines. Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal CuB site with three histidine nitrogens and one oxygen as ligands, the latter with an exchangeable proton(s). The identity and assignment of these ligands are discussed.

Cyanide and carbon monoxide binding to the reduced form of cytochrome bo from Escherichia coli.

Cyanide binds to fully reduced cytochrome bo and induces a blue shift of the Soret absorption band of the high-spin heme o and a change in the visible region spectrum consistent with the expected conversion to a low-spin state. The dissociation constant, determined by titration of the extent of the binding spectrum, is 7.0 +/- 0.6 mM at pH 7.0. In contrast, cyanide does not bind significantly in this concentration range to the reduced form of cytochrome bd. The reduced cyanide compound of cytochrome bo can be laser photolyzed. Typically, less than 20% photolysis was attained with conditions that give essentially full photolysis of the carbon monoxide compound. The subsequent monophasic kinetics of recombination of cyanide at varying cyanide concentrations were used to determine kon, koff, and dissociation constant values at pH 7.0 of 572 +/- 43 M-1 s-1, 4.2 +/- 0.7 s-1, and 7.3 +/- 1.3 mM, respectively. The dissociation constant changes very little in the pH range 6-8, indicating that a proton is bound together with the cyanide anion, as predicted by our recent proposal of a requirement for electroneutrality in the binuclear center [Mitchell, R., & Rich, P. R. (1994) Biochim. Biophys. Acta 1186, 19-26]. Competition studies confirm that cyanide and carbon monoxide cannot bind simultaneously, so that their binding sites must overlap. A small fraction of the reduced unliganded enzyme appears to have a distinct photolysis spectrum in the absence of added ligands, and this is transformed into a typical ferrous cyanide compound only at very high cyanide concentrations. Cyanide binding and photolysis were also examined in a number of mutant forms of cytochrome bo, and in a wild-type form which was partially depleted in CuB. Dramatic changes in rate constants and binding constants were found in several cases. Data from several mutants were compared with analogous data on the binding and photolysis of carbon monoxide, and the effects of mutation were quite different with the two ligands. A model is developed to explain the observed effects of point mutations on the recombination kinetics of both carbon monoxide and cyanide. Overall, the results indicate that the CuB site is required for binding of cyanide, but not carbon monoxide, to the reduced enzyme, possibly by providing the site for binding of the associated proton.

Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand.

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

Magnetization of fast and slow oxidized cytochrome c oxidase.

Oxidized cytochrome c oxidase can now be prepared either to react rapidly (fast) or to react slowly (slow) with cyanide [Baker, G.M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Slow oxidized cytochrome c oxidase is also characterized by an integer spin g = 12 EPR signal which is absent in the fast form. The saturation magnetization of two samples of both forms of cytochrome oxidase has been studied at four applied magnetic fields (0.3125, 1.25, 2.5, and 5.0 T) over a temperature range from 2 to 200 K using a superconducting susceptometer. The saturation magnetization data of the two preparations are readily distinguished. The data for the coupled cytochrome a3: CuB site of both preparations are most simply interpreted as exhibiting S = 2 paramagnetism with D = +13 cm-1 for the fast and D = -7 cm-1 for the slow forms, respectively. However, there is some indication that the fast form is a mixture of both S = 2 and S = 1 paramagnetic species while the slow form is only S = 2.

Infrared evidence of azide binding to iron, copper, and non-metal sites in heart cytochrome c oxidase.

Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch band at 2040, 2016, and 2004 cm-1 for CcO(III)N3 and two at 2040 and 2016 cm-1 for CcO(II)N3 and CcO(I)N3. Resting CcO(IV) reacts with N3- to give one band at 2041 cm-1 assigned to CuB2+N3 and another at 2051 cm-1 to N3- that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N3- binding to CuB as well as Fea3 in partially reduced enzyme, but no binding to Fea3 in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fea or CuA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fea3 or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Fea3 and CuB sites provides further support for Fea3(3)+ OOH, rather than Fea3(3)+ OOCuB2+, as an intermediate in the reduction of O2 to water by the oxidase.

Could CuB be the site of redox linkage in cytochrome c oxidase?

This paper explores the proton pumping function of cytochrome c oxidase [ferrocytochrome-c:oxygen oxidoreductase (EC 1.9.3.1)] based upon redox linkage at the "high-potential" CuB center. A model is proposed that is derived from a redox-linked ligand exchange mechanism previously described for the CuA site. Qualitative analysis of this mechanism indicates that such a mechanism is feasible. However, the relatively short distance between CuB and cytochrome a3 implies that the uncoupling electron transfers are quite facile. In addition, the position of the CuB center with respect to the inner mitochondrial membrane argues against redox linkage at the CuB site.

The structure of molluscan haemocyanins and their homology with tyrosinases

AbstractHaemocyanins, proteins with binuclear copper sites, are dioxygen carriers in four classes of molluscs: gastropods, cephalopods, polyplacophorans (the chitons), and bivalves (in the latter class only in a limited number of primitive species). They show typical quaternary structures and dissociation patterns in the analytical ultracentrifuge and in the electron microscope.The subunit (Mr 450 000) of the βC‐haemocyanin of the gastropod Helix pomatia is composed of eight functional units, which have been separated by chromatography after limited proteolysis. The two types of subunits (Mr 450 000) of the αD‐ and αN‐Hc components are also constituted of eight functional units like the subunit (Mr 390 000) of the haemocyanin of the cephalopod Sepia. The subunit (Mr 350 000) of the haemocyanin of the cephalopod Octopus is composed of only seven functional units.The amino‐acid sequences of a functional unit of Helix and of Octopus haemocyanin show a homology with the CUB site of arthropodan haemocyanins near the ligands of copper, but not with their CUA site. There is a striking homology near the potential copper ligands for both the CUA, and the CUB sites of molluscan haemocyanins with those of tyrosinases from the bacterium Streptomyces to man.