Multicopper oxidases: a workshop on copper coordination chemistry, electron transfer, and metallophysiology

Multicopper oxidases (MCOs) are unique among copper proteins in that they contain at least one each of the three types of biologic copper sites, type 1, type 2, and the binuclear type 3. MCOs are descended from the family of small blue copper proteins (cupredoxins) that likely arose as a complement to the heme-iron-based cytochromes involved in electron transport; this event corresponded to the aerobiosis of the biosphere that resulted in the conversion of Fe(II) to Fe(III) as the predominant redox state of this essential metal and the solubilization of copper from Cu₂S to Cu(H₂O) _n ²⁺. MCOs are encoded in genomes in all three kingdoms and play essential roles in the physiology of essentially all aerobes. With four redox-active copper centers, MCOs share with terminal copper-heme oxidases the ability to catalyze the four-electron reduction of O₂ to two molecules of water. The electron transfers associated with this reaction are both outer and inner sphere in nature and their mechanisms have been fairly well established. A subset of MCO proteins exhibit specificity for Fe²⁺, Cu⁺, and/or Mn²⁺ as reducing substrates and have been designated as metallooxidases. These enzymes, in particular the ferroxidases found in all fungi and metazoans, play critical roles in the metal metabolism of the expressing organism.     

Biomimetic oxidation of glutathione by synthetic analogues of the active centres of ‘blue’ copper proteins

The model process of oxidation of reduced glutathione through chelate copper complexes has been studied, the former being structural analogues of the active centers of ‘blue’ copper proteins. Glutathione forms the relatively stable intermediate CuLSG⁺ with copper complexes in acetonitrile. The intramolecular electron transfer S(glutathione)→Cu(II) is the rate-determining step of the substrate oxidation. On the basis of rate constant (k_obs) values as well as activation energy (E³) values, we have concluded that there is a possibility of functional modelling of active centers of type 1 Cu by copper complexes with thioaza ligands.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

CuA centers and their biosynthetic models in azurin

Cu_A is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)···Cu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper–copper bond, the first reported of its kind in Nature. Such a “diamond-core” Cu₂S(Cys)₂ structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the Cu_A center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the Cu_A center in four different systems—native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands—with a greater emphasis on biosynthetic models of Cu_A, especially on new, deeper insights gained from their studies.     

Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins.

Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Intramolecular electron transfer rate between active-site copper and TPQ in Arthrobacter globiformis amine oxidase

Copper amine oxidases catalyze the oxidative deamination of primary amines operating through a ping-pong bi bi mechanism, divided into reductive and oxidative half-reactions. Considerable debate still exists regarding the role of copper in the oxidative half-reaction, where O₂ is reduced to H₂O₂. Substrate-reduced amine oxidases display an equilibrium between a Cu(II) aminoquinol and a Cu(I) semiquinone, with the magnitude of the equilibrium constant being dependent upon the enzyme source. The initial electron transfer to dioxygen has been proposed to occur from either the reduced Cu(I) center or the reduced aminoquinol cofactor. In order for Cu(I) to be involved, it must be shown that the rate of electron transfer (k _ET) between the aminoquinol and Cu(II) is sufficiently rapid to place the Cu(I) semiquinone moiety on the mechanistic pathway. To further explore this issue, we measured the intramolecular electron transfer rate for the Cu(II) aminoquinol ⇆ Cu(I) semiquinone equilibrium in Arthrobacter globiformis amine oxidase (AGAO) by temperature-jump relaxation techniques. The results presented herein establish that k _ET is greater than the rate of catalysis (k _cat) for the preferred amine substrate β-phenylethylamine at three pH values, thereby permitting the Cu(I) semiquinone to be a viable catalytic intermediate during enzymatic reoxidation in this enzyme. The data show that k _ET is approximately equivalent at pH 6.2 and 7.2, being 2.5 times k _cat for these pH values. At pH 8.2, however, k _ET decreases, becoming comparable to k _cat. Potential reasons for the decreased k _ET at basic pH are presented. The implications of these results in light of a previously published study measuring reoxidation rates of substrate-reduced AGAO are also addressed.     

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQ_amr) that is in equilibrium with a semiquinone radical (TPQ_sq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQ_sq in an “on-copper” conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the “off-copper” conformation of TPQ_amr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQ_amr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQ_amr/TPQ_sq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.     

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.     

A fresh view of the cell biology of copper in enterobacteria

Copper ions are essential but also very toxic. Copper resistance in bacteria is based on export of the toxic ion, oxidation from Cu(I) to Cu(II), and sequestration by copper‐binding metal chaperones, which deliver copper ions to efflux systems or metal‐binding sites of copper‐requiring proteins. In their publication in this issue, Osman et al. ( 2013 ) demonstrate how tightly copper resistance, homeostasis and delivery pathways are interwoven in Salmonella enterica sv. Typhimurium. Copper is transported from the cytoplasm by the two P‐type ATPases CopA and GolT to the periplasm and transferred to SodCII by CueP, a periplasmic copper chaperone. When copper levels are higher, SodCII is also able to bind copper without the help of CueP. This scheme raises the question as to why copper ions present in the growth medium have to make the detour through the cytoplasm. The data presented in the publication by Osman et al. ( 2013 ) change our view of the cell biology of copper in enterobacteria.     

Hemoglobin and myoglobin as reducing agents in biological systems. Redox reactions of globins with copper and iron salts and complexes

In addition to reversible O₂ binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO₂ and MbO₂ are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E ₀, 200-600 mV) and high stability constants, such as [Cu(phen)₂]²⁺, [Cu(dmphen)₂]²⁺, and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu²⁺, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E ₀≤ 100-150 mV) react with HbO₂ and MbO₂ through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO₂ and MbO₂ by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PP_i proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO₂ with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO₂ and cytochrome b ₅ via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO₂ and MbO₂ transformation into met-forms in the presence of small amounts of metal ions or complexes (catalysis), which, until recently, had been demonstrated only for copper compounds with intermediate redox potentials. The main contribution to the reaction rate comes from copper binding to the “inner” histidines, His97 (0.66 nm from the heme) that forms a hydrogen bond with the heme propionate COO^– group, and the distal His64. The affinity of both histidines for copper is much lower than that of the surface histidines residues, and they are inaccessible for modification with chemical reagents. However, it was found recently that the high-potential Fe(3) complex, potassium ferricyanide (400 mV), at a 5 to 20% of molar protein concentration can be an efficient catalyst of MbO₂ oxidation into metMb. The catalytic process includes binding of ferrocyanide anion in the region of the His119 residue due to the presence there of a large positive local electrostatic potential and existence of a “pocket” formed by Lys16, Ala19, Asp20, and Arg118 that is sufficient to accommodate [Fe(CN)₆]^4–. Fast, proton-assisted reoxidation of the bound ferrocyanide by oxygen (which is required for completion of the catalytic cycle), unlike slow [Fe(CN)₆]^4– oxidation in solution, is provided by the optimal location of neighboring protonated His113 and His116, as it occurs in the enzyme active site.     

LccA,an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 ± 10 mU·ml⁻¹) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U·mg⁻¹ with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO₄. Optimal oxidation of ABTS and SGZ was at 45°C and pH 6 and pH 8.4, respectively. The apparent K_m values for SGZ, bilirubin, and ABTS were 35, 236, and 670 μM, with corresponding k_cat values of 22, 29, and 10 s⁻¹, respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50°C of 31.5 h.Multicopper oxidases (MCOs) are a family of enzymes that include laccases (p-diphenol: dioxygen oxidoreductases; EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), ferroxidases (EC 1.16.3.1), bilirubin oxidases (EC 1.3.3.5), and other enzyme subfamilies (27, 65). MCOs couple the oxidation of organic and/or inorganic substrates to the four-electron reduction of molecular oxygen to water. These enzymes often have four Cu atoms classified into type 1 (T1), type 2 (T2), and type 3 (T3) centers, in which a mononuclear T1 center on the surface of the enzyme provides long-range intramolecular one-electron transfer from electron-donating substrates to an internal trinuclear T2-T3 center formed by a T2 Cu coordinated with a T3 Cu pair. The T2-T3 cluster subsequently reduces dioxygen to water.Enzymes of the laccase subfamily oxidize a broad range of compounds, including phenols, polyphenols, aromatic amines, and nonphenolic substrates, by one-electron transfer to molecular oxygen and thus have a wide variety of applications from biofuels to human health. The best-known application is the use of a laccase from the lacquer tree Rhus vernicifera in paint and adhesives for more than 6,000 years in East Asia (29). Laccases have also been used in the delignification of pulp, bleaching of textiles and carcinogenic dyes, detoxification of water and soils, removal of phenolics from wines, improving adhesive properties of lignocellulosic products, determination of bilirubin levels in serum, and transformation of antibiotics and steroids (60). In addition, laccases have demonstrated potential for use in biosensors, bioreactors, and biofuel cells (61).Laccases, once thought to be restricted to eukaryotes (fungi, plants, and insects), appear to be widespread in bacteria (10). Laccase-like MCOs are now known to have numerous biological roles in bacteria, including sporulation, electron transport, pigmentation, metal (copper, iron, and manganese) homeostasis, oxidation of phenolate-siderophores, phenoxazinone synthesis, cell division, and morphogenesis (9). In contrast to the widespread occurrence of laccases in bacteria and eukaryotes, only a few MCOs have been identified in archaea, and this is based only on genome sequences (e.g., the hyperthermophilic crenarchaeote Pyrobaculum aerophilum and the halophilic euryarchaeotes Haloferax volcanii and Halorubrum lacusprofundi). Most archaea with sequenced genomes, however, are anaerobes. Since MCOs reduce molecular oxygen to water, this likely accounts for the limited number of MCOs among archaea.Many archaea thrive under harsh environmental conditions, including high temperature, extreme pH, and/or low water activity. Thus, they have many biochemical and physiological properties that are ideal for industrial applications. Here, we report the identification of a highly thermostable and salt/solvent-tolerant laccase (LccA) from the halophilic archaeon H. volcanii that catalyzed the oxidation of a wide variety of phenolic compounds. LccA was readily secreted and purified from the culture broth as a blue multicopper oxidase that was glycosylated and processed by the removal of 31 amino acid residues from its N terminus.     

Copper Proteins and Oxygen : Correlations between structure and function of the copper oxidases

A comprehensive survey of the interaction of the copper proteins and oxygen is presented including a correlation of structure, function, and other properties of the known copper oxidases and of hemocyanin. The origin of their blue color and the structure of copper complexes and copper proteins are related to the oxidation state of copper ion and relevant electronic transitions probably arising from the formation of charge transfer complexes. The oxygen reactions of hemocyanin, ceruloplasmin, and cytochrome oxidase show half-saturation values far below the other Cu enzymes. The formation of hydrogen peroxide as a reaction product is associated with the presence of one Cu atom per oxidase molecule or catalytic system. Water is the corresponding product of the other Cu oxidases with four or more Cu atoms per molecule, except for monoamine oxidase. Mechanisms for the oxidase action of the two and four electron transfer Cu oxidases and tyrosinase are proposed. These reactions account for the number, the oxidation-reduction potential, and the oxidation state of Cu in the resting enzyme, the cyclical change from Cu(II) to Cu(I), the diatomic nature of O₂, the sequence of the oxidation and reduction reactions, and other salient features. The catalytic reactions involved in the oxidation of ascorbic acid by plant ascorbate oxidase, ceruloplasmin, and Cu(II) are compared. Finally the substrate specificity, inhibitory control, and the detailed mechanism of the oxidase activity of ceruloplasmin are summarized.     

NMR study of the interaction of plastocyanin with chromium(III) analogues of inorganic electron transfer reagents

High resolution nuclear magnetic resonance spectroscopy has been used to examine the interaction of plastocyanins from French bean (Phaseolus vulgaris) and cucumber (Cucumis sativus) with three complexes—potassium hexacyanochromate(III), hexamminechromium(III) nitrate and tris(1,10-phenanthroline)-chromium(III) perchlorate—which are analogues of inorganic electron transfer reagents. The results indicate a high degree of specificity in the binding of these complexes and two binding sites on the protein are identified. One binding site is situated close to the copper atom and is clearly suited to outer sphere electron transfer through one of the histidine ligands. The other binding site is more distant from the copper atom and this mechanism cannot be operative. Electron transfer via hydrophobic channels or electron tunneling are possible mechanisms of electron transfer.     

Real-time detection of Cu2+ sequestration and release by immobilized apo-metallothioneins using SECM combined with SPR

Scanning electrochemical microscopy (SECM) combined with surface plasmon resonance (SPR), SECM-SPR, was applied for real-time detection of the incorporation of Cu(2+) by apo-metallothionein (apo-MT) immobilized on the SPR substrate and release of Cu(2+) from surface-confined metallothionein (MT). Cu(2+) anodically stripped from a Cu-coated SECM Au tip was sequestered by apo-MT upon its diffusion to the SPR substrate, and release of Cu(2+) by MT was accomplished by generating protons via oxidation of hydroquinone at the tip. The high sensitivity of the SPR instrument is capable of following the structural and compositional changes of MT molecules during the metal sequestration and release processes. Due to the enhanced mass transfer rate at the SECM tip, the complication of mass transfer limitation on kinetic measurements, commonly encountered in flow injection SPR, is circumvented. The time-resolved SPR response reveals stepwise changes among three stable MT structures and allows the number of copper ions coordinated in each structure to be determined. The numbers of copper ions incorporated by each MT molecule in the three structures were determined to be 5, 9, and 12. This work expands the SECM-SPR approach to assessments of the dynamics and affinity of binding of small ions to surface-confined proteins and to studies of proteins that do not undergo facile electron transfer reactions.     

The essential role of the Cu(II) state of Sco in the maturation of the Cu<Subscript>A</Subscript> center of cytochrome oxidase: evidence from H135Met and H135SeM variants of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Sco

Sco is a red copper protein that plays an essential yet poorly understood role in the metalation of the Cu_A center of cytochrome oxidase, and is stable in both the Cu(I) and Cu(II) forms. To determine which oxidation state is important for function, we constructed His135 to Met or selenomethionine (SeM) variants that were designed to stabilize the Cu(I) over the Cu(II) state. H135M was unable to complement a scoΔ strain of Bacillus subtilis, indicating that the His to Met substitution abrogated cytochrome oxidase maturation. The Cu(I) binding affinities of H135M and H135SeM were comparable to that of the WT and 100-fold tighter than that of the H135A variant. The coordination chemistry of the H135M and H135SeM variants was studied by UV/vis, EPR, and XAS spectroscopy in both the Cu(I) and the Cu(II) forms. Both oxidation states bound copper via the S atoms of C45, C49 and M135. In particular, EXAFS data collected at both the Cu and the Se edges of the H135SeM derivative provided unambiguous evidence for selenomethionine coordination. Whereas the coordination chemistry and copper binding affinity of the Cu(I) state closely resembled that of the WT protein, the Cu(II) state was unstable, undergoing autoreduction to Cu(I). H135M also reacted faster with H₂O₂ than WT Sco. These data, when coupled with the complete elimination of function in the H135M variant, imply that the Cu(I) state cannot be the sole determinant of function; the Cu(II) state must be involved in function at some stage of the reaction cycle.     

Pathways of Proton Transfer in Cytochrome c Oxidase

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a ₃, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O₂ reduction.     

Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.     

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.