The Bioinorganic Chemistry of Copper

Indian Journal of Chemislry Vol. 42A, September 2003, pp. 2175-2184

The bioinorganic chemistry of copper

R N Mukheljee

Department of Chemistry, lndian In stitute of Technology. Kanpur 208016, India Reccivcd 2 1mlumy 2003

Many enzymes and proteins have copper at their active sites, which plays a key role in biology. An important goal of bioinorganic chemistry is the development of small in organic coordination complexes that reproduce structural, spectroscopic features and functiollal aspects in a manner similar to th eir natural counterparts. To provide an overview of the activities in this field. some results Oil synthetic modelling of a selected number of copper proteins/enzymes are described in this article.

Copper is one of the transItIon elements frequently Correlated with the enzymatic acrivity, the copper found at the active site of proteins. The copper­ proteins exhibit unique spectroscopic properties and, conta111mg enzymes and proteins constitute an accordingly, the proteins are divided in mainly three important class of biologically active compounds. The types. biological functions of copper protei ns/enzymes Type I copper proteins (also called "blue" copper include electron transfer. dioxygcn transport, proteins) are known to have one copper ion in the oxygenation. oxidation, reduction and active site. This copper ion shows some remarkable disproportionation 1.2 . spectroscopic features: an intense absorption around In nature, a variety of copper proteins are essential 600 nm, with an extinction coefficient of about 3000 constituents of aerobic organisms, including ~I em-I. Another characteristic feature of the Type I hemocyanins (arthropodal and molluskan O2 carriers) copper proteins is the extremely small hyperfine 4 I and enzymes that "activate" O2, promoting oxygen splitting in the EPR spectra (A II"" 40-90 x 10- cm- ). atom incorporation into biological sllbstrates. The Type II copper proteins have no distinct unique latter include tyrosinase (a monooxygenase, prop~rties . The spectroscopic data of these proteins incorporating one oxygen atom to the substrate and are comparable to those of "normai" copper reducing th e other to water) and dopamine ~­ compounds. hydroxylase (a mOllooxygenase). "Blue" multicopper Type III copper proteins contain oxidases [e .g., laccase (phenol and diamine antiferromagnetically coupled copper dimers. These oxidation), ascorbate oxidase (oxidation of [­ proteins are diamagnetic and therefore are EPR silent. ascorbate) and ceruloplasmin] promote substrate one­ In some proteins, all three types of copper sites are electron oxidation while reducing O2 to water. present. Such proteins were proposed to classify as Ceruloplasmin may be involved in copper Type IV. In ascorbate oxidase one of the copper ions metabolism. Cytochrome c oxidase transduce energy is found in a distorted tetrahedral (trigonal pyramidal) from the same 4e-/4H+ reduction of O2 occurring at a coordination with two histidines, a methionine and a heme-Cu binuclear centre, and couple this to cysteine. This resembles the active site of the blue membrane proton translocation, utilized in ATP copper protein plastocyanin. Also, a trinuclear copper synthesis. Amine oxidases and galactose oxidase site was found consisting of a Type III copper pair effect amine -7 aldehyde oxidative deaminations and and a "normal" Type II copper ion. alcohol -7 aldehyde oxidative dehydrogenations, Reactions that copper proteins carry out have long respectively. Copper ion reactions with reduced interested inorganic chemists. Copper is an important dioxygen derivatives (e.g., sLlperoxide (02'), hydrogen element in oxidation catalysts for laboratory and peroxide) are essential in Cu-Zn superoxide industrial use. Interest in the copper-dioxygen dismutase, and may be involved in copper-mediated complexes stems from the diverse occurrence of oxidative damage in biological media, including copper proteins which function as highly efficient possibly in Alzheimer's disease. biooxidation catalysts. Copper-dioxygen adducts are 2176 INDIAN J CHEM, SEC A, SEPTEMBER 2003

suggested as key reaction intermediates in these compound. However, when the structure of the biosite enzymatic reactions. The differentiation in the is known, then the complex that it reproduces has, as function of these proteins is attributed primarily to the far as possible, the known structure. A different coordination structure of the copper-dioxygen emphasis is obtained when the action of the metal in intermediate formed in the protein matrices, the protein is reproduced by a model compound and depending on the ligand donors, the geometry, and the the mechanism of a particular reaction is elucidated or coordination mode of the dioxygen. However, the partially explained. correlation between these structural factors and the The purpose of models is not necessarily to function/catalysis of the enzymes remains to be duplicate natural properties but to sharpen or focus elucidated. certain questions. The goal is to elucidate fundamental aspects of structure, spectroscopy, magnetic and electronic structure, reactivity and Importance of inorganic model chemistry chemical mechanism. A synergistic approach to the Investigations of metallobiomolecules have study of metalloenzymes can and has yielded crucial increased markedly during the last two decades. High­ information because synthetic analogues can be used resolution X-ray crystallographic results, in particular, to investigate the effects of systematic variations in have facilitated detailed considerations of structural, coordination chemistry, ligation, local environment electronic and reactivity properties at the molecular and other factors, often providing insights that cannot level. These metallobiomolecules are highly be easily attained from protein studies (Fig. 1). elaborated coordination complexes whose metal­ Reproducing complex biological reactivity within a containing sites (coordination units), termed as simple synthetic molecule is a challenging endeavour "active sites", comprising one or more metal ions and with both intellectual and aesthetic goals. their ligands, are usually the loci of electron transfer, 2 Several researchers ,4 have endeavoured to binding of exogenous molecules and catalysis. The understand the structure and function of copper demonstrated or potential relation between the proteins involved in copper(l)/02 interactions by properties of these sites and those of synthetic studying inorganic models, i.e., synthetically derived coordination complexes has contributed significantly copper(I) complexes, and their O reactivity. Such to~ the emergence of the interdisciplinary field of 2 biomimetic approaches can lead to fundamental bioinorganic chemistry. The complexity of biological insights into the copper-based chemistry. One might systems renders a detailed study of their mechanism also envision the development of reagents or catalysts very difficult. An increasingly popular method of 4f for use in practical oxidation processes • elucidating structures and mechanisms is the use of a 3 It is the purpose of this article to highlight recent simple chemical compound or system . advances in bioinorganic model (structurally Interest in elucidating or mimicking the physico­ characterized) studies on· some selected copper chemical properties of metalloproteins has led to proteins/enzymes, including some results from spurring activity in the synthesis of numerous author's laboratory. interesting coordination complexes. However, recently there has been an increased emphasis upon Protein(s) and synthetic models functional modelling of proteins. While the structural Blue copper proteins: Type I copper and spectroscopic modelling of metalloprotein active "Blue" copper proteins (azurin, plastocyanin)5, sites is an important and ongoing endeavour, the realization that coordination chemists can and should make significant contributions to reactivity studies and mechanism has become apparent. The value of SynthetiC models Metalloprotelns models for metalloproteins will always be relative. One of the difficulties encount~red in simulating a biosite is that, as time p"sses, the objective may I f'---__~I \ Establish relevant Active site change with advancing knowledge. If the structure of coordination chemistry the metal ion environment in the metalloprotein is unknown, the objective may be to reproduce some Fig. I-The synergistic relationship between studies involving property of the system in a similar model coordination metalloprotein biochemistry and inorganic modelling. MUKHERJEE: THE BIOINORGANIC CHEMISTRY OF COPPER 2177 which function as electron transport agents in a (methionine j ~. number of biochemical systems, gain their colour • 0 from an intense electronic absorption band that arises o '.3.12A 2 \ from a charge transfer transition to the Cu + ion at the 2.01A~ 1090 active site from the cysteine thiolate ligand. The (histidine)N'11'ft ~•. '0• unusually low energy of the transition results from the 101\_.'·~cu'f...l.12 A 2 (histidine) "" S (thiolate) coordination geometry about the Cu +, which involves N~"-..Io a nearly trigonal arrangement of two imidazole N 2.081..-.....-/ 135 atoms and the thiolate S atom, as shown in Fig. 2. The (a) methionine S atom is found along the trigonal axis at a long distance, 2.6-3.1 A, reflecting a weak bonding interaction; sometimes a peptide carbonyl oxygen atom is located on the other side of the trigonal plane, at an even longer distance. This bonding arrangement, together with a high degree of covalency associated with the short Cu-S (thiolate) bond, leads to reduced values of the A II Cu hypelfine coupling constant. The coordination geometry stabilizes the copper(I) oxidation state and the redox potentials are unusually high in relation to ordinary copper complexes. The synthesis and structural characterization of a F thiolate-copper(II) complex which closely mimics the spectroscopic characteristics of blue copper proteins have been longtime goals in bioinorganic chemistrl. The main difficulty in synthesizing an accurate model for Type I copper comes from the instability of the [(L)Cu(SC6F 5)] copper(II)-thiolate bond [2Cull-SR -7 2eu' + RSSR]7. (b) Kitajima and co-workers8 were successful in providing structural proof of a copper(IJ) complex Fig. 2--(a) The active -site of azurin, with the indicated bond (trigonal pyramidal) with C6HsS· coordination lengths and angles; (b) The model complex (Fig. 2). The Cu(II)-S(thiolate) distance (2.18 A) is distinctly shorter than those of the reported oxygenated environment, contain at least one complexes. Spectroscopic features of this complex are superoxide dismutase. The one which is pertinent to comparable to those of "blue" copper proteins. The x­ this article is a Cu-Zn protein found in cells that ray analysis (poor data set) of a closely similar contain a nucleus (eukaryotic cells). Its function is to complex with Ph3S· as the thiolate ligand was also catalyze the disproportionation of superoxide ion achieved. (02-), i.e., it is a "superoxide dismutase,,9.

Superoxide dismutase: Type II copper The reduction of dioxygen probably proceeds, in every instance, by a series of one-electron transfer Copper-zinc superoxide dismutase (SOD) contains an reactions. Therefore, unless the intermediate reduction imidazolate-bridged Cu(IJ)-Zn(II) heterodinuclear products are retained within the active site of an metal centre in its active site (Fig. 3). The copper ion enzyme or coordinated to a metal complex, there is is in a distorted square-pyramidal geometry, while the every likelihood that most oxidation reactions will zinc ion located at a distance of 6.2 A from the copper generate superoxide as the initial reduction product. ion is in a distorted tetrahedral structure. The catalytic As superoxide ion is toxic to cells, a defense cycle (Fig. 4) starts with the replacement of a water mechanism must have been initiated by nature. We molecule at the axial position by superoxide ion and now know that essentially all organisms, which use reduces the copper to Cu(l). Concomitantly the bond dioxygen, and many that have to survive an from Cu to imidazolate is broken and O2 is released. 2178 INDIAN J CHEM, SEC A, SEPTEMBER 2003

The Cu-facing nitrogen of histidine becomes second proton from an active-site water, the protonated and a second O2- becomes bound. An uncharged hydrogen peroxide is released. electron is transferred from Cu(l), coupled with a proton transfer from histidine. After addition of a In the copper ions in the imidazolate-bridged Cu(Il)-Zn(Il) heterodinuc1ear complex (Fig. 3) synthesized by Fukuzumi and co-workers 10, the OH2 (histidine) N", /N (histidine) 9 (aspartate) coordimtion site occupied by a solvent can be CU ...... ~ ~z ..-N(histidine) susceptible to ligand substitution, thus providing a / N-- n (histidine) N NO '. binding site for substrate superoxide. The Cu(H)­ ~ 'N (histidine) Zn(II) distance of 6.197(2) A agrees well with that of native enzyme. The complex catalyzed the (a) dismutation of superoxide at biological pH.

Nitrite reductase: Type 1 alld Type 11 copper Denitrification, the dissimilatory transformation of

NO,' and N02' to gaseous N20 or N2, is a central process in the biological nitrogen cycle (Fig. 5) responsible for depletion of nitrogen, necessary for plant growth, from soil I I. The copper-containing nitrite reductases isolated from bacteria and fungi, comprise an important subclass of the set of denitrification enzymes. These enzymes catalyze the

NCMe reduction of N02- to NO, although N20 generation

[(L)CuZn(MeCNhl[CI041J ·2MeCN has been induced under some conditions. The active (b) site (Fig. 5) of this enzyme contains a pair of copper ions, one cf which has been assigned as a "green" Fig. 3----(a) The metal-binding region of Cu, Zn-SOD; (b) The Type I, electron transfer site_ The other site is an model complex. unusual, distorted pseudotetrahedral Type II site.

/arginine /arginine HN HN H 1 "H~ H-Nt+'1 N-H H-N t+' N--H- - -2 H I I I I ("'o:'H H H r. H I "- H,O, -"""':cu N~ / ~

t O,',H+ I::rginine

/arginlne j-j---N1 t+' N-H

HI HI H-N1 t+' N-H---'Q I I ~ - H H "-..~ ~ /

Fig. 4--The catalytic cycle of superoxide dismutase. MUKHERJEE: THE BIOINORGANIC CHEMISTRY OF COPPER 2179

(a)

OH2 N (histidine) . du . (cysteine)S-\U __ N(histidine) (histidine) N-/ 'N.(hlsti~I~) ' } I N (hiItidiDe) ~ S (methionine) [(L)Cu(NO)].O;Smcsityione [(L)Cu(ONO»

Typen "green" Type I 12.5 A • (b)

m+ H 0 NO' . 'f "}' + + 2+ l~' ---.!..- l~'" NO," ~ ""'" NO ="'" "'" • NO (L)CI(N~]

2 2 E-Cu + ~ B-Cu +.N01· (c) Fig. ~The nitrite reductase model compounds.

Fig. 5-(a) The pathway of denitrification; (b) The active site of Hc exhibits two intense bands at ca. 350 nm (-20 nitrite reductase (the two Cu sites are linked by a dipeptide OOO/2Cu) and ca. 580 (-1000), both attributable to 2 fragment); (c) Proposed mechanism for nitrite reduction by nitrite 0 2 . 7 Cu(U) LMCT transitions. reductase. While studying modelling copper-dioxygen Proposed mechanism for reactions of copper­ i3 containing nitrite reductases is presented in Fig. 5. chemistry Kitajima et al. reported the synthesis of a JL-peroxo dinuclear complex with 3,5-dimethyl­ Tolman and co-workers i2 provided examples of a substituted tris(pyrazolyl)borate ligand, which showed number of substrate-bound copper(I) and copper(U) remarkable physicochemical similarities to oxy-Hc complexes (Fig. 6). In an elegant manner they and oxy-Tyr2. Using 3,5-di-isopropyl-substituted terminal ligand they provided the first structural proof modelled N20 generation by copper proteins through 2 2 reductive disproportionation of Cu-NO species. To (Fig. 7) of the existence of JL-11 :11 peroxo prove the reaction sequence they isolated a nitrite­ dicopper(U) core (copper geometry: distorted square bound complex. pyramidal; Cu-Cu: 3.560 A) and reported detailed characterization properties, which eventually led to i4 Hemocyanins: Type III copper the structural characterization of oxy_HC . Hemocyanin2 is a ubiquitous dioxygen carrier for invertebrates, containing a dinuclear copper site to Tolman and co-workers discoveredi5 a novel which dioxygen is bound as peroxide (Fig. 7); the two phenomenon that when copper(I) complex of 1,4,7- copper ions are divalent in the dioxygen binding state triisopropyl-triazacyclononane oxygenated at -78°C, (so-called oxyhemocyanin, oxy-Hc), Oxy-Hc is EPR there exists an eqUilibrium between the two silent and in fact, diamagnetic at room temperature oxygenated species [Cu1I2(JL-112:112 -02)]2+ [side-on due to a very strong antiferromagnetic exchange peroxodicopper(U)] and [CUIlI2(JL-Oh]2+ [bis(JL­ I coupling (-21 > 600 cm- ) between the two Cu(U) oxo)dicopper(ill)] depending on the solvent chosen, ions. Furthermore, instead of d-d bands normally with CH2Clz favouring the former species and THF observed at 600-700 nm for Cu(U) complexes, Oxy- favouring the latter. 2180 INDIAN J CHEM, SEC A, SEPTEMBER 2003

. N (histidine) N (histidine) N (histidine) (histidine) NIl. . . dine) N 11 I (his II 1I11 I I Ii'~ . 1 ",. n .\\\~O/t.'CIl .,,\\\N (histidine) "'Gu Cu 'Cit'V U" (histidine) . (histidine) / I'N (histidine) ~'O I 'N (histidine) ~ N (histidine) N (hlsUdine) o o Cu ... Cu=-4.6A Cu ... Cu .. j.6A (a) (b)

R R=iPr. LiPr

Fig. 7-{a) The active site of deoxy-Hc; (b) The active site of oxy-Hc; (c) The model complex of oxy-Hc.

Tyrosinase: Type III copper It is known that when potatoes, apples, bananas, OH T~ Jvoo_._. :m +C(~ sweet potatoes or mushrooms are injured they turn brown. This is due to the conversion of tyrosine to the H, y9H, .1 ~ 9-9H-NH, 9H-NH, pigment melanin, by the sequence of reactions shown COOH COOH melanin pigments in Fig. 8. The same process cau~es skin tanning, (a) following exposure to ultraviolet radiation. The 2 enzymatic reactions are catalyzed by tyrosinase • The enzyme is present in the interior of the plant material .g; ~8' ,2+ and since the reaction requires molecular oxygen, the . \ /0,/ pigmentation does not occur until the interior is CtrW/·,/c.m exposed. Tyrosinase catalyzes (i) the o-hydroxylation of monophenols to o-diphenols (cresolase activity) ,"""# and the further oxidation of these to o-diquinones [(L)CuiOMe)][pP6h (catecholase activity). These qui nones undergo further (b) enzymatic and nonenzymatic reactions that lead to polymeric pigmented material. Thus, tyrosinases possess both monooxygenase and oxidase activity. In . Fig. 8--{a) The metabolism of tyrosine; (b) The model complex animals, these reactions give skin, eyes and hair their of Karlin and co-workers; (c) The model complex from author's distinctive pigmentation. In order to deduce the laboratory . MUKHERJEE: THE BIOlNORGANIC CHEMISTRY OF COPPER 2181

HZO ' 0

. ~ CCo

(a)

Oz -cat

(b)

Fig. 9--{a) Mechanism of cresolase and catecholase activity of tyrosinase and/or catechol oxidase; (b) Catechol oxidase model reaction structures and mechanism of action of the protein­ A considerable number of ligand oxidations has active sites, a major focus of research has utilized the been reported, where aerobic treatment of a copper(I) biomimetic approach. complex, mostly dinuclear, yields a dinuclear Comparisons of chemical and spectroscopic copper(lI) complex with an oxidized ligand, which properties of tyrosinase and its derivatives with those can be isolated. Using tailor-made binucleating N­ of hemocyanin, establish a close similarity of the donor ligands having m-CH2C6~CH2 spacers active sites structures in these two proteins. The active between the coordination units, Karlin and co-workers site of tyrosinase apparently has greater accessibility reported l6 the first model (Fig. 8) consisting of a to exogenous ligands, including substrate molecules, ligand that provides two tridentate bis[2-(2- by comparison to the active site in hemocyanin. The pyridylethyl)amine] donor units to each copper ion. A similarity of the oxy-states of hemocyanin and mechanism was proposed which involves an tyrosinase points to the probable close relationship electrophilic attack of a bent 1l-11 2:112-peroxide to the between the binding of dioxygen and the ability to CH bond of the aromatic ring. Interestingly, when 1- activate it for incorporation into organic substrates. pyrazolyl or 2-imidazolyl donor groups fully or 2182 INDIAN J CHEM, SEC A, SEPTEMBER 2003

HO H

H H (a)

(tyrosinatc) 0 (histidine) N 11,1. I . "'·"Cu .••• \\\\N (hislldine)

.. H20'~" 0 (tyrosinate) (b)

[Cu(L)]

(c)

Fig. 10-{a) The reaction catalyzed by galactose oxidase; (b) The active site of galactose oxidase at pH 7.0; (c) The model complex. partially replace the 2-pyridyl ligands hydroxylation in contrast to tyrosinases, catalyze exclusively the does not occur. However, when Schiff base ligands oxidation of catechols to the corresponding o-quinone providing three or even only two nitrogen donors are by molecular oxygen without acting on monophenols. used, hydroxylation takes place. We demonstrated4 Thus, catechol oxidase lacks · hydroxylase activity. the synthesis of the first m-CH2C6~CH2 The resulting highly reactive quinones auto­ hydroxylation ligand system, · within the non-Schiff polymerize to form brown polyphenolic catechol base family, providing only two nitrogen melanins, a process thought to protect the damage4 coordinations to each copper centre (Fig. 8). While plant from pathogens or insects. The enzyme contains the Il-peroxo intermediate could not be identified an antiferromagnetically coupled (EPR silent) spectroscopically owing to its instability, on the basis dicopper centre. Three-dimensional X-ray crystal of the closely related ligand structure to our ligand, it structural analysis of catechol oxidase, from sweet was demonstrated that the reaction proceeds via a Il­ potato, in the resting Cu(II)-Cu(II) state, the reduced peroxo intermediate. Cu(l)-Cu(l) form, in complex with the inhibitor have been achieved. Both copper centres have three Catechol oxidase: Type III copper histidine ligands. In the oxidized catechol oxidase 17 The ubiquitous plant enzyme catechol oxidases , structure the two Cu(II) ions are 2.9 A apart. In MUKHERJEE: THE BIOINORGANIC CHEMISTRY OF COPPER 2183 addition to the six histidine ligands, a bridging hydroxide ion completes the four-coordinate trigonal pyramidal coordination sphere for each Cu(II) ion. - Mechanism of cresolase and catecholase activity of tyrosinase and/or catechol oxidase is presented in Fig. 9. Recently we have shown l8 that the phenoxo­ Ihydroxo-bridged dicopper(II) complex [Fig. 8(c)] acts as an efficient catalyst for catechol oxidase-like activity (Fig. 9).

Galactose oxidase: Type II copper Galactose oxidasel9 is a fungal enzyme that catalyzes the oxidation of galactose and a number of other primary alcohols to the corresponding aldehyde, Fig. Il-Postulated reaction mechanism for galactose oxidase. a reaction in which dioxygen is reduced to hydrogen peroxide (Fig. 10). The active site structure is presented schematically in Fig. 10. A unique feature (hiItidiue) N I~ N (histidine) of this active site embodies the modification of the --Cu tyrosinate residue located in the equatorial plane by a covalent linkage to the sulphur atom of a nearby 3.68 X1\3 .841 cysteine residue. 20 (hiI1idi.ae) ~ 13.71 J..\ /N (histidine) Stack et al. were able to synthesize nonplanar Cu....-.- Cu 10) copper(II) complexes (Fig. from which they could (hiItidiue) N/II '0/H '\' N (hlstldlne) obtain the corresponding copper(I) complexes and ,...... ",,-,.1.:...._) N . N (histidiDe relatively stable phenoxyl-radical copper(II) (a) complexes by reduction and oxidation, respectively. The complex can act as a catalyst or as a precursor for a catalyst in the reaction of benzylic and allylic NMez . 0./01) 3+ alcohols with molecular oxygen at room temperature, · C)/\R)i yielding the respective aldehyde and hydrogen ...... "I'. 'N peroxide. Turnover numbers as high as 1300 are a ~NMez 0 reported for the catalytic cycles. Most noteworthy, the [(LhCul~[CF1S03h · 4CH2CI2 catalytic oxidation seems to proceed by the same (b) mechanism as the enzyme-catalyzed 'reaction (Fig. 11). Fig. 12-(a) The trinuclear Cu cluster in the multicopper oxidase. ascorbate oxidase. The Type I Cu site (not shown) is -12.5 A from the Type ill Cu atoms. opposite the Type II centre. (b) The "Blue" multicopper oxidase - ascorbate oxidase: Type model compound. Ncopper 2c d Ascorbate oxidase • catalyzes the oxidation of l­ (Cu-Cu: 2.641 and 2.704 A). The relevance of this ascorbate with concomitant reduction of O2 to water. synthetic complex to the reduction of O2 at the The trinuclear Cu site is shown in Fig. 12. Stack and trinuclear active sites of multicopper oxidases was 21 co-workers reported an unusual 3:1 (copper: O2 discussed (three copper(l) centres produce 4e- to stoichiometry) reaction between a mononuclear reduce O2 to H20). copper(I) complex of a N-permethylated (IR, 2R)­ cyclohexanediamine ligand with dioxygen. The end Acknowledgement product of this reaction, stable at only low­ Research on copper bioinorganic chemistry carried temperatures (X-ray structure at -40°C) , is a discrete out in author's laboratory has been supported by the mixed-valence trinuclear copper cluster (Fig. 12), Council of Scientific & Industrial Research, with two terminal Cu(II) and a central Cu(III) centre Department of Science & Technology, Government of 2184 INDIAN J CHEM, SEC A, SEPTEMBER 2003

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