Design parameters for tuning the type 1 Cu multicopper oxidase redox potential: insight from a combination of first principles and empirical molecular dynamics simulations

The redox potentials and reorganization energies of the type 1 (T1) Cu site in four multicopper oxidases were calculated by combining first principles density functional theory (QM) and QM/MM molecular dynamics (MD) simulations. The model enzymes selected included the laccase from Trametes versicolor, the laccase-like enzyme isolated from Bacillus subtilis, CueO required for copper homeostasis in Escherichia coli, and the small laccase (SLAC) from Streptomyces coelicolor. The results demonstrated good agreement with experimental data and provided insight into the parameters that influence the T1 redox potential. Effects of the immediate T1 Cu site environment, including the His(N(δ))-Cys(S)-His(N(δ)) and the axial coordinating amino acid, as well as the proximate H(N)(backbone)-S(Cys) hydrogen bond, were discerned. Furthermore, effects of the protein backbone and side-chains, as well as of the aqueous solvent, were studied by QM/MM molecular dynamics (MD) simulations, providing an understanding of influences beyond the T1 Cu coordination sphere. Suggestions were made regarding an increase of the T1 redox potential in SLAC, i.e., of Met198 and Thr232 in addition to the axial amino acid Met298. Finally, the results of this work presented a framework for understanding parameters that influence the Type 1 Cu MCO redox potential, useful for an ever-growing range of laccase-based applications.     

Paramagnetic properties of the halide-bound derivatives of oxidised tyrosinase investigated by 1H NMR spectroscopy

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms.     

Axial ligand modulation of the electronic structures of binuclear copper sites: analysis of paramagnetic 1H NMR spectra of Met160Gln Cu(A).

Cu(A) is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu(A) sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu(A) centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta-CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C(beta)-H(beta) dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys beta-CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu(A) excited states by tuning axial ligand interactions.     

A spectroscopic investigation of the interaction between nitrogen monoxide and copper sites of the fungal laccase from Rigidoporus lignosus

The interaction of NO, with the copper centres of the laccase secreted by Rigidoporus lignosus was studied under both aerobic or anaerobic conditions. The reduction of the T1 site was always observed, as detected by the disappearance of the characteristic optical band at 604 nm (T3 presents probably the same behaviour because of the decreasing of the band at 330 nm) and the absence of its characteristic EPR signal, while T2 undergoes an initial partial and transitory reduction, its EPR signal intensity totally restoring after 24 h interaction. Different magnetic parameters of the T2 site have been detected, evidencing an increase of the hyperfine coupling constant. Furthermore, the number of superhyperfine lines on the fourth line of T2 copper was also found to increase from seven in the native to nine in the NO-treated laccase, this fact implying the coordination of a nitrogenous species to the T2 site. It was also shown that nitrite can be a source of NO, thus, paralleling the behaviour of NO-donor molecules or NO gas, but after longer interaction times. The nitrogenous species coordinated to T2 site is probably NO2-, which arises indirectly by NO oxidation. In order to understand the mechanistic pathway of this interaction, some experiments were also carried out in the presence of azide to study the interaction of NO with this laccase having its trinuclear cluster blocked by the presence of an exogenous ligand as N3-. After the addition of NO-donor molecules to the azide-treated laccase, a new EPR signal appeared at low temperatures, which is ascribable to the partially reduced T3 site, while the T1 and T2 sites were found to be totally reduced. The mechanistic pathway of the NO interaction seems to proceed through the reduction of T1 and T3 copper sites, followed by the coordination of nitrogenous species to T2.     

On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

The catalytic cycle of multicopper oxidases (MCOs) involves intramolecular electron transfer (IET) from the Cu‐T1 copper ion, which is the primary site of the one‐electron oxidations of the substrate, to the trinuclear copper cluster (TNC), which is the site of the four‐electron reduction of dioxygen to water. In this study we report a detailed characterization of the kinetic and electrochemical properties of bilirubin oxidase (BOx) – a member of the MCO family. The experimental results strongly indicate that under certain conditions, e.g. in alkaline solutions, the IET can be the rate‐limiting step in the BOx catalytic cycle. The data also suggest that one of the catalytically relevant intermediates (most likely characterized by an intermediate oxidation state of the TNC) formed during the catalytic cycle of BOx has a redox potential close to 0.4 V, indicating an uphill IET process from the T1 copper site (0.7 V) to the Cu‐T23. These suggestions are supported by calculations of the IET rate, based on the experimentally observed Gibbs free energy change and theoretical estimates of reorganization energy obtained by combined quantum and molecular mechanical (QM/MM) calculations.     

Spectroscopic and kinetic studies of perturbed trinuclear copper clusters: the role of protons in reductive cleavage of the O-O bond in the multicopper oxidase Fet3p

The multicopper oxidase Fet3p couples four 1e(-) oxidations of substrate to the 4e(-) reduction of O2 to H2O. Fet3p uses four Cu atoms to accomplish this reaction: the type 1, type 2, and coupled binuclear type 3 sites. The type 2 and type 3 sites together form a trinuclear Cu cluster (TNC) which is the site of O2 reduction. This study focuses on mutants of two residues, E487 and D94, which lie in the second coordination sphere of the TNC and defines the role that each plays in the structural integrity of the TNC, its reactivity with O2, and in the directional movement of protons during reductive cleavage of the O-O bond. The E487D, E487A, and D94E mutants have been studied in the holo and type 1 depleted (T1D) forms. Residue E487, located near the T3 center, is found to be responsible for donation of a proton during the reductive cleavage of the O-O bond in the peroxide intermediate and an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. Residue D94, near the T2 site, plays a key role in the reaction of the reduced TNC with O2 and drives electron transfer from the T2 Cu to cleave the O-O bond by deprotonating the T2 Cu water ligand. A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC.     

Active site comparison of CoII blue and green nitrite reductases

Copper-containing nitrite reductases (NiRs) possess type 1 (T1) and type 2 (T2) copper sites and can be either green or blue in color owing to differences at their T1 centers. The active sites of a green and a blue NiR were studied by utilizing their T1CuI/T2CoII and T1CoII/T2CoII-substituted forms. The UV/Vis spectra of these derivatives highlight the similarity of the T2 centers in these enzymes and that T1 site differences are also present in the CoII forms. The paramagnetic NMR spectra of T1CuI/T2CoII enzymes allow hyperfine shifted resonances from the three T2 His ligands to be assigned: these exhibit remarkably similar positions in the spectra of both NiRs, emphasizing the homology of the T2 centers. The addition of nitrite results in subtle alterations in the paramagnetic NMR spectra of the T1CuI/T2CoII forms at pH<7, which indicate a geometry change upon the binding of substrate. Shifted resonances from all of the T1 site ligands have been assigned and the CoII--N(His) interactions are alike, whereas the CbetaH proton resonances of the Cys ligand exhibit subtle chemical shift differences in the blue and green NiRs. The strength of the axial CoII--S(Met) interaction is similar in the two NiRs studied, but the altered conformation of the side chain of this ligand results in a dramatically different chemical shift pattern for the CgammaH protons. This indicates an alteration in the bonding of the axial ligand in these derivatives, which could be influential in the CuII proteins.     

Determinants of the relative reduction potentials of type-1 copper sites in proteins

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.     

Electrochemical Studies of Intramolecular Electron Transfer in Laccase from Trametes versicolor

This paper describes electrochemical behavior of laccase from the fungus Trametes versicolor. The issues related to discrimination of the redox potentials corresponding to copper centers T1 and T2/T3 in the active site and possible mechanism of intramolecular electron transfer have been discussed. The electron‐transfer rate constant for laccase immobilized on carbon electrode is 3.4 s^?1. The bioelectrocatalytic activity of the enzyme was studied in the presence of 1,4‐hydroquinone (HQ). The kinetics of HQ oxidation is very fast (K_M=3.8 μM). However, the catalytic activity of laccase in the presence of high concentration of HQ decreases drastically. It is suggested that the T2/T3 copper center is able to accept electrons from HQ molecules directly via intramolecular channel.     

Spectroelectrochemical investigation of intramolecular and interfacial electron-transfer rates reveals differences between nitrite reductase at rest and during turnover

A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Fo?rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 μM), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer.     

Intermolecular photoinduced electron-transfer of 1,8-naphthalimides in protic polar solvents

The intermolecular photoinduced electron transfer (PET) processes of 1,8-naphthalimide (NI) derivatives including NI-linker-phenothiazine dyads were investigated in a protic H(2)O/CH(3)CN (v/v=1:1) solvent using ns-laser flash photolysis with 355 nm-laser excitation. NI derivatives are surrounded by H(2)O in the ground state in H(2)O/CH(3)CN. The T(1)-T(n) absorption band of (3)NI* was observed at around 470 nm. The transient absorption band at around 410 nm increased concomitantly with the decay of (3)NI* in H(2)O/CH(3)CN. This implies that hydrated NI anion radical (NI*(-)) is primarily generated via the quenching of (3)NI* by NI at the diffusion control rate. This intermolecular PET did not occur in aprotic CH(3)CN. The formation and decay times of NI*(-) showed strong dependence on the concentration of NI. Then, we suggest that NI*(-) could undergo proton abstraction to give ketyl radical species of NI [NI(H)*] in H(2)O/CH(3)CN.     

Intramolecular excimer formation and photoinduced electron-transfer process in bis-1,8-naphthalimide dyads depending on the linker length

The photophysical properties of bis-1,8-naphthalimide (NI-L-NI) dyads with different linkers ( L = -C 3H 6-, -C 4H 8-, -C 6H 12-, -C 8H 16-, and -C 9H 18-) as well as the reference NI derivative (NI-C 7H 15) were investigated in CH 3CN and H 2O/CH 3CN (v/v = 1:9). The normal fluorescence peak of (1)NI*-L-NI was observed at 379 nm together with a broad emission at longer wavelength both in aprotic CH 3CN and in H 2O/CH 3CN, which is assigned to an excimer, (1)(NI-L-NI)*. The excimer emission maximum was blue-shifted with increasing length of the linker. The photoinduced electron-transfer process of NI-L-NI was also investigated in both solvents by using nanosecond-laser flash photolysis. The T 1-T n absorption band for (3)NI*-L-NI was observed around 470 nm in both solvents. In H 2O/CH 3CN, NI-L-NI is solvated with H 2O in the ground state to exist as solvated NI-L-NI. In the excited triplet state, the NI radical anion (NI (*-)) was generated via the intramolecular quenching of (3)NI*-L-NI by another NI moiety. The solvated NI (*-)-L-NI may undergo the proton abstraction process to give NI(H) (*)-L-NI, which can be confirmed by the transient absorption band at 410 nm. This band was not observed in pure aprotic CH 3CN.     

Ligand and loop variations at type 1 copper sites: influence on structure and reactivity

Type 1 (T1) copper sites promote biological electron transfer and are found in the cupredoxins and a number of copper-containing enzymes including the multi-copper oxidases. A T1 copper site usually has a distorted tetrahedral geometry with strong ligands provided by the thiolate sulfur of a Cys and the imidazole nitrogens of two His residues. The active site structure is typically completed by a weak axial Met ligand (a second weak axial interaction is found in azurin resulting in a trigonal bipyramidal geometry). The axial Met is not conserved and Gln, Phe, Leu and Val are also found in this position. Three of the four ligands at a T1 copper site are situated on a single C-terminal loop whose length and structure varies. Studies are discussed which investigate both the influence of physiologically relevant axial ligand alterations, and also of mutations to the length and structure of the ligand-containing loop, on the properties of T1 copper sites.     

聚甲基丙烯酸-对-甲氧基偶氮苯烷氧基酯的合成及柔性间隔基对液晶行为的影响

通过甲基丙烯酸钾盐与溴化物的反应合成了一系列柔性间隔基长度不同的甲基丙烯酸 对 甲氧基偶氮苯烷氧基酯（ＰＭＡＡＺＯｎ,ｎ＝２,３,４,５,６）,然后聚合成相应的聚合物．用１Ｈ ＮＭＲ和ＩＲ表征了聚合物的结构,ＧＰＣ测定了分子量,ＤＳＣ和偏光显微镜观测了液晶高分子的相行为和织构．研究了间隔基长度对织构、相变和热力学性质的影响,观测到聚合物的ＴＮＩ随间隔基的长度变化表现出的奇偶效应．     

The reaction of M(CO)3(Ph2PCH2CH2PPh2)(M = Fe, Ru) with parahydrogen: probing the electronic structure of reaction intermediates and the internal rearrangement mechanism for the dihydride products

The photochemical reaction of Ru(CO)(3)(dppe) and Fe(CO)(3)(dppe)(dppe = Ph(2)PCH(2)CH(2)PPh(2)) with parahydrogen has been studied by in situ-photochemistry resulting in NMR spectra of Ru(CO)(2)(dppe)(H)(2) that show significant enhancement of the hydride resonances while normal signals are seen in Fe(CO)(2)(dppe)(H)(2). This effect is associated with a singlet electronic state for the key intermediate Ru(CO)(2)(dppe) while Fe(CO)(2)(dppe) is a triplet. DFT calculations reveal electronic ground states consistent with this picture. The fluxionality of Ru(CO)(2)(dppe)(H)(2) and Fe(CO)(2)(dppe)(H)(2) has been examined by NMR spectroscopy and rationalised by theoretical methods which show that two pathways for ligand exchange exist. In the first, the phosphorus and carbonyl centres interchange positions while the two hydride ligands are unaffected. A second pathway involving interchange of all three ligand sets was found at slightly higher energy. The H-H distances in the transition states are consistent with metal-bonded dihydrogen ligands. However, no local minimum (intermediate) was found along the rearrangement pathways.     

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.     

Comparative Spectroelectrochemical Studies of Lyophilized and Nonlyophilized Laccases from Cerrena unicolor Basidiomycete

The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET‐based biocatalytic reduction of O₂ by C. unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady‐state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3‐D structures of laccase molecules, conformational changes, and partial denaturation of the enzyme, which appeared after enzyme isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high K_M‐value towards O₂, and the unusual electrochemical behavior of C. unicolor laccase used in the present study. In spite of the comparable specific activity and long‐term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of enzyme than its lyophilized counterpart.     

A simultaneous reduction, substitution, and self-assembly reaction under hydrothermal conditions afforded the first diiodopyridine copper(I) coordination polymer

A simultaneous reduction of copper(II) to copper(I) by pyridinecarboxylate and the substitution of carboxylato groups by iodo nucleophiles in a self-assembly process under hydrothermal conditions afforded a new iodine-inclusion coordination polymer [CuI(C5H3NI2)*1/2I2] 1. The synthetic studies of the substitution process produced a new supramolecular compound [IC5H3NCOOH] 2 and revealed that the catalytic properties of copper ions in redox and substitution reactions under hydrothermal conditions are attractive. Crystal data for [CuI(C5H3NI2)*1/2I2]: triclinic, space group P1; cell dimensions a = 4.216(1) A, b = 11.254(2) A, c = 12.196(2) A, alpha = 80.34(3) degrees, beta = 88.44(3) degrees, gamma = 83.10(3); V = 566.2(2) A(3), Z = 2. Crystal data for [IC(5)H(3)NCOOH]: monoclinic, space group P2(1)/c; cell dimensions a = 5.041(1) A, b = 17.313(2) A, c = 8.639(1) A, beta = 95.042(2) degrees; V = 751.02(13) A(3), Z = 4.     

Catalysis of novel enzymatic iodide oxidation by fungal laccase

A fungal laccase (Myceliophthom thermophila) has been shown to function as an iodide oxidase. Unlike other halides which interact with the type 2 copper site and are inhibitors for the laccase, iodide interacts with the type 1 copper site and serves as a substrate capable of donating an electron to the laccase. Under anaerobic conditions, the interaction between the laccase and iodide results in the reduction of the laccase type 1 copper and the concomitant oxidation of iodide to form iodide. In aerated solutions, the laccase catalyzes the oxidation of iodide to iodine and the concomitant reduction of dioxygen to water. The reaction exhibits typical Michaelis kinetics with aK _m of 0.16 ± 0.02M and ak _cat of 2.7 ± 0.2 turnovers per min at the optimal pH (3.4). The catalysis can be enhanced by 2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid), which shuttles electrons rapidly between iodide and the laccase. Bilirubin oxidase also demonstrates significant iodide oxidase activity, suggesting that the property could be a common feature for copper-containing oxidases. Possible industrial and medicinal applications for a laccase-based iodine production system are discussed.