Multicopper Protein Research Articles

The green cupredoxin CopI is a multicopper protein able to oxidize Cu(I)

Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development

SummaryAccess to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 μM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

A Nonsense Variant in Hephaestin Like 1 (HEPHL1) Is Responsible for Congenital Hypotrichosis in Belted Galloway Cattle.

Genodermatosis such as hair disorders mostly follow a monogenic mode of inheritance. Congenital hypotrichosis (HY) belong to this group of disorders and is characterized by abnormally reduced hair since birth. The purpose of this study was to characterize the clinical phenotype of a breed-specific non-syndromic form of HY in Belted Galloway cattle and to identify the causative genetic variant for this recessive disorder. An affected calf born in Switzerland presented with multiple small to large areas of alopecia on the limbs and on the dorsal part of the head, neck, and back. A genome-wide association study using Swiss and US Belted Galloway cattle encompassing 12 cases and 61 controls revealed an association signal on chromosome 29. Homozygosity mapping in a subset of cases refined the HY locus to a 1.5 Mb critical interval and subsequent Sanger sequencing of protein-coding exons of positional candidate genes revealed a stop gain variant in the HEPHL1 gene that encodes a multi-copper ferroxidase protein so-called hephaestin like 1 (c.1684A>T; p.Lys562*). A perfect concordance between the homozygous presence of this most likely pathogenic loss-of-function variant and the HY phenotype was found. Genotyping of more than 700 purebred Swiss and US Belted Galloway cattle showed the global spread of the mutation. This study provides a molecular test that will permit the avoidance of risk matings by systematic genotyping of relevant breeding animals. This rare recessive HEPHL1-related form of hypotrichosis provides a novel large animal model for similar human conditions. The results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 002230-9913).

Application of laccases for mycotoxin decontamination

Several food commodities can be infected by filamentous fungi, both in the field and during storage. Some of these fungi, under appropriate conditions, are capable of producing a wide range of secondary metabolites, including mycotoxins, which may resist food processing and arise in the final feed and food products. Contamination of these products with mycotoxins still occurs very often and that is why research in this area is valuable and still evolving. The best way to avoid contamination is prevention; however, when it is not possible, remediation is the solution. Enzymatic biodegradation of mycotoxins is a green solution for removal of these compounds that has attracted growing interest over recent years. Due to their ability to detoxify a wide variety of recalcitrant pollutants, laccases have received a lot of attention. Laccases are multi-copper proteins that use molecular oxygen to oxidise various aromatic and non-aromatic compounds, by a radical-catalysed reaction mechanism. Being non-specific, they are capable of degrading a wide range of compounds and the radical species formed can evolve towards both synthetic and degradative processes. The present review provides an overview of structural features, biological functions and catalytic mechanisms of laccases. The utilisation of laccases for mycotoxin degradation is reviewed, as well as shortcomings and future needs related with the use of laccases for mycotoxin decontamination from food and feed.

Effects of Succinic Anhydride Modification on Laccase Stability and Phenolics Removal Efficiency

Chemical modification is a useful method to change the properties of enzymes. Laccase is a phenol oxidase belonging to a multicopper protein, which catalyzes the oxidation of many phenolics. DeniLite IIS, a commercial laccase preparation from the Novozymes China Company, was purified by ammonium sulfate fractional precipitation. Succinic anhydride (SA) was used as a modifier for the chemical modification of the purified laccase. The effects of modification were characterized using the 6-trinitrobenzene sulfonic acid method, ultraviolet spectroscopy, and fluorescence spectroscopy. The pH stability, thermal stability, and the phenolics removal efficiency for the native and modified laccases were compared. The results showed that the average amino modification yield of the modified laccase was 85% and the modified laccase had a blue shifted ultraviolet peak and fluorescence emission peak as well as a decrease in the ultraviolet absorbance and an increase in the fluorescence intensity. Although chemical modification with SA did not change the optimum temperature for the catalysis of the laccase, it caused the optimum pH of the catalysts to shift from 4.5 to 5.5 and the enzymatic activity increased by 60%. Compared with the native laccase, the modified laccase exhibited remarkably higher pH stability and thermal stability and its catalysis efficiency ( k cat ) and substrate affinity ( k cat / K m ) increased by 53% and 122%, respectively. The phenolics removal efficiency ( o -, m -, p -dihydroxybenzene) of the modified laccase increased by 48%, 57%, and 18%, respectively. These results indicate that the modified laccase with higher stability and higher efficiency is suitable for application in industrial production and for the treatment of phenolics-polluted water. 采用 (NH 4 ) 2 SO 4 分步沉淀法对诺维信中国公司生产的漆酶制剂 DeniLite IIS 进行了纯化, 并用丁二酸酐 (SA) 对纯化酶进行了化学修饰, 运用三硝基苯磺酸法、紫外光谱法及荧光光谱法对修饰效果进行了初步表征, 比较了天然酶和修饰酶的 pH 稳定性、热稳定性及除酚效率. 结果表明, 修饰酶的平均氨基修饰度为 85%, 其紫外吸收峰和荧光发射峰均出现蓝移, 而且紫外吸收减小、荧光强度增加. 尽管采用 SA 化学修饰未能改变漆酶的最适反应温度, 但使其最适反应 pH 值由 4.5 提高到 5.5, 并且使酶活提高 60%. 与天然酶相比, 修饰酶的 pH 稳定性和热稳定性更高, 催化效率 ( k cat ) 和酶与底物的亲和力 ( k cat / K m ) 分别提高了 53% 和 122%, 对邻、间和对苯二酚的除酚效率分别提高了 48%, 57% 和 18%. 这预示着这些修饰漆酶可望应用于工业生产和酚类污染废水的治理. Laccase was purified and modified with succinic anhydride. Chemical modification increased the optimum pH, enhanced the substrate affinity and activity, improved the pH and thermal stability, and increased the phenolics removal efficiency.

Laccase: A Review of Its Past and Its Future in Bioremediation

Laccases are multicopper proteins that use molecular oxygen to oxidize a broad spectrum of organic compounds by a radical-catalyzed reaction mechanism. Many articles over the past 15 years have touted the diverse potential applications of laccase in various biotechnological processes. This review covers the natural roles of the enzyme, its structural properties, substrates, reaction mechanism, and inhibitors, as well as its applications regarding the detoxification and bioremediation of polluted wastewaters and soils. Other applications are briefly covered as well. The authors critically assess the advantages, shortcomings, and future needs relating to laccase availability, effectiveness, and cost-efficiency, and also provide numerous references to the many divisions of research into this multifaceted enzyme.

Homologues of nitrite reductases in ammonia‐oxidizing archaea: diversity and genomic context

Ammonia-oxidizing archaea are frequent and ubiquitous inhabitants of terrestrial and marine environments. As they have only recently been detected, most aspects of their metabolism are yet unknown. Here we report on the occurrence of genes encoding potential homologues of copper-dependent nitrite reductases (NirK) in ammonia-oxidizing archaea of soils and other environments using metagenomic approaches and PCR amplification. Two pairs of highly overlapping 40 kb genome fragments, each containing nirK genes of archaea, were isolated from a metagenomic soil library. Between 68% and 85% of the open reading frames on these genome fragments had homologues in the genomes of the marine archaeal ammonia oxidizers Nitrosopumilus maritimus and Cenarchaeum symbiosum. Extensions of NirK homologues with C-terminal fused amicyanin domains were deduced from two of the four fosmids indicating structural variation of these multicopper proteins in archaea. Phylogenetic analyses including all major groups of currently known NirK homologues revealed that the deduced protein sequences of marine and soil archaea were separated into two highly divergent lineages that did not contain bacterial homologues. In contrast, another separated lineage contained potential multicopper oxidases of both domains, archaea and bacteria. More nirK gene variants directly amplified by PCR from several environments indicated further diversity of the gene and a widespread occurrence in archaea. Transcription of the potential archaeal nirK in soil was demonstrated at different water contents, but no significant increase in transcript copy number was observed with increased denitrifying activity.

X-ray structure of a two-domain type laccase: A missing link in the evolution of multi-copper proteins

A multi-copper protein with two cupredoxin-like domains was identified from our in-house metagenomic database. The recombinant protein, mgLAC, contained four copper ions/subunits, oxidized various phenolic and non-phenolic substrates, and had spectroscopic properties similar to common laccases. X-ray structure analysis revealed a homotrimeric architecture for this enzyme, which resembles nitrite reductase (NIR). However, a difference in copper coordination was found at the domain interface. mgLAC contains a T2/T3 tri-nuclear copper cluster at this site, whereas a mononuclear T2 copper occupies this position in NIR. The trimer is thus an essential part of the architecture of two-domain multi-copper proteins, and mgLAC may be an evolutionary precursor of NIR.

カーボンナノ素材と酵素との電子移動反応に及ぼすカーボン表面官能基の効果

Bilirubin oxidase (BOD) contains multi active center copper sites (type 1, 2 and 3) and catalyzes the oxidation of bilirubin to biliverdin (at the type 1 site) with the concomitant four-electron reduction of dioxygen to water (at type 2-3 sites). BOD electron transfer reaction investigations at carbon electrodes are attractive studies, not only from the point of view for the basic understanding of multi-copper protein reactions, but also for the application of dioxygen biocathodes for biofuel cells operating near neutral pH solutions. Several studies on the mediated electrochemistry of BOD using monomeric and redox polymer mediators have been reported. Recently, the direct electron transfer reaction of BOD at carbon electrodes under anaerobic and aerobic conditions has been reported. In the present study, we investigated electrode reactions of BOD adsorbed on carbon black (CB). CB has an advantage for large surface area-to-volume ratios. Heterogeneous electron transfer rate constants (k°) between BOD and CB were evaluated by analyzing steady-state catalytic voltammograms under aerobic conditions. We found that k° values improved with the use of UV-ozone treated CB.

Controlled Oxidation of Hydrocarbons by the Membrane-Bound Methane Monooxygenase: The Case for a Tricopper Cluster

[Figure: see text]. The growing need for inexpensive methods to convert methane to methanol has sparked considerable interest in methods that catalyze this process. The integral membrane protein particulate methane monooxygenase (pMMO) mediates the facile conversion of methane to methanol in methanotrophic bacteria. Most evidence indicates that pMMO is a multicopper enzyme, and these copper ions support redox, dioxygen, and oxo-transfer chemistry. However, the exact identity of the copper species that mediates the oxo-transfer chemistry remains an area of intense debate. This highly complex enzyme is notoriously difficult to purify because of its instability outside the lipid bilayer and tendency to lose its essential metal cofactors. For this reason, pMMO has resisted both initial identification and subsequent isolation and purification for biochemical and biophysical characterization. In this Account, we describe evidence that pMMO is a multicopper protein. Its unique trinuclear copper cluster mediates dioxygen chemistry and O-atom transfer during alkane hydroxylation. Although a recent crystal structure did not show this tricopper cluster, we provide compelling evidence for such a cluster through redox potentiometry and EPR experiments on the "holo" enzyme in pMMO-enriched membranes. We also identify a site in the structure of pMMO that could accommodate this cluster. A hydrophobic pocket capable of harboring pentane, the enzyme's largest known substrate, lies adjacent to this site. In addition, we have designed and synthesized model tricopper clusters to provide further chemical evidence that a tricopper cluster mediates the enzyme's oxo-transfer chemistry. These biomimetic models exhibit similar spectroscopic properties and chemical reactivity to the putative tricopper cluster in pMMO. Based on computational analysis using density functional theory (DFT), triangular tricopper clusters are capable of harnessing a "singlet oxene" upon activation by dioxygen. An oxygen atom is then inserted via a concerted process into the C-H bond of an alkane in the transition state during hydroxylation. The turnover frequency and kinetic isotope effect predicted by DFT show excellent agreement with experimental data.

Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes

Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 % similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.

Evidence that OmcB and OmpB ofGeobacter sulfurreducensare outer membrane surface proteins

The c-type cytochrome (OmcB) and the multicopper protein (OmpB) required for Fe(III) oxide reduction by Geobacter sulfurreducens were predicted previously to be outer membrane proteins, but it is not clear whether they are positioned in a manner that permits the interaction with Fe(III). Treatment of whole cells with proteinase K inhibited Fe(III) reduction, but had no impact on the inner membrane-associated fumarate reduction. OmcB was digested by protease, resulting in a smaller peptide. However, immunogold labeling coupled with transmission electron microscopy did not detect OmcB, suggesting that it is only partially exposed on the cell surface. In contrast, OmpB was completely digested with protease. OmpB was loosely associated with the cell surface as a substantial portion of it was recovered in the culture supernatant. Immunogold labeling demonstrated that OmpB associated with the cell was evenly distributed on the cell surface rather than localized to one side of the cell like the conductive pili. Although several proteins required for Fe(III) oxide reduction are shown to be exposed on the outer surface of G. sulfurreducens, the finding that OmcB is also surface exposed is the first report of a protein required for optimal Fe(III) citrate reduction at least partially accessible on the cell surface.

Modular syntheses of multidentate ligands with variable N-donors: applications to tri- and tetracopper(i) complexes

A general method for the preparation of multidentate ligands comprised of a multi-imine platform derived from 1,1,1-tris(aminomethyl)ethane or tris(aminoethyl)amine connected to bi- and tridentate N-donor chelates has been developed. The feasibility of the method has been demonstrated through the synthesis and characterization of a large set of these ligand types. Complexation to Cu(I) was accomplished for several cases, yielding tri- and tetracopper(I) complexes that have been characterized in solution by NMR spectroscopy and conductivity, and in the solid state by elemental analysis, mass spectrometry, and/or X-ray crystallography. These complexes are potentially useful for modeling multicopper protein active sites.

A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to L-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.

Reduction thermodynamics of the T1 Cu site in plant and fungal laccases

The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV-vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the Eo' of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same Eo', as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.

Function and molecular evolution of multicopper blue proteins

Function and molecular evolution of multicopper blue proteins

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ⇔ U ⇒ F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria.

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.

Laccases: structure, reactions, distribution

Laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various aromatic and non-aromatic compounds by a radical-catalyzed reaction mechanism. The enzymes are involved in the pathogenicity, immunity and morphogenesis of organisms and in the metabolic turnover of complex organic substances such as lignin or humic matter. Owing to their high non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. Until recently, laccases were only found in eukaryotes (fungi, higher plants, insects), but now there is strong evidence for their widespread distribution in prokaryotes and the first crystal structure of a bacterial laccase is already available. Phylogenetically, laccases are members of the multi-copper protein family including ascorbate oxidase, ceruloplasmin and bilirubin oxidase.