Bacterial Mn 2+ Oxidizing Systems and Multicopper Oxidases: An Overview of Mechanisms and Functions

Geomicrobiology Journal 171ndash24 2000Copyright Cdeg 2000 Taylor amp Francis0149-0451 00 $1200 + 00

Bacterial Mn2+ Oxidizing Systems andMulticopper Oxidases An Overview

of Mechanisms and Functions

G J BROUWERSE VIJGENBOOMP L A M CORSTJENSJ P M DE VRINDE W DE VRIND-DE JONG

Leiden Institute of ChemistryLeiden UniversityLeiden The Netherlands

Manganese is oxidized by a wide variety of bacteria The current state of knowledgeon mechanisms and functions of Mn2+ oxidation in two strains of Pseudomonas putidain Leptothrix discophora SS-1 and in Bacillus sp strain SG-1 is reviewed In all threespecies proteins bearing resemblance to multicopper oxidases appear to be involved inthe oxidation process A short description of the classi cation of Cu centers is followedby a more detailed review of properties and postulated functions of some well-knownmulticopper oxidases Finally suggestions are made for future research to assess thepotential role of multicopper oxidases in bacterial Mn2+ oxidation

Keywords Mn2+ oxidation multicopper oxidases Leptothrix discophoraPseudomonas putida Bacillus sp SG-1

Manganese an essential element for all living organisms can occur in oxidation states rang-ing from iexcl 3 in some organometallic compounds to +7 in the permanganate ion only the +2+3 and +4 states are of biological signi cance however Trace quantities of manganese arecommon throughout the microbial plant and animal kingdoms Manganese can act as an ac-tivator of enzymes for instance DNA polymerase and phosphoenolpyruvate carboxykinase(see Schramm and Wedler 1986) Because the transition metal is redox-active under physi-ological conditions it plays an important role in biological redox reactions especially thoseinvolving interactions with oxygen Well-known examples of manganese redox enzymesinclude the water-oxidizing complex of photosynthesis (Barber 1984 Dismukes 1986) Mnsuperoxide dismutase (Beyer and Fridovich 1986) and Mn pseudocatalase (Dubinina 1978)

The bioavailability of manganese is strongly in uenced by the factors that determineits oxidation state Mn(II) is usually soluble as an ion or complexed to organic or inorganicligands Mn(III) unless complexed to ligands or incorporated in enzymes is unstable in

Received 27 July accepted 23 September 1999The authors are grateful to Nora Goosen for critically reading the manuscript and fruitful discussions on its

contents and are indebted to Michiel de Kuijper and Walbert Bakker for their assistance during the preparation ofthe manuscript

Address correspondence to Dr Liesbeth W de Vrind-de Jong Leiden Institute of Chemistry GorlaeusLaboratories Leiden University PO Box 9502 2300 RA Leiden The Netherlands E-mail Vrind echemleidenunivnl

1

2 G J Brouwers et al

aequous environments and readily disproportionates to Mn2+ and MnO2 Mn(IV) formshighly insoluble oxyhydroxide s and oxides In natural systems Mn2+ oxidation is ther-modynamically favorable but often proceeds at very low rates (Diem and Stumm 1984Nealson et al 1988) Abiotic Mn2+ oxidation can be catalyzed by extreme environmentalconditions (high pH and high O2 pressure) and by adsorption of the ions on mineral surfacessuch as Fe oxides and silicates (Morgan and Stumm 1964 Sung and Morgan 1981 Hemand Lind 1983 Murray et al 1985 Davies and Morgan 1989) Mn(IV) reduction is favoredby the presence of reducing agents under anaerobic conditions low pH or the presence ofMn2+ -complexing agents

The conversions of Mn between oxidation states can be catalyzed by living organ-isms especially microbes Many anaerobic bacterial species are able to reduce Mn oxideseither by production of acids or reducing substances such as sul des or by using the oxi-dized metal as an electron acceptor in respiration (Lovley 1991 Nealson and Myers 1992Nealson and Little 1997) In aerobic environments a wide variety of microorganisms cat-alyze the oxidation of Mn2+ (Ehrlich 1984 Ghiorse 1984) Compared with abiotic Mn2+

oxidation microorganisms can accelerate the oxidation by as much as ve orders of mag-nitude (Nealson et al 1988 Tebo 1991 Wehrli 1990) In strati ed environments reducingand oxidizing microbial species can provide a rapid cycling of Mn around the oxicanoxicboundary In these milieus Mn can serve as a redox shuttle in the oxidation and (potentially)reduction of organic carbon (Figure 1) (Nealson and Myers 1992)

Microbial Mn2+ oxidation proceeds through indirect or direct mechanisms Indirectmechanisms include the production of O2 (in photosynthesis) and of alkaline or oxidizingmetabolites Direct Mn2+ oxidation involves the microbial production of speci c macro-molecules (polysaccharides or proteins) catalyzing the process In several bacterial generaenzymes have been shown to be involved in Mn2+ oxidation (Ehrlich 1968 1983 Jung andSchweisfurth 1979 Douka 1980 de Vrind et al 1986a Adams and Ghiorse 1987 Boogerdand de Vrind 1987 Okazaki et al 1997) Three Mn2+ -oxidizing species have been studied in

FIGURE 1 Schematic representation of the cycling of Mn around the oxicanoxic boundaryand the potential selection between Mn cycling and the cycling of carbon As has beenshown (see text for references) organic carbon can be oxidized during Mn(IIIIV) reductionCarbon dioxide xation during Mn(II) oxidation is still a topic of discussion

Bacterial Mn2+ Oxidation and Multicopper Oxidases 3

detail in several laboratories including our own the fresh-water Gram-negative organismsLeptothrix discophora and Pseudomonas putida and the marine Gram-positive bacteriumBacillus species SG-1 Until recently common principles underlying Mn2+ oxidation in thedifferent species could not be recognized and the functional signi cance of the process hasremained obscure Six years ago molecular biological techniques were introduced in the eld of microbial Mn2+ oxidation an approach that has led to identi cation of several genesinvolved in the oxidation of Mn2+ The most exciting outcome of these investigations is the nding that in all three species mentioned above copper-dependent enzymes appear to playa role in Mn2+ oxidation This is the rst evidence that different species use similar toolsin the oxidizing process In this review we aim to summarize possible functions current ndings and properties of these (and related) copper proteins

Possible Functions of Bacterial Mn2+ Oxidation

Many studies have been devoted to the question of whether bacteria can grow with Mn2+

as an energy source Because the reaction Mn2+ + 12O2 + H2O MnO2 + 2H+ has astandard free energy change at pH 7 of iexcl 70 kJ mol iexcl 1 in principle Mn2+ oxidation cansustain bacterial growth Models have been proposed in which electron transport by wayof an electron transport chain generates a proton gradient that permits ATP formation byoxidative phosphorylation (Figure 2) (Ehrlich 1976 1996 1999 Tebo et al 1997) ATP

FIGURE 2 Schematic representation of a model suggesting the generation of energy (ATP)as the result of bacterial manganese oxidation Electrons from Mn2+ enter the electrontransport chain via a Mn oxidase Protons released during the oxidation of Mn2+ togetherwith proton consumption upon oxygen reduction generate a proton gradient across themembrane which results in the generation of ATP via ATPase Dotted arrow In view of thedifference in redox potential between the Mn(IV)Mn(II) and cytochrome c oxred redoxcouples electrons from Mn(IV) will have to travel through reverse electron transport toreduce cytochrome c


synthesis coupled to Mn2+ oxidation has been reported in the Gram-negative marine strainSSW22 (Ehrlich 1983 Ehrlich and Salerno 1990) and involvement of proteins containingc-type hemes such as cytochromes in Mn2+ oxidation has been proposed (Arcuri and Ehrlich1979 Ehrlich 1983 Tebo et al 1997 Caspi et al 1998 de Vrind et al 1998) A ribulose-15-biphosphate carboxylase gene suggesting the potential for autotrophic growth has beenidenti ed in a marine Mn2+ -oxidizing species (Caspi et al 1996) To date however nobacterial species has been shown to be capable of autotrophic growth on Mn2+ Perhapssome mixotrophic bacteria derive supplemental energy from Mn2+ oxidation especiallyin carbon-limited environments (Ehrlich 1976) So far the question as to whether Mn2+

oxidation yields useful energy can not be decisively answeredThe Mn oxides produced by Mn2+ -oxidizing organisms have been ascribed a protective

function They may protect cells from UV damage or because of their strong adsorptiveproperties from toxicity by heavy metals Mn2+ -oxidizing enzymes may be involved indetoxi cation of such harmful oxygen species as superoxide and peroxide (Archibald andFridovich 1981) in analogy with superoxide dismutases and catalases (Dubinina 1978) Theprolonged viability of Mn oxide-encrusted L discophora cells has been ascribed to one or acombination of these effects (Adams and Ghiorse 1985) Mn oxides may also defend againstpredation or viral attack (Emerson 1989) Insight into the possible protective function ofMn2+ oxidation calls for detailed studies on the resistance of oxidizing wild-type strainsagainst harmful environmental factors as compared with nonoxidizing mutant strains Sofar such studies have not been performed

Recently many microbial species have been described that are able to couple anaerobicrespiration of organic carbon with reduction of metals including oxidized Mn (Lovley andPhillips 1988 Myers and Nealson 1990 Nealson and Myers 1992) Some investigators havespeculated that Mn2+ -oxidizing organisms accumulate Mn oxides as electron acceptors forsurvival under anaerobic or microaerophilic conditions (Tebo 1983 de Vrind et al 1986b)Various Mn2+ -oxidizing species have been shown to reduce the Mn oxides produced aero-bically under low oxygen or anaerobic circumstances (Brom eld and David 1976 de Vrindet al 1986b Ehrlich 1988) Because anaerobic growth of these species with Mn oxide asan electron acceptor has not been demonstrated the functional signi cance of the reductionprocesses in these organisms is not resolved

An interesting possibility for the function of microbial Mn2+ oxidation has been pro-posed by Sunda and Kieber (1994) They showed that Mn oxides oxidize complex humicsubstances releasing low molecular mass organic compounds (eg pyruvate) that can serveas substrates for bacterial growth This implies that Mn2+ -oxidizing bacteria may survivein nutrient-poor environments by producing a strong oxidizing agent able to degrade bio-logically recalcitrant carbon pools (cf Tebo et al 1997) In this respect Mn2+ -oxidizingbacteria may be compared with wood-degrading fungi which secrete peroxidases and lac-cases able to lyse lignin (see later sections) Fungal manganese peroxidases catalyze theformation of Mn(III) complexes which can subsequently oxidize phenolic compounds

Despite numerous investigations pointing to speci c roles for enzymatic Mn2+ oxi-dation by bacteria unequivocal evidence for the various functional models is still lackingOnly elucidation of the mechanisms of the oxidation process in individual bacterial speciesand identi cation of the cellular components involved will eventually give insight in thefunction(s) of Mn2+ oxidation

Mn2+ Oxidation in Pseudomonas putida

P putida is a heterotrophic aerobic fresh-water and soil proteobacterial species Two Mn2+ -oxidizing strains have been studied with biochemical and genetic techniques P putida


MnB1 (formerly called P manganoxydans) was isolated from a manganese crust in a waterpipeline by Schweisfurth (1973) P putida GB-1 isolated by Nealson (cf Corstjens 1993)was previously called P uorescens based on partial 16S rRNA gene sequencing (Okazakiet al 1997) More extensive 16S rRNA gene sequencing and physiological characteristicshave con rmed its identi cation as a putida strain (de Vrind et al 1998)

Mn2+ oxidation in strains MnB1 and GB-1 shows similar characteristics The highestactivity is obtained in the early stationary growth phase (DePalma 1993 Okazaki et al 1997)possibly induced by nutrient starvation (Jung and Schweisfurth 1979 DePalma 1993) Theoxidizing activity also appears to depend on the oxygen concentration in the culture duringgrowth In strain GB-1 the activity per cell doubled when the oxygen concentration wasincreased from 20 to 30 saturation whereas the growth rates of the cells were not affected(Okazaki et al 1997) Both strains deposit the oxides on the cell surface an indication thatone or more components of the oxidizing apparatus are localized at the outer membrane(Okazaki et al 1997) Mn2+ oxidation depends on one or more enzymes as indicated by itskinetics (Km raquo 10 l M) pH optimum (pH 7) temperature optimum (35plusmnC) and inhibitionby HgCl2 The oxidizing activity is also inhibited by NaN3 KCN EDTA Tris and o-phenanthroline indicating that a redox protein containing metal cofactors is involved in theprocess (Okazaki et al 1997)

After partial puri cation of the oxidizing activity of strain GB-1 polyacrylamide gelelectrophoresis of the puri ed preparation revealed oxidizing factors of raquo 250 and 180 kDa(Okazaki et al 1997) An oxidizing factor of 130 kDa has also been reported (Corstjens1993) and the Mn2+ -oxidizing enzyme has been suggested to be part of a complex that candisintegrate into smaller fragments at least some of which retain oxidizing activity (Okazakiet al 1997) Incorporation of the Mn2+ -oxidizing protein in a complex may explain whythe activity is enhanced by treatment of the crude preparation with sodium dodecyl sulfate(SDS) and proteases at low concentrations These agents possibly expose active sites thatwere formerly less accessible for Mn2+

Both P putida MnB1 and GB-1 are readily accessible to genetic manipulation Transpo-son mutagenesis has been applied to isolate mutants defective in Mn2+ oxidation (Figure 3A)(Corstjens 1993 Brouwers et al 1998 1999 Caspi et al 1998 de Vrind et al 1998) Thereader is referred to these studies for detailed information Here we will summarize thecharacterization of nonoxidizing mutants of P putida MnB1 and GB-1 and highlight someof the conclusions drawn from the mutagenesis experiments P putida GB-1 lost its abilityto oxidize Mn2+ by transposon insertion into a gene called cumA that encodes a proteinrelated to multicopper oxidases (Brouwers et al 1999) That the CumA protein could bea component of the putative Mn2+ -oxidizing complex is supported by the observation thatCu2+ ions added during growth of bacteria stimulated the oxidizing activity The genecumA with cumB occurs in a two-gene operon that encodes a potential membrane protein(Brouwers et al 1999) Mutation of cumB did not affect Mn2+ oxidation (Brouwers et al1999) but did result in decreased growth

In both P putida MnB1 and GB-1 mutations in genes of the cytochrome c maturationoperon (ccm) abolished the synthesis of c-type cytochromes as well as the Mn2+ -oxidizingactivity (Brouwers et al 1998 Caspi et al 1998) This may point to involvement of theelectron transport chain in Mn2+ oxidation Alternatively a c-type heme is next to CumApart of the putative Mn2+ -oxidizing complex mentioned above It is also possible that oneof the enzymes from the cytochrome c biogenesis pathway has a dual function and plays arole in Mn2+ oxidation in an unknown manner (cf Yang et al 1996 Gaballa et al 1998)For instance two of the ccm genes encode thioredoxin proteins with CXXC motifs that caninteract with Cu ions These proteins have been proposed to play a role in Cu metabolism inaddition to cytochrome c biogenesis (Yang et al 1996) Interference with their expression


FIGURE 3 (A) Transposon insertion in several genes of the Mn2+ -oxidizing P putidaGB-1 results in a change of oxidizing brown colonies (left) into a nonoxidizing whitephenotype (right) For details see text (B) Sodium dodecyl sulfatendashpolyacrylamide gelelectrophoresis of spent medium of L discophora SS-1 Lanes (a) molecular mass markers(1 205000 2 116000 3 97000 4 66000 5 45000 Da) (b) spent medium stained forprotein with Coomassie brillant blue (c) spent medium stained for Fe2+ -oxidizing activityby immersion of the gel in (NH4)2Fe(SO4)2 solution (d) spent medium stained for Fe2+ -oxidizing activity and then with Coomassie brillant blue (the arrow indicates the Fe-stainedband) (e) spent medium stained for Mn2+ -oxidizing activity by immersion of the gel inMnCl2 solution Figure 3B is reprinted with permission from Corstjens et al (1992)

may affect Mn2+ -oxidizing activity if the multicopper oxidase is part of the oxidizingcomplex

Mutations in genes involved in the general secretion pathway (GSP) for protein se-cretion across the outer membrane resulted in a nonoxidizing phenotype (Brouwers et al1998) Full activity was recovered after disruption of the cell walls This indicates that atleast one of the components of the oxidizing complex is a substrate of the GSP machinerycon rming its location in the outer membrane

Mn2+ Oxidation in Leptothrix discophora

Bacterial species belonging to the genus Leptothrix are able to oxidize Fe2+ as well asMn2+ (Dondero 1975 Van Veen et al 1978) One of them L discophora is a sheath-forming heterotrophic fresh-water bacterium commonly found in habitats with an aerobicndashanaerobic interface where manganese and iron cycle between their oxidized and reducedforms During its stationary growth phase L discophora oxidizes Mn2+ and Fe2+ (Adamsand Ghiorse 1987 Boogerd and de Vrind 1987) In its natural environment L discophoradeposits the oxides on the sheaths surrounding the cells (van Veen et al 1978) and at


least part of the Mn2+ -oxidizing system is thought to consist of sheath components Theability of Leptothrix species to form a structured sheath is easily lost when cultured in thelaboratory One such sheathless strain L discophora SS-1 secretes its Fe2+ - and Mn2+ -oxidizing factors into the culture medium (Figure 3B) (Adams and Ghiorse 1987) In thisstrain both Fe2+ and Mn2+ oxidation appear to be enzymatically catalyzed (Adams andGhiorse 1987 Boogerd and de Vrind 1987 de Vrind-de Jong et al 1990) The Km valuesfor Fe2+ and Mn2+ are both raquo 10 l M comparable with the Km of Mn2+ oxidation in Pputida Both processes are heat-sensitive and inhibited by enzyme poisons such as HgCl2KCN and NaN3 Inhibition of Mn2+ oxidation by o-phenanthroline points to involvementof a metal cofactor in this process (Adams and Ghiorse 1987)

Whether Mn2+ and Fe2+ are oxidized by different enzymes or by identical or closelyrelated components is not known The secreted activities have shown different behaviorsfor instance upon ultra ltration (de Vrind-de Jong et al 1990) and isoelectric focusing(de Vrind-de Jong unpublished observations) Analysis of concentrated spent medium bydenaturing gel electrophoresis revealed several discrete factors ranging from 50 to 180 kDaSome of these factors oxidize Fe2+ some oxidize Mn2+ and some are able to oxidize bothmetals (Boogerd and de Vrind 1987 Corstjens et al 1992) These results may indicate thatdifferent factors are involved in Mn2+ and Fe2+ oxidation However the data may also beexplained by assuming that the oxidizing components are part of a complex consisting ofsimilar or related components in variable ratios (Brouwers 1999)

Secretion of the oxidizing factor(s) as part of a complex is consistent with the ob-servation that electrophoretic analysis of concentrated spent medium under nondenaturingconditions does not reveal discrete oxidizing factors (Adams and Ghiorse 1987 Corstjens1993) The proposed complex has been suggested to originate from membranous blebs(Adams and Ghiorse 1986) Incorporation of the active oxidizing sites in a membranecomplex can explain their relative insensitivity to or even slight stimulation by SDS andproteases As already described a similar situation exists in P putida GB-1

The oxidizing enzymes are dif cult to isolate probably because they are secretedas part of a complex (Adams and Ghiorse 1987) Only microgram amounts of a 110-kDa Mn2+ -oxidizing factor (called MOF) have been isolated (Corstjens 1993 Corstjenset al 1997) it consists of protein and probably polysaccharide (Adams and Ghiorse 1987Emerson and Ghiorse 1992) This partially puri ed preparation has been used to raiseantibodies ( a -MOF) Screening an L discophora expression library with a -MOF resultedin the identi cation of a single gene called mofA Because this gene was detected withantibodies against an isolated Mn2+ -oxidizing factor it very likely encodes a structuralcomponent of the Mn2+ -oxidizing system of L discophora Interestingly mofA appears toencode a protein (of raquo 180 kDa) related to multicopper oxidases (Corstjens et al 1997) andaddition of 40 l M Cu2+ to a growing culture increased Mn2+ oxidation by raquo 80 whencorrected for equal cell numbers (Brouwers et al 1999)

The mofA gene was suggested to be part of an operon (Corstjens et al 1997) anassumption supported by recent data (Brouwers 1999) One of the genes belonging tothis operon encodes a protein with a potential heme-binding site which may be a furtherindication that c-type hemes play a role in Mn2+ oxidation in addition to the putativemulticopper oxidase

Mn2+ Oxidation in Bacillus SG-1

Bacillus strain SG-1 a Gram-positive marine organism isolated from a near-shore manga-nous sediment (Nealson and Ford 1980) forms inert endospores upon nutrient limitationThe germ cells of the spores are surrounded by a peptidoglycan cortex covered by highly


cross-linked spore coat proteins The outermost spore layer is an exosporium a membranousstructure differing in composition from the spore coat (Francis et al 1997) The sporescatalyze the oxidation of Mn2+ (Rosson and Nealson 1982) their oxidative properties haverecently been reviewed (Francis and Tebo 1999) The oxidizing activity has been localizedin the exosporium (Francis et al 1997) and its kinetics heat sensitivity and inhibition byprotein poisons such as NaN3 and HgCl2 suggest that an exosporium protein is involved inthe process (Rosson and Nealson 1982 de Vrind et al 1986a)

The highly cross-linked and resistant structure of outer spore coverings has prohibitedthe isolation of Mn2+ -oxidizing factors from spores (Tebo et al 1988) A Mn2+ -oxidizingprotein band of raquo 205 kDa was identi ed after gel electrophoresis of spore coatexosporiumextracts but these results have been dif cult to reproduce (Tebo et al 1997)mdashprobablybecause of the separation during gel electrophoresis of the diverse components necessaryfor Mn2+ oxidation

Transposon mutagenesis has been more successful in identifying components involvedin Mn2+ oxidation (Van Waasbergen et al 1993 1996) Mutants still able to form en-dospores but de cient in Mn2+ oxidation appear to contain transposon insertions in genesof an operon called mnx The mnx genes are transcribed during midsporulation when sporecoat protein production is initiated The operon consists of seven genes (mnxA to mnxG)two of which encode proteins with sequence similarity to other proteins found in databasesMnxG shares sequence similarity with members of the family of multicopper oxidasesits subdomain structure is typical of multicopper oxidases ceruloplasmin in particular(Solomon et al 1996 see also later sections) Stimulation of Mn2+ oxidation by Cu2+

ions (Van Waasbergen et al 1996) indicates that MnxG is an essential component of theMn2+ -oxidizing complex

MnxC shows limited sequence similarity to redox-active proteins (Tebo et al 1997) Itcontains the sequence CXXXC which resembles the thioredoxin motif CXXC ThereforeMnxC has been suggested to play a role in a redox process An alternative function of theconserved cysteine residues of MnxC in the transport of Cu2+ possibly to the multicopperoxidase MnxG has also been envisaged (Tebo et al 1997)

Because Bacillus spores are metabolically inert their Mn2+ -oxidizing capacity is notdirectly related to metabolic function Bacillus SG-1 is one of the organisms for whichthe vegetative cells have been shown to reduce the manganese oxide upon germinationof the spores (de Vrind et al 1986b cf section ldquoPossible functions of manganese oxida-tionrdquo) Manganese oxide reduction appeared to be coupled to oxidation of b- and c-typecytochromes (de Vrind et al 1986b) However as also stated above de nite proof that cellsof Bacillus SG-1 can grow with manganese oxide as a terminal electron acceptor is stilllacking

In summary the studies on the three Mn2+ -oxidizing species described suggest thatmulticopper oxidases form an essential component of the Mn2+ -oxidizing systems in oxi-dizing bacterial species in general By using different approaches proteins with sequencesimilarity to multicopper oxidases were shown to be involved in the oxidizing process in allthree species Moreover in all three species Mn2+ oxidation could be stimulated by Cu2+

ions either added during growth of the bacteria (P putida and L discophora) or addedto partly puri ed Mn2+ -oxidizing preparations (spore coats of Bacillus SG-1) RecentlyCu2+ was shown to enhance the oxidizing activity of yet another Mn2+ -oxidizing speciesPedomicrobium sp ACM 3076 (Larsen et al 1999) Although multicopper oxidases as acommon theme in bacterial Mn2+ oxidation seems well established at present commonmechanisms or functions cannot be distinguished except for the possible involvement ofc-type hemes in P putida and L discophora The potential multicopper oxidases do notshow sequence similarity outside the putative Cu-binding domains Moreover the molecular


FIGURE 4 Comparison of the genomic regions anking the putative multicopper oxidase-encoding genes from P putida GB-1 (cumA) L discophora SS-1 (mofA) and Bacillus SG-1(mnxG) Vertical bars designated A B C and D indicate the Cu-binding regions Note thatin the MnxG protein the C- and N-terminal Cu-binding domains are reversed in comparsionwith the other putative multicopper oxidases and that an extra Cu-binding domain appearsto be present For descriptions of the diverse gene products see text

organizations of the operons to which the encoding genes apparently belong differ greatlyfrom one another (Figure 4) Can this imply that the ability to oxidize Mn2+ evolved froman oxidation process with a primary function other than metal oxidation This question ledus to review brie y the properties and functions of Cu-dependent proteins in particular thewell-known multicopper oxidases

Copper Proteins

Copper became available as a cofactor in proteins during oxygenation of the atmosphere Inthe rst two billion years of life the reducing environment locked the metal in its reducedform which precipitated as highly insoluble sul des Copper proteins contain one or moreCu ions as a cofactor and generally function in redox reactions with one or more Cu centersas the active sites sometimes in cooporation with other redox centers such as heme Insome proteins with sequence similarities to Cu redox proteins the Cu ions are bound butnot redox-active Such proteins function in Cu metabolism transport and resistance

Cu sites have historically been divided into three classes based on their coordinationenvironment and geometry (Canters and Gilardi 1993 Solomon et al 1996) The differ-ences in coordination characteristics are re ected in their spectroscopic properties such asabsorption and electron paramagnetic resonance (EPR) spectra In type I sites one Cu ionis coordinated with three ligands (one sulfur and two nitrogens) in an equatorial planedonated by one cysteine and two histidine residues (Figure 5A) In some type I Cu sites afourth axial ligand is provided by the thioether sulfur of a methionine In the oxidized statetype I Cu proteins have a strong absorption maximum at raquo 600 nm giving them an intenseblue color hence they are called ldquoblue copper proteinsrdquo The small blue copper proteinsare also referred to as cupredoxins and are characterized by a speci c EPR signal TypeI Cu proteins generally serve in intermolecular electron transport and are not catalyticallyactive They occur in all three kingdoms of life and both sequence and structure analysisshow that they derive from a common ancestor (Ryden and Hunt 1993) Some examplesare listed in Table 1

Type II Cu proteins also contain one Cu ion which is coordinated with four N or Oligands in a square planar con guration around the metal (Figure 5A) They do not absorbin the visible light spectrum and their EPR spectrum discriminates them from type I sitesThey can be involved in the catalytic oxidation of substrate molecules and can act in isolationor in cooporation with type III sites (Table 1 see also below) Type II Cu proteins show littleor no phylogenetic homology and are thought to be the products of convergent evolution(Abolmaali et al 1998)


TABLE 1 Examples of copper-binding proteins and thedifferent types of copper-binding sites

Number ofCopper proteins coppers Type

Azurin plastocyanin amicyanin 1 ISuperoxide dismutase galactose 1 II

oxidase amine oxidaseTyrosinase hemocyanin 2 IIINitrite reductase 2

1 I1 II

Cytochrome c oxidase 32 CuA

1 heme a3ndashCuB

Laccases ascorbate oxidase 4Fet3p PcoA CopAa 1 I

1 II2 III

Ceruloplasmin 63 I1 II2 III

aCopA binds 11 coppers in total four as shown here and seven by anunknown manner (Mellano and Cooksey 1988) For detailed informa-tion and functions see text and Solomon et al 1996 Pouderoyen 1996Kroes 1997

FIGURE 5 (A) Types I II and III copper centers in multicopper oxidases R = methioninein several type I sites (eg azurin see also Figure 4) L = O or N ligands (B) Schematicrepresentation of the orientation of the type II copper with respect to type III dimer in atrinuclear cluster also called type IV


Type III Cu sites are dinuclear sites containing two closely situated Cu ions (Figure5A) they do not exhibit an EPR signal because of their antiferromagnetic coupling Theycan serve as oxygen carriers or can activate oxygen during the hydroxylation of phenolicsubstrates (Table 1) In the oxygen-bound state they are characterized by a strong absorptionmaximum at 330 nm Type III Cu proteins are thought to have evolved by combination ofsimple as yet unknown mononuclear Cu centers (Abolmaali et al 1998)

In the 1990s additional Cu sites have been de ned on the basis of spectroscopic dataincluding the dinuclear CuA and CuZ centers and the CuBndashhemeA site (Table 1) (Malmstrom1990 Dennison and Canters 1996 Farrar et al 1998) A trinuclear site consisting of type IIand type III centers is sometimes called type IV (Messerschmidt et al 1992) (Figure 5B)Trinuclear sites often occur in proteins with a type I site as well The latter proteins aregenerally called multicopper oxidases or blue oxidases

Multicopper oxidases display internal sequence homology allowing the identi cationof subdomains The subdomain structure of some oxidases has been con rmed by X-raycrystallography (Zaitseva et al 1996) Each domain is characterized by an eight-strandedGreek b -barrel rst observed in the small blue copper proteins azurin and plastocyanin(cf Murphy et al 1997) hence the name for this structure the ldquocupredoxin foldrdquo The phy-logenetic relationship between the individual domains of multicopper oxidases and the bluecopper proteins indicates that the multicopper oxidases evolved from the latter by domainduplication (Ryden and Hunt 1993 Murphy et al 1997 Abolmaali et al 1998) Extensionand modi cation of domains resulted in the rearrangement of Cu-binding sites and enabledthe formation of new types of Cu centers with catalytic activity (Abolmaali et al 1998) Thetype I center in multicopper oxidases still serves its ancestral function in that it shuttles elec-trons from the electron donor to the catalytic center (also see below) Small modi cationsin the protein environment of the Cu sites allowed the ne-tuning of their redox potentials tospeci c redox partners (Messerschmidt 1998) This generated a family of oxidase enzymeswith a large variety of redox potentials and substrate speci cities potentially including mul-ticopper oxidases that are able to oxidize transition metals such as Mn The properties andpostulated functions of well-known multicopper oxidases are the subject of the next section

Multicopper Oxidases

Multicopper oxidases are a class of Cu enzymes that can be de ned by their spectroscopiccharacteristics sequence similarity and reactivity Members of this family include plant andfungal laccase plant and bacterial ascorbate oxidase human ceruloplasmin yeast Fet3p andbacterial CopA Recently additional members have been isolated and partly characterizedas discussed below

In general multicopper oxidases contain one type I Cu site and a type II and typeIII center organized in a trinuclear cluster (Solomon et al 1996) Ceruloplasmin is uniqueamong the multicopper oxidases in that it contains two additional type I centers All mul-ticopper oxidases have absorption maxima at raquo 600 nm (type I) or 330 nm (type III) andshow EPR signals characteristic of type I and type II sites

The amino acid sequence similarity in the regions containing Cu-binding ligands issubstantial (Figure 6) Generally two of these regions are situated near the C terminusand separated by 35ndash75 amino acid residues the other two are near the N terminus andseparated by 35ndash60 residues Each of the N- and C-terminal regions contains a conservedHXH motif The eight histidines within the four motifs are the conserved peptide ligands ofthe trinuclear center The type II Cu is coordinated by two of these histidines and each ofthe two Cu ions belonging to the type III site is coordinated by three histidines (Figure 6)


FIGURE 6 Alignment of the (putative) copper binding regions of different multicopperoxidases The binding regions A B C and D correspond to those shown in Figure 4 Thecopper-binding residues are designated I II IIIa or IIIb on the basis of the types of copperthey can potentially bind Abbreviations used and Genbank accession number LacF(ungus)Trametes versicolor laccase (X84683 Jonsson et al 1995) LacP(lant) Acer pseudopla-tanus laccase (U12757 Sterjiades et al 1992) CpAO Cucurbita pepo medullosa ascorbateoxidase (J04494 Ohkawa et al 1989) HsCp Human ceruloplasmin (M13699 Koschinskyet al 1986) Fet3 Saccharomyces cerevisiae Fet3p (L25090 Askwith et al 1994) CopAcopper resistance protein Pseudomonas syringae (M19930 Mellano and Cooksey 1988)PcoA copper resistance protein plasmid pRJ1004 Escherichia coli (X83541 Brown et al1995) CumA Pseudomonas putida GB-1 (AF086638 Brouwers et al 1999) MofA Lep-thothrix discophora SS-1 (Z25774 Corstjens et al 1997) MnxG marine Bacillus sp SG-1(U31081 Van Waasbergen et al 1996)

(Solomon et al 1996) The remaining ligand positions are often occupied by another his-tidine nitrogen and a water molecule The conserved ligands of the type I Cu are locatedin the two C-terminal regions and consist of one cysteine and two histidines (Figure 6)Some of the multicopper oxidase type I Cu atoms are also coordinated by a methionine10 amino acids distant from the cysteine residue Others contain a noncoordinating leucineor phenylalanine at this position (cf Figure 6) The cysteine residue is anked by two ofthe histidines coordinating the trinuclear cluster (Figure 6) This arrangement provides afunctional proximity between the type I Cu and the trinuclear cluster Type I Cu is currentlythought to oxidize the substrate by four subsequent one-electron oxidations The electronsare passed to the trinuclear cluster through the cysteinendashhistidine pathway (Lowery et al1993) The trinuclear cluster is the site of the four-electron reduction of oxygen to waterprobably through a peroxide intermediate (Sundaram et al 1997) Virtually all multicopperoxidases consist of three cupredoxin domains whereas ceruloplasmin consists of six (theputative multicopper oxidase from Bacillus SG-1 also contains six subdomains see above)

Most multicopper oxidases oxidize organic substrates At present only ceruloplasminand Fet3p are known to directly catalyze the oxidation of a metal ion in this case Fe2+ (seebelow) One fungal laccase has recently been shown to be able to directly use Mn2+ as asubstrate (Hofer and Schlosser 1999 see also below) Some multicopper oxidase homologssuch as CopA do not seem to be redox-active at all (see below) Multicopper oxidases occuras enzymes with low (Km in the millimolar range) or high (Km in the micromolar range)substrate speci city Laccases generally fall into the former category and are not supposedto contain a substrate-binding pocket in contrast to the multicopper oxidases with highsubstrate speci city such as ascorbate oxidase and ceruloplasmin


Laccase

Laccase is a polyphenol oxidase (p-diphenoldioxygen oxidoreductase) rst identi ed inthe Japanese lacquer tree Rhus vernicifera at the end of the nineteenth century (Yoshida1883) Since then it has been detected in a variety of plants (OrsquoMalley et al 1993) and shownto be a ubiquitous fungal enzyme as well (Thurston 1994) Laccase activity has also beenfound in insects (Binnington and Barrett 1988) and in one bacterium Azospirillum lipoferum(Givaudan et al 1993) Recently a laccase-like pluripotent polyphenol oxidase was iden-ti ed in a marine Alteromonas species (Sanchez-Amat and Solano 1997) Laccases havereceived a great deal of attention in view of their application in industrial oxidative processessuch as deligni cation dye bleaching and ber modi cation (Xu et al 1996 Xu 1996)

Laccases can oxidize a broad spectrum of aromatic substrates including o- and p-diphenols methoxy-substituted phenols and methoxy-substituted diamines and substratespeci cities vary from one laccase to another (Thurston 1994) Laccases have been shownto catalyze the formation of Mn(III) chelates from Mn2+ either through oxidized phenolicintermediates (Archibald and Roy 1992) or directly in the presence of the chelator sodiumpyrophosphate (Hofer and Schlosser 1999)

Many plant and fungal species produce isoforms of laccase encoded by multigenefamilies (Mansur et al 1998 Ranocha et al 1999) The functions of the different laccasesare as yet unresolved and may depend on the organism or type of tissue in which they areexpressed (Ranocha et al 1999) Most notably they are implicated in the biosynthesis (byplants) or degradation (by fungi) of lignin a complex aromatic biopolymer

Plant laccases have been puri ed from several species allowing biochemical charac-terization immunolocalization and measurements of in vitro activity Laccase has beenimmunolocalized in the cell walls of lignifying cells of Acer stems (Driouich et al 1992)and laccase-like activity has been detected histochemically in lignifying xylem tissue ofherbaceous and woody species (Bao et al 1993 OrsquoMalley et al 1993 Liu et al 1994McDouglas et al 1994 McDouglas and Morrison 1996) Several isolated plant laccaseshave been shown to catalyze the dehydrogenative polymerization of monolignols (ligninmonomers) (Sterjiades et al 1992 Bao et al 1993) Laccase genes were preferentiallyexpressed in differentiating xylem tissue of poplar (Ranocha et al 1999) These data con-vincingly point to involvement of laccase in lignin formation incontrast to previous opinionsproposing an exclusive role for lignin peroxidases in lignin biosynthesis (Nakamura 1967Harkin and Obst 1973) However neither laccase nor peroxidase alone can account forthe stereospeci city of the monolignol polymerization reaction Sterjiades et al (1993) hy-pothesized that laccases and peroxidases work in a coordinated manner in lignin formationwith laccases producing oligolignols and peroxidases catalyzing the formation of ligninfrom oligolignols In some species moreover eg the lacquer tree laccase does not seemto be involved in ligni cation at all (Nakamura 1967) At present studies are underway todownregulate laccase genes in transgenic plants and to study the effect on lignin contentand composition (Ranocha et al 1999) These experiments may clarify the speci c role ofdifferent laccases in lignin biosynthesis

Some proposed functions of fungal laccases are pigment formation lignin degrada-tion and detoxi cation of phenoxy radicals produced during deligni cation or of phenolsproduced by other organisms (Thurston 1994) The best established function is pigment for-mation A laccase-negative mutant of Aspergillus nidulans produced white spores instead ofthe green spores formed by the wild type Addition of partly puri ed laccase to the growthmedium of the mutant restored the wild-type phenotype (Clutterbuck 1972) The bacterialAzospirillum lipoferum laccase also has a function in pigment formation (Givaudan et al1993) The pigments may protect the microbial cells from radiation damage or from attackby cell wallndashdegrading enzymes (Givaudan et al 1993)


Because laccases are ubiquitous in wood-rotting fungi they are assigned a role inlignin degradation along with hemoprotein peroxidases They are assumed to take part inthe oxidative cleavage of some of the numerous structures in the complex biopolymer Thebest evidence in support of such a role is that a laccase-negative mutant of Sporotrichumpulverulentum was unable to degrade lignin The lignin-degrading ability was restored inlaccase-positive revertants (Ander and Eriksson 1976) Mn(III)-catalyzed degradation oflignin by puri ed laccase in concert with manganese peroxidase has been demonstratedin vitro (Sterjiades et al 1993) As stated above laccase can catalyze the formation ofstrongly oxidizing Mn(III) chelates (Archibald and Roy 1992 Hofer and Schlosser 1999)These Mn(III) chelates like the Mn(III) chelates produced by manganese peroxidase areable to oxidatively degrade lignin However many lignin-degrading fungal strains do notproduce laccase (Thurston 1994) Perhaps a combination of oxidative enzymes manganeseperoxidase and laccase or manganese peroxidase and lignin peroxidase is the minimumrequirement for lignin breakdown (de Jong et al 1992) In conclusion the functions offungal laccases like those of plant laccases are as yet not well established

Ascorbate Oxidase

Ascorbate oxidase which catalyzes the oxidation of ascorbate to dehydroascorbate ismainly found in higher plants especially in members of the Cucurbitaceae (eg cucumbersquash and melon Ohkawa et al 1989) It has been studied extensively biochemically andvarious genes encoding ascorbate oxidases have been isolated and sequenced (Nakamuraet al 1968 Ohkawa et al 1989 1990 Esaka et al 1992 Moser and Kanellis 1994) Insome plants various isoforms are encoded by a multigene family The enzymes are as-sociated with the cell wall (Lin and Varner 1991 Esaka et al 1992) Ascorbate oxidaseis one of the few multicopper oxidases that have been crystallographically characterized(Messerschmidt et al 1992)

Although ascorbate oxidase has been characterized in detail with regard to enzymestructure and expression its role in plant metabolism is still obscure Ascorbate oxidasetranscripts and activities are greatest in actively growing tissues (Ohkawa et al 1989 Linand Varner 1991 Esaka et al 1992) Consequently the enzyme has been suggested to playa role in cell elongation possibly by cell wall loosening as a result of the interaction ofdehydroascorbate with cell wall components (Lin and Varner 1991) Alternatively ascorbateoxidase may function in regulation of the cell cycle by controlling the concentration ofcellular ascorbic acid [ascorbic acid indirectly promotes the progression from the G1 tothe S phase in the cell cycle (Arrigoni 1994)] Ascorbate oxidase may induce cell cyclearrest by lowering the ascorbic acid concentration for instance during early stages of fruitdevelopment when cells do not divide (Diallinas et al 1997) Finally ascorbate is one of thecompounds involved in plant oxidative defense systems (Foyer et al 1994) often activatedunder stress conditions This may explain why ascorbate oxidase expression is repressedunder stress conditions such as wounding (Diallinas et al 1997)

Multicopper Ferrooxidases

Two multicopper oxidases apparently involved in the oxidation of a metal ion instead of anorganic substrate have thus far been characterized ceruloplasmin ubiquitous in vertebratesand Fet3p from the yeast Saccharomyces cerevisiae Both enzymes catalyze the oxidationof Fe2+ (Km lt 10 l M) and their main function is thought to be the mediator of Fe transport(Askwith and Kaplan 1998) They are functional homologs although structurally Fet3p ismore closely related to laccase and ascorbate oxidase than to ceruloplasmin (see below)


Fet3p is an integral membrane protein with anextracellular multicopper oxidase domain(Askwith et al 1994 Stearmann et al 1996) Spectroscopic analysis of isolated recombinantFet3p indicates the presence of one type I Cu site and a trinuclear cluster similar toamong other things laccase (Hassett et al 1998) Fet3p mediates Fe transport by convertingferrous iron into ferric iron the substrate for the permease Ftr1p (Kaplan and OrsquoHalloran1996 Stearman et al 1996) Ferrous iron is generated by cell surface ferrireductaseswhich solubilize extracellular ferric iron pools to make them available for transport Theinvolvement of the multicopper oxidase Fet3p in Fe metabolism is illustrated by the fact thatmutations in Cu transport proteins such as the Fet3p Cu loader Ccc2p result in a de ciencyof Fet3p activity and of high-af nity Fe transport (Dancis 1998) All genes involved in Culoading of Fet3p have human homologs with high degrees of sequence conservation (seebelow)

Ceruloplasmin is to date unique among the multicopper oxidases in that it contains threetype I Cu sites next to a trinuclear cluster (Zaitseva et al 1996) One of the type I sites appearsto be permanently reduced (Machonkin et al 1998) and thus is catalytically irrelevant Oneof the remaining type I sites is connected through a cysteinendashhistidine pathway to thetrinuclear cluster similar to the type I sites in laccase and ascorbate oxidase The functionof the third type I site is still uncertain Recently Machonkin et al (1998) suggested thatthe resting form of ceruloplasmin in plasma under aerobic conditions is a four-electronoxidized form consistent with its function in the four-electron reduction of O2 to H2O

Although isolated ceruloplasmin is able to oxidize aromatic substrates (Solomon et al1996) it oxidizes Fe2+ with much higher af nity Its main function is thought to be the mo-bilization of cellular iron by acting as a ferrooxidase The ferric iron thus produced is boundto plasma transferrin and transported to body tissues The link between iron metabolism andcopper was established years ago in Cu-de cient swine which had low plasma ceruloplas-min activity developed anemia and accumulated iron in several tissues (Lee et al 1968)The human genetic disease aceruloplasminemia (absence of ceruloplasmin) results in de-fects in iron homeostasis including iron accumulation in tissues (Harris et al 1995) Defectsin the MenkesWilson disease protein a Cu transporter result in low concentrations of ac-tive plasma ceruloplasmin and anemia similar to the symptoms in Cu-de cient swine (Bullet al 1993) The MenkesWilson disease protein is highly similar to the yeast Cu-transporterCcc2p substituted for defective Ccc2p it can restore the Cu-loading and activity of Fet3pin ccc2p mutants (Payne and Gitlin 1998) This illustrates the evolutionary conservation ofproteins responsible for homeostasis of transition metals (Askwith and Kaplan 1998)

In contrast to the proposed role of ceruloplasmin in stimulation of iron egress fromcells recent studies with isolated cell cultures have indicated a function in cellular Fe up-take (Attieh et al 1999) Alternatively ceruloplasmin could be involved in Cu metabolismrather than in that of Fe controlling the concentration of Cu and maintaining the redoxbalance in plasma (Leung 1998) or delivering Cu to Cu-dependent enzymes such as cy-tochrome c oxidase and superoxide dismutase (Marceau and Aspin 1973a 1973b Hsieh andFrieden 1975) Studies indicating involvement of ceruloplasmin in superoxide-dependentoxidative modi cation of plasma lipoproteins (Mukhopadhyay and Fox 1998) have furthercomplicated our insights into the physiological role of this multicopper oxidase

Putative Multicopper Oxidase Homologs

Several other multicopper oxidases involved in the oxidation of organic compounds have re-cently been described and partially characterized Bilirubin oxidase sulochrin oxidase anddihydrogeodin oxidase have been identi ed in various fungi and phenoxazinone synthasehas been found in several Streptomyces species (see Solomon et al 1996 for an overview)


The amino acid sequences of these proteins show the conserved Cu-binding domains presentin all multicopper oxidases Studies on overproduced wild-type and mutant bilirubin oxidasehave indicated intramolecular electron transport from the type I Cu to the trinuclear clustervia a cysteinendashhistidine pathway (Shimizu et al 1999) Bilirubin oxidase and phenoxazinoneoxidase show a small but important sequence similarity outside the Cu-binding domains tothe putative multicopper oxidase MofA from L discophora SS-1 (Corstjens et al 1997)

Some multicopper oxidase homologs identi ed in bacterial species do not seem to beredox-active They appear to be involved in Cu resistance for instance CopA a plasmid-borne homolog of multicopper oxidases isolated from P syringae pv tomato (Mellano andCooksey 1988) The copA gene is part of an operon consisting of four genes copA throughcopD All four gene products are involved in Cu resistance but only CopA and CopB areessential Mutation analysis indicates that CopC and CopD are required for full activitybut low-level resistance can be conferred in their absence (Mellano and Cooksey 1988)CopA is localized in the periplasmic space and can bind 11 Cu ions per molecule Cu ionsentering the bacterial cells are thus accumulated in the periplasm Cooksey (1993) proposedthat four Cu atoms are bound to the type I II and III Cu sites and that additional Cu ionsare bound in octapeptide motifs Homologs of the cop gene cluster have been identi edon the chromosome of Xanthomonas campestris pv juglandis (Lee et al 1994) and on theEscherichia coli plasmid pRJ1004 (Brown et al 1995) In the latter organism resistance isbased on reduced Cu uptake instead of Cu accumulation (Cervantes and Gutierrez-Corona1994) Multicopper oxidases involved in Cu resistance appear to depend on their Cu2+

binding not their oxidative potential The possibility cannot be excluded however thatthey serve an as yet unknown oxidative purpose as well

Concluding Remarks and Future Prospects

The oxidation of Mn can be catalyzed by a wide variety of bacterial species The variousoxidizing systems differ in many respects For instance the process can be catalyzed bymetabolically inert spores by cellular outer membrane components or by bacterial sheathsExcept for the intracellular oxidation of Mn2+ by Lactobacillus plantarum which usesMn2+ ions in scavinging superoxide radicals (Archibald and Fridovich 1981) bacterial Mnoxidation appears to be con ned to outer surface coverings Because of diversity in oxidizingsystems it has been hard to formulate unifying concepts on the mechanisms and functionsof the process Only recently has a common theme been revealed Proteins with sequencesimilarity to multicopper oxidases play a role in Mn2+ oxidation in a marine Gram-positiveand two fresh-water Gram-negative species These proteins MnxG MofA and CumA allcontain the highly conserved Cu-binding ligands characteristic of multicopper oxidasesMnxG appears to consist of subdomains The multicopper oxidases ceruloplasmin laccaseand ascorbate oxidase contain subdomains with a speci c so-called cupredoxin fold BothMofA and CumA can also be expected to contain cupredoxin subdomains This can beveri ed by a computerized search for internal homology regions in the proteins The aminoacid sequences can be analyzed for their potential to adapt a cupredoxin fold Cloning therespective genes allows overproduction and puri cation of the proteins For instance partsof MofA have already been produced and puri ed in substantial quantities after expressionin E coli (Brouwers 1999) Optimization of the overproduction systems will open theway for spectroscopic and eventually crystallographic characterization of these putativemulticopper oxidases

The question arises as to whether MnxG MofA and CumA are directly or indirectlyinvolved in Mn2+ oxidation The genes encoding MnxG and CumA have been identi ed bymutagenesis and characterization of nonoxidizing mutants This approach will also detect


genes encoding indirectly involved products for example regulatory genes genes encodingenzymes involved in transport genes responsible for biosynthesis of potential cofactorsand so forth MofA on the other hand has been identi ed by antibodies against an activeMn2+ -oxidizing factor which supports a direct involvement of the multicopper oxidases inMn2+ oxidation

If MnxG MofA and CumA are directly involved in metal oxidation they can becompared with the metal-oxidizing multicopper oxidases ceruloplasmin and Fet3p whichoxidize Fe2+ with a Km comparable with that of bacterial Mn2+ oxidation However neitherMnxG MofA nor CumA is able to oxidize Mn2+ when produced from an expression vectorin E coli (Tebo et al 1997 Brouwers 1999) Spectroscopic analysis of the overproducedproteins should indicate whether Cu ions are correctly incorporated in the protein structureAnalysis of Cu incorporation in Fet3p has shown that speci c genes of the Cu metabolismhave to be operative for Fet3p to attain activity Such genes may be absent or silent in Ecoli To circumvent potential dif culties in the production of active multicopper oxidasesbecause of the absence of metal cofactors expression of for instance CumA in closelyrelated organisms such as the nonoxidizing P putida strains may also be attempted

In principle multicopper oxidases oxidize their substrates directly with the concomitantreduction of oxygen to water Except for multicopper oxidases with low substrate speci citysuch as laccases the oxidases are proposed to contain speci c substrate-binding pocketsConsequently MnxG MofA and CumA may contain Mn2+ -binding sites This can becon rmed by Mn2+ -binding studies and the potential binding sites may be identi ed bysite-directed mutagenesis

An alternative approach to assessing the role of the multicopper oxidases in Mn2+ oxi-dation is to localize the proteins in subcellular fractions with the use of speci c antibodies inwild-type and mutant cells Production of such antisera has become feasible by the develop-ment of overproduction and puri cation protocols for recombinant proteins For instance ifCumA represents the structural Mn2+ -oxidizing enzyme in P putida GB-1 it should be lo-calized at the outer membrane of wild-type cells The protein may be found in the periplasmplasma membrane or cytosol of the transport mutants Localization can also be performedwith GFP (green uorescent protein) or other uorescent protein fusions Antibodies may beused to attempt puri cation of the native multicopper oxidases by af nity chromatographyTo date puri cation by other biochemical methods has been very problematic

Studies on the Mn2+ -oxidizing factors as they are electrophoretically detected in ex-tracts of Bacillus SG-1 spores Pseudomonas putida cells and Leptothrix discophora spentmedia suggest that these factors are not isolated as single proteins They appear to residein complexes originating from spore coats (Bacillus) sheath remnants (Leptothrix) or theouter membrane (Pseudomonas) Because these putative complexes display the oxidizingactivity the Mn2+ -oxidizing multicopper oxidases may depend on additional factors forMn2+ oxidation For instance attached saccharides may assist in binding Mn2+ Isolationof the native multicopper oxidases will allow characterization of potential nonprotein com-ponents Because inhibition of cytochrome c synthesis in P putida GB-1 and MnB-1 resultsin loss of oxidizing activity and the mof operon of L discophora appears to encode a pro-tein with a potential heme-binding site c-type hemes may play a role in Mn2+ oxidationHowever the genes of the cytochrome c operon have been shown to ful ll dual functionsand the determinants for the different functions appear to reside in different regions of thegenes For instance the 3 0 end of ccmI was shown to be essential for Cu resistance andnot for cytochrome c synthesis in P uorescens 09906 (Yang et al 1996) Different aminoacid residues in the CcmC protein were found to function in pyoverdine production andcytochrome c biogenesis in P uorescens ATCC 17400 (Gaballa et al 1998) Thus pos-sibly one or more of the ccm gene products are involved in the Mn2+ -oxidizing process


for instance in the metabolism of Cu on which the Mn2+ -oxidizing enzyme is supposed todepend Studies of the effect of site-directed mutagenesis of the cytochrome c biogenesisgenes on Mn2+ oxidation and on the production of CumA will resolve this question Unfor-tunately studies on the effects of speci c mutagenesis of the mof operon are not feasiblebecause no transformation system is available for L discophora

Finally Mn2+ may not be the primary substrate of the potential multicopper oxidasesMnxG MofA and CumA Instead they might be primarily involved in the oxidative cross-linking of cell surface structures such as spore coat proteins or sheath components with theability to oxidize Mn2+ beinga (bene cial) side effect If so these oxidases maybe comparedwith laccases which have been proposed to function among other processes in the oxidativepolymerization of lignin monomers However unpublished results in our laboratory showedthat neither the spent medium of L discophora SS-1 nor cells of P putida GB-1 were ableto directly oxidize syringaldazine a model laccase substrate Determining the substratespeci city of the multicopper oxidases MnxG MofA and CumA isof utmost importance foridentifying their physiologic role This underlines the necessity of overproduction systemsand puri cation protocols for active proteins

References

Abolmaali B Taylor HV Weser U 1998 Evolutionary aspects of copper binding centers in copperproteins Struct Bonding 9191ndash190

Adams LF Ghiorse WC 1985 In uence of manganese on growth of a sheathless strain of Leptothrixdiscophora Appl Environ Microbiol 49556ndash562

Adams LF Ghiorse WC 1986 Physiology and ultrastructure of Leptothrix discophora SS-1 ArchMicrobiol 145126ndash135

Adams LF Ghiorse WC 1987 Characterization of extracellular Mn2+ -oxidizing activity and isola-tion of an Mn2+ -oxidizing protein from Leptothrix discophora SS-1 J Bacteriol 1691279ndash1285

Ander P Eriksson K-E 1976 The importance of phenol oxidase activity in lignin degradation by thewhite rot fungus Sporotrichum pulverulentum Arch Microbiol 1091ndash8

Archibald FS Fridovich I 1981 Manganese and defense against oxygen toxicity in Lactobacillusplantarum J Bacteriol 145442ndash451

Archibald F Roy B 1992 Production of manganic chelates by laccase from lignin-degrading fungusTrametes (Coriolus) versicolor Appl Environ Microbiol 581496ndash1499

Arcuri EJ Ehrlich HL 1979 Cytochrome involvement in Mn(II) oxidation by two marine bacteriaAppl Environ Microbiol 37916ndash923

Arrigoni O 1994 Ascorbate system in plant development J Bionerget Biomembr 26407ndash419Askwith C Eide D Van Ho A Bernard PS Li L Davis-Kaplan S Sipe DM Kaplan J 1994 The

fet3 gene of S cerevisiae encodes a multicopper oxidase required for ferrous iron uptake Cell76403ndash410

Askwith C Kaplan J 1998 Iron and copper transport in yeast and its relevance to human diseaseTrends Biochem Sci 23135ndash138

Attieh ZK Muhkopadhyay CK Seshadri V Tripoulas NA Fox PL 1999 Ceruloplasmin ferroxidaseactivity stimulates cellular iron uptake by a trivalent cation-speci c transport mechanism J BiolChem 2741116ndash1123

Bao W OrsquoMalley DM Whetten R Sederoff RR 1993 A laccase associated with ligni cation inloblolly pine xylem Science 260672ndash674

Barber J 1984 Has the mangano-protein of the water splitting reaction of photosynthesis beenisolated Trends Biochem Sci 999ndash100

Beyer WF Fridovich I 1986 Manganese-catalase and manganese superoxide dismutase spectro-scopic similarity with functional diversity In VL Schramm FC Wedler editors Manganese inmetabolism and enzyme function Orlando Academic Press p 193ndash219


Binnington KC Barrett FM 1988 Ultrastructural localization of phenoloxidases in cuticle andhematopoietic tissue of the blow y Lucilia cuprina J Insect Physiol 34405ndash419

Boogerd FC de Vrind JPM 1987 Manganese oxidation by Leptothrix discophora J Bacteriol169489ndash494

Brom eld SM David DJ 1976Sorption and oxidation of manganous ions and reduction of manganeseoxide by cell suspensions of manganese oxidizing bacterium Soil Biol Biochem 837ndash43

Brouwers GJ 1999 Molecular genetic aspects of microbial manganese oxidation a geophysiologicalstudy PhD dissertation Leiden University Leiden The Netherlands 149 pp

Brouwers GJ de Vrind JPM Corstjens PLAM de Vrind-de Jong EW 1998 Genes of the two-stepprotein secretion pathway are involved in the transport of the manganese-oxidizing factor acrossthe outer membrane of Pseudomonas putida strain GB-1 Am Mineral 831573ndash1582

Brouwers GJ de Vrind JPM Corstjens PLAM Cornelis P Baysse C de Vrind-de Jong EW 1999CumA a gene encoding a multicopper oxidase is involved in Mn2+ -oxidation in Pseudomonasputida GB-1 Appl Environ Microbiol 651762ndash1768

Brown NL Barrett SR Camakaris J Lee TO Rouch DA 1995 Molecular genetics and transportanalysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004 MolMicrobiol 171153ndash1166

Bull PC Thomas GR Rommens JM Forbes JR Cox DW 1993 The Wilson disease gene is a putativecopper transporting P-type ATPase similar to the Menkes gene Nat Genet 5327ndash337

Canters GW Gilardi G 1993 Engineering type I copper sites in proteins FEBS Lett 32539ndash48Caspi R Haygood MG Tebo BM 1996 Unusual ribulose 15-biphosphate carboxylaseoxygenase

genes from a marine-oxidizing bacterium Microbiology 1422549ndash2559Caspi R Tebo BM Haygood MG 1998 c-type cytochromes and manganese oxidation in Pseu-

domonas putida strain MnB1 Appl Environ Microbiol 643549ndash3555Cervantes C Gutierrez-Corona F 1994 Copper resistance mechanisms in bacteria and fungi FEMS

Microbiol Rev 14121ndash138Clutterbuck AJ 1972 Absence of laccase from yellow spored mutants of Aspergillus nidulans J Gen

Microbiol 70423ndash435Cooksey DA 1993 Copper uptake and resistance in bacteria Mol Microbiol 71ndash5Corstjens PLAM 1993 Bacterial oxidation of iron and manganese a molecular-biological approach

PhD dissertation Leiden University Leiden The NetherlandsCorstjens PLAM de Vrind JPM Westbroek P de Vrind-de Jong EW 1992 Enzymatic iron oxidation

by Leptothrix discophora identi cation of an iron-oxidizing protein Appl Environ Microbiol58450ndash454

Corstjens PLAM de Vrind JPM Goosen T de Vrind-de Jong EW 1997 Identi cation and molecularanalysis of the Leptothrix discophora SS-1 mofA gene a gene putatively encoding a manganeseoxidizing protein with copper domains Geomicrobiol J 1491ndash108

Dancis A 1998 Genetic analysis of iron uptake in the yeast Saccharomyces cerevisiae J Pediatr132s24ndashs29

Davies SHR Morgan JJ 1989 Manganese oxidation kinetics on metal oxide surfaces J ColloidInterface Sci 12963ndash77

Dawson CR 1966 In J Peisach P Aisen W Blumberg editors The biochemistry of copper NewYork Academic Press p 305

de Jong E de Vries FP Field JA van de Zwan RP de Bont JAM 1992 Isolation of basidiomyceteswith high peroxidative activity Mycol Res 961098ndash1104

Dennison C Canters GW 1996 The CuA site in cytochrome c oxidase Recl Trav Chim Pays-Bas115345ndash351

DePalma SR 1993 Manganese oxidation by Pseudomonas putida PhD dissertation Harvard Uni-versity Cambridge Massachusetts

de Vrind JPM de Vrind-de Jong EW de Voogt J-WH Westbroek P Boogert FC Rosson RA 1986aManganese oxidation by spores and the spore coats of a marine Bacillus species Appl EnvironMicrobiol 521096ndash1100

de Vrind JPM Boogerd FC de Vrind-de Jong EW 1986b Manganese reduction by a marine Bacillusspecies J Bacteriol 16730ndash34


de Vrind JPM Brouwers GJ Corstjens PLAM de Dulk J de Vrind-de Jong EW 1998 A geneinvolved in the manganese oxidation and regulation of siderophore production is part of thecytochrome c biogenesis operon in the manganese-oxidizing Pseudomonas putida strain GB-1Appl Environ Microbiol 643556ndash3562

de Vrind-de Jong EW Corstjens PLAM Kempers ES Westbroek P de Vrind JPM 1990 Ox-idation of manganese and iron by Leptothrix discophora use of N N N 0 N 0 -tetramethyl-p-phenylenediamine as an indicator of metal oxidation Appl Environ Microbiol 563458ndash3462

Diallinas G Pateraki I Sanmartin M Scossa A Stilianou E Panopoulos NJ Kanellis AK 1997Melon ascorbate oxidase cloning of a multigene family induction during fruit development andrepression by wounding Plant Mol Biol 34759ndash770

Diem D Stumm W 1984 Is dissolved Mn2+ being oxidized by O2 in absence of Mn-bacteria orsurface catalysts Geochim Cosmochim Acta 481571ndash1573

Dismukes GC 1986 The organisation and function of manganese in the water-oxidizing complex ofphotosynthesis In VL Schramm FC Wedler editors Manganese in metabolism and enzymefunction Orlando Academic Press p 275ndash309

Dondero NC 1975 The SphaerotilusndashLeptothrix group Annu Rev Microbiol 29407ndash428Douka CE 1980 Kinetics of manganese oxidation by cell-free extracts of bacteria isolated from

manganese concretions from soil Appl Environ Microbiol 3974ndash80Driouich A Laine AC Vian B Faye L 1992 Characterization and localization of laccase forms in

stem and cell cultures of sycamore Plant J 213ndash24Dubinina GA 1978 Mechanism of the oxidation of divalent iron and manganese by iron bacteria

growing at neutral pH of the medium Microbiology 47471ndash478Ehrlich HL 1968 Bacteriology of manganese nodules II Manganese oxidation by cell-free extracts

from a manganese nodule bacterium Appl Microbiol 16197ndash202Ehrlich HL 1976 Manganese as an energy source for bacteria In JO Nriagu editor Environmental

biogeochemistry 2 Metal transfer and ecological mass balances Ann Arbor MI Ann ArborScience Publishers p 633ndash644

Ehrlich HL 1983 Manganese-oxidizing bacteria from a hydrothermally active area on the GalapagosRift Ecol Bull (Stockholm) 35357ndash366

Ehrlich HL 1984 Different forms of bacterial manganese oxidation In WR Strohl H Tuovinen edi-tors Microbial chemoautrophy Ohio State Univ Colloq 8 Columbus OH Ohio State UniversityPress p 47ndash56

Ehrlich HL 1988 Bioleaching of manganese by marine bacteria In Proc 8th Int Biotechnol SympJuly 17ndash22 1988 Paris France

Ehrlich HL 1996 Geomicrobiology New York Marcel DekkerEhrlich HL 1999 Microbes as geological agents their role in mineral formation Geomicrobiol J

16135ndash153Ehrlich HL Salerno JC 1990 Energy coupling in Mn2+ oxidation by a marine bacterium Arch

Microbiol 15412ndash17Emerson D 1989 PhD dissertation Cornell University Ithaca New YorkEmerson D Ghiorse WC 1992 Isolation cultural maintenance and taxonomy of a sheath-forming

strain of Leptothrix discophora and characterization of manganese-oxidizing activity associatedwith the sheath Appl Environ Microbiol 584001ndash4010

Esaka M Fujisawa K Goto M Kisu Y 1992 Regulation of ascorbate oxidase expression in pumpkinby auxin and copper Plant Physiol 100231ndash237

Farrar JA Zumft WG Thomson AJ 1998 CuA and CuZ are variants of the electron transfer centerin nitrous oxide reductase Proc Natl Acad Sci USA 959891ndash9896

Foyer CH Descourvieres P Kunert KJ 1994 Protection against oxygen radicals an improved defensemechanism study in transgenic plants Plant Cell Envir 17507ndash523

Francis CA Casciotti KL Tebo BM 1997 Manganese oxidizing activity localized to the exosporiumof the spores of the marine Bacillus sp strain SG-1 In Ann Mtg Am Soc Microbiol Miami FLMay 1997 Washington DC American Society of Microbiology

Francis CA Tebo BM 1999 Marine Bacillus spores as catalysts for oxidative precipitation andsorption of metals J Mol Microbiol Biotechnol 171ndash78


Gaballa A Baysse C Koedam N Muyldermans S Cornelis P 1998 Different residues in periplas-mic domains of the CcmC inner membrane protein of Pseudomonas uorescens ATCC 17400are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake Mol Microbiol30547ndash555

Ghiorse WC 1984 Biology of iron- and manganese-depositing bacteria Annu Rev Microbiol 38515ndash550

Givaudan A Effosse A Faure D Potier P Bouillant ML 1993 Polyphenol oxidase from Azospir-illum lipoferum isolated from the rhizosphere evidence for a laccase in non-motile strains ofAzospirillum lipoferum FEMS Microbiol Lett 108205ndash210

Harkin JM Obst TR 1973 Ligni cation in trees indication of exclusive peroxidase participationScience 180296ndash297

Harris ZL Takahashi Y Miyajima H Serizawa M MacGillivray RT Gitlin JD 1995 Aceruloplas-minemia molecular characterization of this disorder of iron metabolism Proc Natl Acad SciUSA 922539ndash2543

Hassett RF Yuan DS Kosman DJ 1998 Spectral and kinetic properties of the Fet3 protein fromSaccharomyces cerevisiae a multinuclear copper ferroxidase enzyme J Biol Chem 27323274ndash23282

Hem JD Lind CJ 1983 Nonequilibrium models for predicting forms of precipitated manganeseoxides Geochim Cosmochim Acta 472037ndash2046

Hofer C Schlosser D 1999 Novel enzymatic oxidation of Mn2+ to Mn3+ catalyzed by a fungallaccase FEBS Lett 451186ndash190

Hsieh HS Frieden E 1975 Evidence for ceruloplasmin as a copper transport protein Biophys ResCommun 671326ndash1331

Jonsson L Sjostrom K Haggstrom I Nyman PO 1995 Characterization of a laccase gene from thewhite-rot fungus Trametes versicolor and structural features of basidiomycete laccases BiochimBiophys Acta 1251210ndash215

Jung WK Schweisfurth R 1979 Manganese oxidation by an intacellular protein of a Pseudomonasspecies Z Allg Microbiol 19107ndash115

Kaplan J OrsquoHalloran TV 1996 Iron metabolism in eukaryotes Mars and Venus at it again Science2711510ndash1512

Koschinsky ML Funk WD van Oost BA MacGillivray RT 1986 Complete cDNA sequence ofhuman preceruloplasmin Proc Natl Acad Sci USA 835086ndash5090

Kroes S 1997 Structural analysis of mutants of the blue copper protein azurin PhD dissertationLeiden University Leiden The Netherlands

Larsen EI Sly LI McEwan AG 1999 Manganese(II) adsorption and oxidation by whole cells and amembrane fraction of Pedomicrobium sp ACM 3067 Arch Microbiol 171257ndash264

Lee GR Nacht S Lukens JN Cartwright GE 1968 Iron metabolism in copper-de cient swine J ClinInvest 472058ndash2069

Lee Y-A Hendson M Panopoulos NJ Schroth MN 1994 Molecular cloning chromosomal mappingand sequence analysis of copper resistance genes from Xanthomonas campestris pv juglandishomology with small blue copper proteins and multicopper oxidase J Bacteriol 176173ndash188

Leung FY 1998 Trace elements that act as antioxidants in parenteral micronutrition J Nutr Biochem9304ndash307

Lin LS Varner JE 1991 Expression of ascorbic acid oxidase in zucchini squash (Cucurbita pepoL) Plant Physiol 96159ndash165

Liu L Dean JFD Friedman WE Eriksson K-EL 1994 A laccase-like phenoloxidase is correlatedwith lignin biosynthesis in Zinnia elegans stem tissues Plant J 6213ndash224

Lovley DR 1991 Dissimilatory Fe(III) and Mn(IV) reduction Microbiol Rev 55259ndash287Lovley DR Phillips EJP 1988 Novel mode of microbial energy metabolism organic carbon oxidation

coupled to dissimilatory reduction of iron or manganese Appl Environ Microbiol 541472ndash1480

Lowery MD Guckert JA Gebhard MS Solomon EI 1993 Active-site electronic structure contribu-tions to electron-transfer pathways in rubredoxin and plastocyanin direct versus superexchangeJ Am Chem Soc 1153012ndash3013


Machonkin TE Zhang HH Hedman B Hodgson KO Solomon EI 1998 Spectroscopic and magneticstudies of human ceruloplasmin identi cation of a redox-inactive reduced type I copper siteBiochemistry 379570ndash9578

Malmstrom BG 1990 Cytochrome oxidase some unsolved problems and controversial issues ArchBiochem Biophys 280233ndash241

Mansur M Suarez T Gonzalez AE 1998 Differential gene expression in the laccase gene familyfrom Basidiomycete I-62 (CECT 20197) Appl Environ Microbiol 64771ndash774

Marceau N Aspin N 1973a The intracellular distribution of the radiocopper derived from cerulo-plasmin and from albumin Biochim Biophys Acta 328338ndash350

Marceau N Aspin N 1973b The association of the copper derived from ceruloplasmin with cy-tocuprein Biochim Biophys Acta 328351ndash358

McDouglas GJ Morrison IM 1996 Extraction and partial puri cation of cell-wall-associatedconiferyl alcohol oxidase from developing xylem of sitka spuce Holzforschung 50549ndash553

McDouglas GJ Steward D Morrison IM 1994 Cell-wall-bound oxidases from tobacco (Nicotianatabacum) xylem participate in lignin formation Planta 1949ndash14

Mellano MA Cooksey DA 1988 Nucleotide sequencing and organisation of copper resistance genesfrom Pseudomonas syringae pv tomato J Bacteriol 1702879ndash2883

Messerschmidt A 1998 Metal sites in small blue copper proteins blue copper oxidases and vanadium-containing enzymes Struct Bonding 9037ndash68

Messerschmidt A Ladenstein R Huber R Bolognesi M Avigliano L Petruzzelli R Rossi A Finazzi-Agro A 1992 Re ned crystal structure of ascorbate oxidase at 19 EcircA resolution J Mol Biol224179ndash205

Morgan JJ Stumm W 1964 Colloid-chemical properties of manganese dioxide J Colloid Sci 19347ndash359

Moser O Kanellis AK 1994 Ascorbate oxidase of Cucumis melo L var reticulatus puri cationcharacterization and antibody production J Exp Bot 45717ndash724

Mukhopadhyay CK Fox PL 1998 Ceruloplasmin copper induces oxidant damage by a redox processutilizing cell-derived superoxide as reductant Biochemistry 3714222ndash14229

Murphy MEP Lindley PF Adman ET 1997 Structural comparison of cupredoxin domains domainrecycling to construct proteins with novel functions Protein Sci 6761ndash770

Murray JW Dillard JG Giovanoli R Moers H Stumm W 1985 Oxidation of Mn(II) initial miner-alogy oxidation state and aging Geochim Cosmochim Acta 49463ndash470

Myers CR Nealson KH 1990 Respiration-linked proton translocation coupled to anaerobic reduc-tion of manganese(IV) and iron(III) in Shewanella putrefaciens MR-1 J Bacteriol 1726232ndash6238

Nakamura W 1967 Studies on the biosynthesis of lignins I Disproof against the catalytic activityof laccase in the oxidation of coniferyl alcohol J Biochem 6254ndash61

Nakamura T Makino N Ogura Y 1968 Puri cation and properties of ascorbate oxidase from cu-cumber J Biochem 64189ndash195

Nealson KH Ford J 1980 Surface enhancement of bacterial manganese oxidation implications foraquatic environments Geomicrobiol J 221ndash37

Nealson KH Little B 1997 Breathing manganese and iron solid-state respiration Adv Appl Micro-biol 45213ndash239

Nealson KH Myers CR 1992 Microbial reduction of manganese and iron new approaches to carboncycling Appl Environ Microbiol 58439ndash443

Nealson KH Tebo BM Rosson RA 1988 Occurrence and mechanisms of microbial oxidation ofmanganese Adv Appl Microbiol 33299ndash318

Ohkawa J Okada N Shinmyo A Takano M 1989 Primary structure of cucumber (Cucumis sativus)ascorbate oxidase deduced from cDNA sequence homology with blue copper proteins andtissue-speci c expression Proc Natl Acad Sci USA 861239ndash1243

Ohkawa J Shinmyo A Kanchanapoon M Okada N Takano M 1990 Structure and expression ofthe gene coding for a multicopper enzyme ascorbate oxidase of cucumber Ann NY Acad Sci613483ndash488


Okazaki M Sugita T Shimizu M Ohode Y Iwamoto K de Vrind-de Jong EW de Vrind JPMCorstjens PLAM 1997 Partial puri cation and characterization of manganese oxidizing factorsof Pseudomonas uorescens GB-1 Appl Environ Microbiol 634793ndash4799

OrsquoMalley DM Whetten R Bao W Chen C-H Sederoff RR 1993 The role of laccase in ligni cationPlant J 4751ndash757

Payne AS Gitlin JD 1998 Functional expression of the Menkes disease protein reveals common bio-chemical mechanisms among the copper-transporting P-type ATPases J Biol Chem 2733765ndash3770

Pouderoyen G 1996 The effect of metal ligand and surface mutations on spectroscopy and reactivityof azurin PhD dissertation Leiden University Leiden The Netherlands

Ranocha P McDougall G Hawkins S Sterjiades R Borderies G Stewart D Cabanes-Macheteau MBoudet A-M Goffner D 1999 Biochemical characterization molecular cloning and expressionof laccasesmdasha divergent gene familymdashin poplar Eur J Biochem 259485ndash495

Rosson RA Nealson KH 1982 Manganese binding and oxidation by spores of a marine Bacillus JBacteriol 1511027ndash1034

Ryden LG Hunt LT 1993 Evolution of protein complexity the blue copper-containing oxidases andrelated proteins J Mol Evol 3641ndash66

Sanchez-Amat A Solano F 1997 A pluripotent polyphenol oxidase from the melanogenic marineAlteromonas sp shares catalytic capabilities of tyrosinases and laccases Biochem Biophys ResCommun 240787ndash792

Schramm VL Wedler FC 1986 Manganese in metabolism and enzyme function Orlando AcademicPress Inc

Schweisfurth R 1973 Manganoxydierende Bacterien I Isolierung und Bestimmung einiger Stammevon Manganbakterien Z Allg Mikrobiol 13341ndash347

Shimizu A Kwon JH Sasaki T Satoh T Sakurai N Sakurai T Yamaguchi S Samejima T 1999 My-rothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands Biochemistry383034ndash3042

Solomon EI Sundaram UM Machonkin TE 1996 Multicopper oxidases and oxygenases Chem Rev962563ndash2605

Stearmann R Yuan DS Yamaguchi-Iwai Y Klausner RD Dancis A 1996 A permeasendashoxidasecomplex involved in high-af nity iron uptake in yeast Science 2711552ndash1557

Sterjiades R Dean JFD Eriksson K-EL 1992 Laccase from sycamore maple (Acer pseudoplatanus)polymerizes monolignols Plant Physiol 991162ndash1168

Sterjiades R Dean JFD Eriksson K-EL 1993 Extracellular laccases and peroxidases from sycamoremaple (Acer pseudoplatanus) cell suspension culturesmdashreactions with monolignols and ligninmodel compounds Planta 19075ndash87

Sunda WG Kieber DJ 1994 Oxidation of humic substances by manganese oxides yields low-molecular-weight organic substrates Nature 36762ndash64

Sundaram UM Zhang HH Hedman B Hodgson KO Solomon EI 1997 Spectroscopic investigationof peroxide binding to the trinuclear copper cluster site in laccase correlation with the peroxylevelintermediate and relevance to catalysis J Am Chem Soc 11912525ndash12540

Sung W Morgan JJ 1981 Oxidative removal of Mn(II) from solution catalysed by the c -FeOOH(lepidocrocite) surface Geochim Cosmochim Acta 452377ndash2383

Tebo BM 1983 The ecology and ultrastructure of marine manganese oxidizing bacteria PhD disser-tation University of California San Diego California

Tebo BM 1991 Manganese(II) oxidation in the suboxic zone of the Black Sea Deep Sea Res38S883ndashS905

Tebo BM Mandernack K Rosson RA 1988 Manganese oxidation by a spore coat or exosporiumprotein from spores of manganese(II) oxidizing marine Bacillus abstr I-121 In Abstr 88th AnnMtg Am Soc Microbiol p 201

Tebo BM Ghiorse WC van Waasbergen LG Siering PL Caspi R 1997 Bacterial mediated min-eral formation insights into manganese(II) oxidation from molecular genetic and biochemicalstudies Rev Mineral 35225ndash266

Thurston CF 1994 The structure and function of fungal laccases Microbiology 14019ndash26


Van Veen WL Mulder EG Deinema MH 1978 The SphaerotilusndashLeptothrix group of bacteriaMicrobiol Rev 42329ndash626

Van Waasbergen LG Hoch JA Tebo BM 1993 Genetic analysis of the marine manganese-oxidizingBacillus sp strain SG-1 protoplast transformation Tn917 mutagenesis and identi cation ofchromosomal loci involved in manganese oxidation J Bacteriol 1757594ndash7603

Van Waasbergen LG Hildebrand M Tebo BM 1996 Identi cation and characterization of a genecluster involved in manganese oxidation by spores of the marine Bacillus sp strain SG-1 JBacteriol 1783517ndash3530

Wehrli B 1990 Redox reactions of metal ions at mineral surfaces In W Stumm editor Aquaticchemical kinetics New York John Wiley amp Sons p 311ndash336

Xu F 1996 Oxidation of phenols anilines and benzenethiols by fungal laccases correlation betweenactivity and redox potentials as well as halide inhibition Biochemistry 357608ndash7614

Xu F Shin W Brown SH Wahleithner JA Sundaram UM Solomon EI 1996 A study of a series ofrecombinant fungal laccases and bilirubin oxidases that exhibit signi cant differences in redoxpotential substrate speci city and stability Biochim Biophys Acta 1292303ndash311

Yang C-H Delgado M-J Wexler M Allan Downie J Johnson AWB 1996 A chromosomal locusrequired for copper resistance competetive tness and cytochrome c biogenesis in Pseudomonas uorescens Proc Natl Acad Sci USA 937315ndash7320

Yoshida H 1883 Chemistry of lacquer (Urushi) part 1 J Chem Soc 43472ndash486Zaitseva I Zaitseva V Card G Moshov K Bax B Ralph A Lidley P 1996 The X-ray structure of

human serum ceruloplasmin at 31 EcircA nature of the copper centers J Biol Inorg Chem 115ndash23


aequous environments and readily disproportionates to Mn2+ and MnO2 Mn(IV) formshighly insoluble oxyhydroxide s and oxides In natural systems Mn2+ oxidation is ther-modynamically favorable but often proceeds at very low rates (Diem and Stumm 1984Nealson et al 1988) Abiotic Mn2+ oxidation can be catalyzed by extreme environmentalconditions (high pH and high O2 pressure) and by adsorption of the ions on mineral surfacessuch as Fe oxides and silicates (Morgan and Stumm 1964 Sung and Morgan 1981 Hemand Lind 1983 Murray et al 1985 Davies and Morgan 1989) Mn(IV) reduction is favoredby the presence of reducing agents under anaerobic conditions low pH or the presence ofMn2+ -complexing agents

The conversions of Mn between oxidation states can be catalyzed by living organ-isms especially microbes Many anaerobic bacterial species are able to reduce Mn oxideseither by production of acids or reducing substances such as sul des or by using the oxi-dized metal as an electron acceptor in respiration (Lovley 1991 Nealson and Myers 1992Nealson and Little 1997) In aerobic environments a wide variety of microorganisms cat-alyze the oxidation of Mn2+ (Ehrlich 1984 Ghiorse 1984) Compared with abiotic Mn2+

oxidation microorganisms can accelerate the oxidation by as much as ve orders of mag-nitude (Nealson et al 1988 Tebo 1991 Wehrli 1990) In strati ed environments reducingand oxidizing microbial species can provide a rapid cycling of Mn around the oxicanoxicboundary In these milieus Mn can serve as a redox shuttle in the oxidation and (potentially)reduction of organic carbon (Figure 1) (Nealson and Myers 1992)

Microbial Mn2+ oxidation proceeds through indirect or direct mechanisms Indirectmechanisms include the production of O2 (in photosynthesis) and of alkaline or oxidizingmetabolites Direct Mn2+ oxidation involves the microbial production of speci c macro-molecules (polysaccharides or proteins) catalyzing the process In several bacterial generaenzymes have been shown to be involved in Mn2+ oxidation (Ehrlich 1968 1983 Jung andSchweisfurth 1979 Douka 1980 de Vrind et al 1986a Adams and Ghiorse 1987 Boogerdand de Vrind 1987 Okazaki et al 1997) Three Mn2+ -oxidizing species have been studied in

FIGURE 1 Schematic representation of the cycling of Mn around the oxicanoxic boundaryand the potential selection between Mn cycling and the cycling of carbon As has beenshown (see text for references) organic carbon can be oxidized during Mn(IIIIV) reductionCarbon dioxide xation during Mn(II) oxidation is still a topic of discussion
















































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References



































































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References





























































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References




















































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References














































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References








































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References
































































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References























































































































































Copper Proteins









1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References

























































































































































1 I1 II


1 heme a3ndashCuB


1 II2 III

















Laccase








Ascorbate Oxidase




























References

































































































































































Laccase








Ascorbate Oxidase




























References

























































































































































Laccase








Ascorbate Oxidase




























References





















































































































































Laccase








Ascorbate Oxidase




























References






















































































































































Ascorbate Oxidase




























References











































































































































































References




































































































































































References





























































































































































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References





























































































































































































































































































































































































































































































































































































Documents

Bacterial Mn 2+ Oxidizing Systems and Multicopper Oxidases: An Overview of Mechanisms and Functions