Bacterial Mn2+ Oxidizing Systems and Multicopper

Geomicrobiology Journal, 17:1–24, 2000 Copyright ° C 2000 Taylor & Francis 0149-0451 /00 $12.00 + .00

Bacterial Mn2 + Oxidizing Systems and Multicopper Oxidases: An Overview of Mechanisms and Functions G. J. BROUWERS E. VIJGENBOOM P. L. A. M. CORSTJENS J. P. M. DE VRIND E. W. DE VRIND-DE JONG Leiden Institute of Chemistry Leiden University Leiden, The Netherlands Manganese is oxidized by a wide variety of bacteria. The current state of knowledge on mechanisms and functions of Mn 2 + oxidation in two strains of Pseudomonas putida, in Leptothrix discophora SS-1, and in Bacillus sp. strain SG-1 is reviewed. In all three species, proteins bearing resemblance to multicopper oxidases appear to be involved in the oxidation process. A short description of the classi cation of Cu centers is followed by a more detailed review of properties and postulated functions of some well-known multicopper oxidases. Finally, suggestions are made for future research to assess the potential role of multicopper oxidases in bacterial Mn 2 + oxidation. Keywords Mn2 + oxidation, multicopper oxidases, Leptothrix discophora, Pseudomonas putida, Bacillus sp. SG-1

Manganese, an essential element for all living organisms, can occur in oxidation states ranging from ¡ 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 are common throughout the microbial, plant, and animal kingdoms. Manganese can act as an activator of enzymes, for instance, DNA polymerase and phosphoenolpyruvate carboxykinase (see Schramm and Wedler 1986). Because the transition metal is redox-active under physiological conditions, it plays an important role in biological redox reactions, especially those involving interactions with oxygen. Well-known examples of manganese redox enzymes include the water-oxidizing complex of photosynthesis (Barber 1984; Dismukes 1986), Mn superoxide dismutase (Beyer and Fridovich 1986), and Mn pseudocatalase (Dubinina 1978). The bioavailability of manganese is strongly in uenced by the factors that determine its oxidation state. Mn(II) is usually soluble as an ion or complexed to organic or inorganic ligands. Mn(III), unless complexed to ligands or incorporated in enzymes, is unstable in Received 27 July; accepted 23 September 1999. The 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 of the manuscript. Address correspondenc e to Dr. Liesbeth W. de Vrind-de Jong, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. E-mail: Vrind e@chem. leidenuniv.nl

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G. J. Brouwers et al.

aequous environments and readily disproportionates to Mn2 + and MnO2 . Mn(IV) forms highly insoluble oxyhydroxide s and oxides. In natural systems, Mn2 + oxidation is thermodynamically favorable but often proceeds at very low rates (Diem and Stumm 1984; Nealson et al. 1988). Abiotic Mn2 + oxidation can be catalyzed by extreme environmental conditions (high pH and high O2 pressure) and by adsorption of the ions on mineral surfaces such as Fe oxides and silicates (Morgan and Stumm 1964; Sung and Morgan 1981; Hem and Lind 1983; Murray et al. 1985; Davies and Morgan 1989). Mn(IV) reduction is favored by the presence of reducing agents under anaerobic conditions, low pH, or the presence of Mn2 + -complexing agents. The conversions of Mn between oxidation states can be catalyzed by living organisms, especially microbes. Many anaerobic bacterial species are able to reduce Mn oxides, either by production of acids or reducing substances such as sul des or by using the oxidized metal as an electron acceptor in respiration (Lovley 1991; Nealson and Myers 1992; Nealson and Little 1997). In aerobic environments a wide variety of microorganisms catalyze 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 magnitude (Nealson et al. 1988; Tebo 1991; Wehrli, 1990). In strati ed environments, reducing and oxidizing microbial species can provide a rapid cycling of Mn around the oxic/anoxic boundary. 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. Indirect mechanisms include the production of O2 (in photosynthesis) and of alkaline or oxidizing metabolites. Direct Mn2 + oxidation involves the microbial production of speci c macromolecules (polysaccharides or proteins) catalyzing the process. In several bacterial genera, enzymes have been shown to be involved in Mn2 + oxidation (Ehrlich 1968, 1983; Jung and Schweisfurth 1979; Douka 1980; de Vrind et al. 1986a; Adams and Ghiorse 1987; Boogerd and 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 oxic/anoxic boundary and the potential selection between Mn cycling and the cycling of carbon. As has been shown (see text for references), organic carbon can be oxidized during Mn(III/IV) reduction. Carbon dioxide xation during Mn(II) oxidation is still a topic of discussion.

Bacterial Mn 2 + Oxidation and Multicopper Oxidases

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detail in several laboratories, including our own: the fresh-water Gram-negative organisms Leptothrix discophora and Pseudomonas putida, and the marine Gram-positive bacterium Bacillus species SG-1. Until recently, common principles underlying Mn2 + oxidation in the different species could not be recognized, and the functional signi cance of the process has remained 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 genes involved 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 play a role in Mn2 + oxidation. This is the rst evidence that different species use similar tools in 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 + + 1/2 O2 + H2 O ! MnO2 + 2H + has a ¡ 1 standard free energy change at pH 7 of ¡ 70 kJ mol , in principle Mn2 + oxidation can sustain bacterial growth. Models have been proposed in which electron transport by way of an electron transport chain generates a proton gradient that permits ATP formation by oxidative 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 electron transport chain via a Mn oxidase. Protons released during the oxidation of Mn2 + , together with proton consumption upon oxygen reduction, generate a proton gradient across the membrane, which results in the generation of ATP via ATPase. Dotted arrow: In view of the difference in redox potential between the Mn(IV)/Mn(II) and cytochrome c ox/red redox couples, electrons from Mn(IV) will have to travel through reverse electron transport to reduce cytochrome c.

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synthesis coupled to Mn2 + oxidation has been reported in the Gram-negative marine strain SSW22 (Ehrlich 1983; Ehrlich and Salerno 1990), and involvement of proteins containing c-type hemes such as cytochromes in Mn2 + oxidation has been proposed (Arcuri and Ehrlich 1979; Ehrlich 1983; Tebo et al. 1997; Caspi et al. 1998; de Vrind et al. 1998). A ribulose-1,5biphosphate carboxylase gene, suggesting the potential for autotrophic growth, has been identi ed in a marine Mn2 + -oxidizing species (Caspi et al. 1996). To date, however, no bacterial species has been shown to be capable of autotrophic growth on Mn2 + . Perhaps some mixotrophic bacteria derive supplemental energy from Mn2 + oxidation, especially in carbon-limited environments (Ehrlich 1976). So far, the question as to whether Mn2 + oxidation yields useful energy can not be decisively answered. The 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 adsorptive properties, from toxicity by heavy metals. Mn2 + -oxidizing enzymes may be involved in detoxi cation of such harmful oxygen species as superoxide and peroxide (Archibald and Fridovich 1981), in analogy with superoxide dismutases and catalases (Dubinina 1978). The prolonged viability of Mn oxide-encrusted L. discophora cells has been ascribed to one or a combination of these effects (Adams and Ghiorse 1985). Mn oxides may also defend against predation or viral attack (Emerson 1989). Insight into the possible protective function of Mn2 + oxidation calls for detailed studies on the resistance of oxidizing wild-type strains against harmful environmental factors as compared with nonoxidizing mutant strains. So far, such studies have not been performed. Recently, many microbial species have been described that are able to couple anaerobic respiration of organic carbon with reduction of metals, including oxidized Mn (Lovley and Phillips 1988; Myers and Nealson 1990; Nealson and Myers 1992). Some investigators have speculated that Mn2 + -oxidizing organisms accumulate Mn oxides as electron acceptors for survival 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 aerobically, under low oxygen or anaerobic circumstances (Brom eld and David 1976; de Vrind et al. 1986b; Ehrlich 1988). Because anaerobic growth of these species with Mn oxide as an electron acceptor has not been demonstrated, the functional signi cance of the reduction processes in these organisms is not resolved. An interesting possibility for the function of microbial Mn2 + oxidation has been proposed by Sunda and Kieber (1994). They showed that Mn oxides oxidize complex humic substances, releasing low molecular mass organic compounds (e.g., pyruvate) that can serve as substrates for bacterial growth. This implies that Mn2 + -oxidizing bacteria may survive in nutrient-poor environments by producing a strong oxidizing agent able to degrade biologically recalcitrant carbon pools (cf. Tebo et al. 1997). In this respect, Mn2 + -oxidizing bacteria may be compared with wood-degrading fungi, which secrete peroxidases and laccases able to lyse lignin (see later sections). Fungal manganese peroxidases catalyze the formation of Mn(III) complexes, which can subsequently oxidize phenolic compounds. Despite numerous investigations pointing to speci c roles for enzymatic Mn2 + oxidation by bacteria, unequivocal evidence for the various functional models is still lacking. Only elucidation of the mechanisms of the oxidation process in individual bacterial species and identi cation of the cellular components involved will eventually give insight in the function(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


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MnB1 (formerly called P. manganoxydan s) was isolated from a manganese crust in a water pipeline 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 (Okazaki et al. 1997). More extensive 16S rRNA gene sequencing and physiological characteristics have 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 highest activity 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). The oxidizing activity also appears to depend on the oxygen concentration in the culture during growth. In strain GB-1, the activity per cell doubled when the oxygen concentration was increased 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 that one 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 its kinetics (K m » 10 l M), pH optimum (pH 7), temperature optimum (35±C), and inhibition by HgCl2 . The oxidizing activity is also inhibited by NaN3 , KCN, EDTA, Tris, and ophenanthroline, indicating that a redox protein containing metal cofactors is involved in the process (Okazaki et al. 1997). After partial puri cation of the oxidizing activity of strain GB-1, polyacrylamide gel electrophoresis of the puri ed preparation revealed oxidizing factors of » 250 and 180 kDa (Okazaki et al. 1997). An oxidizing factor of 130 kDa has also been reported (Corstjens 1993), and the Mn2 + -oxidizing enzyme has been suggested to be part of a complex that can disintegrate into smaller fragments, at least some of which retain oxidizing activity (Okazaki et al. 1997). Incorporation of the Mn2 + -oxidizing protein in a complex may explain why the 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 that were formerly less accessible for Mn2 + . Both P. putida MnB1 and GB-1 are readily accessible to genetic manipulation. Transposon 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). The reader is referred to these studies for detailed information. Here we will summarize the characterization of nonoxidizing mutants of P. putida MnB1 and GB-1 and highlight some of the conclusions drawn from the mutagenesis experiments. P. putida GB-1 lost its ability to oxidize Mn2 + by transposon insertion into a gene, called cumA, that encodes a protein related to multicopper oxidases (Brouwers et al. 1999). That the CumA protein could be a component of the putative Mn2 + -oxidizing complex is supported by the observation that Cu2 + ions, added during growth of bacteria, stimulated the oxidizing activity. The gene cumA, 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 al. 1999) but did result in decreased growth. In both P. putida MnB1 and GB-1, mutations in genes of the cytochrome c maturation operon (ccm) abolished the synthesis of c-type cytochromes as well as the Mn2 + -oxidizing activity (Brouwers et al. 1998; Caspi et al. 1998). This may point to involvement of the electron transport chain in Mn2 + oxidation. Alternatively, a c-type heme is next to CumA, part of the putative Mn2 + -oxidizing complex mentioned above. It is also possible that one of the enzymes from the cytochrome c biogenesis pathway has a dual function and plays a role 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 can interact with Cu ions. These proteins have been proposed to play a role in Cu metabolism in addition to cytochrome c biogenesis (Yang et al. 1996). Interference with their expression

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FIGURE 3 (A) Transposon insertion in several genes of the Mn2 + -oxidizing P. putida GB-1 results in a change of oxidizing brown colonies (left) into a nonoxidizing white phenotype (right). For details, see text. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of spent medium of L. discophora SS-1. Lanes: (a) molecular mass markers (1, 205,000; 2, 116,000; 3, 97,000; 4, 66,000; 5, 45,000 Da); (b) spent medium, stained for protein with Coomassie brillant blue; (c) spent medium, stained for Fe2 + -oxidizing activity by immersion of the gel in (NH4 )2 Fe(SO4 )2 solution; (d) spent medium, stained for Fe2 + oxidizing activity and then with Coomassie brillant blue (the arrow indicates the Fe-stained band); (e) spent medium, stained for Mn2 + -oxidizing activity by immersion of the gel in MnCl2 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 oxidizing complex. Mutations in genes involved in the general secretion pathway (GSP) for protein secretion across the outer membrane resulted in a nonoxidizing phenotype (Brouwers et al. 1998). Full activity was recovered after disruption of the cell walls. This indicates that at least one of the components of the oxidizing complex is a substrate of the GSP machinery, con 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 as Mn2 + (Dondero 1975; Van Veen et al. 1978). One of them, L. discophora, is a sheathforming, heterotrophic fresh-water bacterium, commonly found in habitats with an aerobic– anaerobic interface, where manganese and iron cycle between their oxidized and reduced forms. During its stationary growth phase, L. discophora oxidizes Mn2 + and Fe2 + (Adams and Ghiorse 1987; Boogerd and de Vrind 1987). In its natural environment, L. discophora deposits the oxides on the sheaths surrounding the cells (van Veen et al. 1978), and at


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least part of the Mn2 + -oxidizing system is thought to consist of sheath components. The ability of Leptothrix species to form a structured sheath is easily lost when cultured in the laboratory. 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 this strain, both Fe2 + and Mn2 + oxidation appear to be enzymatically catalyzed (Adams and Ghiorse 1987; Boogerd and de Vrind 1987; de Vrind-de Jong et al. 1990). The K m values for Fe2 + and Mn2 + are both » 10 l M, comparable with the K m of Mn2 + oxidation in P. putida. Both processes are heat-sensitive and inhibited by enzyme poisons such as HgCl2 , KCN, and NaN3 . Inhibition of Mn2 + oxidation by o-phenanthroline points to involvement of a metal cofactor in this process (Adams and Ghiorse 1987). Whether Mn2 + and Fe2 + are oxidized by different enzymes or by identical or closely related components is not known. The secreted activities have shown different behaviors, for 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 by denaturing gel electrophoresis revealed several discrete factors, ranging from 50 to 180 kDa. Some of these factors oxidize Fe2 + , some oxidize Mn2 + , and some are able to oxidize both metals (Boogerd and de Vrind 1987; Corstjens et al. 1992). These results may indicate that different factors are involved in Mn2 + and Fe2 + oxidation. However, the data may also be explained by assuming that the oxidizing components are part of a complex, consisting of similar or related components in variable ratios (Brouwers 1999). Secretion of the oxidizing factor(s) as part of a complex is consistent with the observation that electrophoretic analysis of concentrated spent medium under nondenaturing conditions does not reveal discrete oxidizing factors (Adams and Ghiorse 1987; Corstjens 1993). The proposed complex has been suggested to originate from membranous blebs (Adams and Ghiorse 1986). Incorporation of the active oxidizing sites in a membrane complex can explain their relative insensitivity to or even slight stimulation by SDS and proteases. As already described, a similar situation exists in P. putida GB-1. The oxidizing enzymes are dif cult to isolate, probably because they are secreted as part of a complex (Adams and Ghiorse 1987). Only microgram amounts of a 110kDa Mn2 + -oxidizing factor (called MOF) have been isolated (Corstjens 1993; Corstjens et al. 1997); it consists of protein and probably polysaccharide (Adams and Ghiorse 1987; Emerson and Ghiorse 1992). This partially puri ed preparation has been used to raise antibodies (a -MOF). Screening an L. discophora expression library with a -MOF resulted in the identi cation of a single gene, called mofA. Because this gene was detected with antibodies against an isolated Mn2 + -oxidizing factor, it very likely encodes a structural component of the Mn2 + -oxidizing system of L. discophora. Interestingly, mofA appears to encode a protein (of » 180 kDa) related to multicopper oxidases (Corstjens et al. 1997), and addition of 40 l M Cu2 + to a growing culture increased Mn2 + oxidation by » 80% when corrected for equal cell numbers (Brouwers et al. 1999). The mofA gene was suggested to be part of an operon (Corstjens et al. 1997), an assumption supported by recent data (Brouwers 1999). One of the genes belonging to this operon encodes a protein with a potential heme-binding site, which may be a further indication that c-type hemes play a role in Mn2 + oxidation, in addition to the putative multicopper oxidase.

Mn2+ Oxidation in Bacillus SG-1 Bacillus strain SG-1, a Gram-positive marine organism isolated from a near-shore manganous sediment (Nealson and Ford 1980), forms inert endospores upon nutrient limitation. The germ cells of the spores are surrounded by a peptidoglycan cortex covered by highly

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cross-linked spore coat proteins. The outermost spore layer is an exosporium, a membranous structure differing in composition from the spore coat (Francis et al. 1997). The spores catalyze the oxidation of Mn2 + (Rosson and Nealson 1982); their oxidative properties have recently been reviewed (Francis and Tebo 1999). The oxidizing activity has been localized in the exosporium (Francis et al. 1997), and its kinetics, heat sensitivity, and inhibition by protein poisons such as NaN3 and HgCl2 suggest that an exosporium protein is involved in the process (Rosson and Nealson 1982; de Vrind et al. 1986a). The highly cross-linked and resistant structure of outer spore coverings has prohibited the isolation of Mn2 + -oxidizing factors from spores (Tebo et al. 1988). A Mn2 + -oxidizing protein band of » 205 kDa was identi ed after gel electrophoresis of spore coat/exosporium extracts, but these results have been dif cult to reproduce (Tebo et al. 1997)—probably because of the separation during gel electrophoresis of the diverse components necessary for Mn2 + oxidation. Transposon mutagenesis has been more successful in identifying components involved in Mn2 + oxidation (Van Waasbergen et al. 1993, 1996). Mutants, still able to form endospores but de cient in Mn2 + oxidation, appear to contain transposon insertions in genes of an operon called mnx. The mnx genes are transcribed during midsporulation when spore coat 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 databases. MnxG shares sequence similarity with members of the family of multicopper oxidases; its 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 the Mn2 + -oxidizing complex. MnxC shows limited sequence similarity to redox-active proteins (Tebo et al. 1997). It contains the sequence CXXXC, which resembles the thioredoxin motif CXXC. Therefore, MnxC has been suggested to play a role in a redox process. An alternative function of the conserved cysteine residues of MnxC in the transport of Cu2 + , possibly to the multicopper oxidase MnxG, has also been envisaged (Tebo et al. 1997). Because Bacillus spores are metabolically inert, their Mn2 + -oxidizing capacity is not directly related to metabolic function. Bacillus SG-1 is one of the organisms for which the vegetative cells have been shown to reduce the manganese oxide upon germination of the spores (de Vrind et al. 1986b; cf. section “Possible functions of manganese oxidation”). Manganese oxide reduction appeared to be coupled to oxidation of b- and c-type cytochromes (de Vrind et al. 1986b). However, as also stated above, de nite proof that cells of Bacillus SG-1 can grow with manganese oxide as a terminal electron acceptor is still lacking. In summary, the studies on the three Mn2 + -oxidizing species described suggest that multicopper oxidases form an essential component of the Mn2 + -oxidizing systems in oxidizing bacterial species in general. By using different approaches, proteins with sequence similarity to multicopper oxidases were shown to be involved in the oxidizing process in all three 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 added to partly puri ed Mn2 + -oxidizing preparations (spore coats of Bacillus SG-1). Recently, Cu2 + was shown to enhance the oxidizing activity of yet another Mn2 + -oxidizing species, Pedomicrobium sp. ACM 3076 (Larsen et al. 1999). Although multicopper oxidases as a common theme in bacterial Mn2 + oxidation seems well established at present, common mechanisms or functions cannot be distinguished, except for the possible involvement of c-type hemes in P. putida and L. discophora. The potential multicopper oxidases do not show sequence similarity outside the putative Cu-binding domains. Moreover, the molecular


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FIGURE 4 Comparison of the genomic regions anking the putative multicopper oxidaseencoding 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 that in the MnxG protein the C- and N-terminal Cu-binding domains are reversed in comparsion with the other putative multicopper oxidases, and that an extra Cu-binding domain appears to be present. For descriptions of the diverse gene products, see text. organizations of the operons to which the encoding genes apparently belong differ greatly from one another (Figure 4). Can this imply that the ability to oxidize Mn2 + evolved from an oxidation process with a primary function other than metal oxidation? This question led us to review brie y the properties and functions of Cu-dependent proteins, in particular the well-known multicopper oxidases.

Copper Proteins Copper became available as a cofactor in proteins during oxygenation of the atmosphere. In the rst two billion years of life, the reducing environment locked the metal in its reduced form, which precipitated as highly insoluble sul des. Copper proteins contain one or more Cu ions as a cofactor and generally function in redox reactions with one or more Cu centers as the active sites, sometimes in cooporation with other redox centers such as heme. In some proteins with sequence similarities to Cu redox proteins, the Cu ions are bound but not redox-active. Such proteins function in Cu metabolism, transport, and resistance. Cu sites have historically been divided into three classes, based on their coordination environment and geometry (Canters and Gilardi 1993; Solomon et al. 1996). The differences in coordination characteristics are re ected in their spectroscopic properties such as absorption and electron paramagnetic resonance (EPR) spectra. In type I sites, one Cu ion is coordinated with three ligands (one sulfur and two nitrogens) in an equatorial plane, donated by one cysteine and two histidine residues (Figure 5A). In some type I Cu sites, a fourth axial ligand is provided by the thioether sulfur of a methionine. In the oxidized state, type I Cu proteins have a strong absorption maximum at » 600 nm, giving them an intense blue color; hence they are called “blue copper proteins.” The small blue copper proteins are also referred to as cupredoxins and are characterized by a speci c EPR signal. Type I Cu proteins generally serve in intermolecular electron transport and are not catalytically active. They occur in all three kingdoms of life, and both sequence and structure analysis show that they derive from a common ancestor (Ryden and Hunt 1993). Some examples are listed in Table 1. Type II Cu proteins also contain one Cu ion, which is coordinated with four N or O ligands in a square planar con guration around the metal (Figure 5A). They do not absorb in the visible light spectrum, and their EPR spectrum discriminates them from type I sites. They can be involved in the catalytic oxidation of substrate molecules and can act in isolation or in cooporation with type III sites (Table 1; see also below). Type II Cu proteins show little or no phylogenetic homology and are thought to be the products of convergent evolution (Abolmaali et al. 1998).

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G. J. Brouwers et al. TABLE 1 Examples of copper-binding proteins and the different types of copper-binding sites Copper proteins Azurin, plastocyanin, amicyanin Superoxide dismutase, galactose oxidase, amine oxidase Tyrosinase, hemocyanin Nitrite reductase

Cytochrome c oxidase

Number of coppers

Type

1 1

I II

2 2

III 1 1

I II

2 1

CuA heme a3 –CuB

1 1 2

I II III

3 1 2

I II III

3

Laccases, ascorbate oxidase, Fet3p, PcoA, CopAa

4

Ceruloplasmin

6

a

CopA binds 11 coppers in total, four as shown here and seven by an unknown manner (Mellano and Cooksey 1988). For detailed information and functions see text and Solomon et al. 1996; Pouderoyen 1996; Kroes 1997.

FIGURE 5 (A) Types I, II, and III copper centers in multicopper oxidases. R = methionine in several type I sites (e.g., azurin; see also Figure 4); L = O or N ligands. (B) Schematic representation of the orientation of the type II copper with respect to type III dimer in a trinuclear cluster, also called type IV.


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Type III Cu sites are dinuclear sites containing two closely situated Cu ions (Figure 5A); they do not exhibit an EPR signal because of their antiferromagnetic coupling. They can serve as oxygen carriers or can activate oxygen during the hydroxylation of phenolic substrates (Table 1). In the oxygen-boun d state they are characterized by a strong absorption maximum at 330 nm. Type III Cu proteins are thought to have evolved by combination of simple, 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 data, including the dinuclear CuA and CuZ centers and the CuB –hemeA site (Table 1) (Malmström 1990; Dennison and Canters 1996; Farrar et al. 1998). A trinuclear site, consisting of type II and 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 are generally called multicopper oxidases or blue oxidases. Multicopper oxidases display internal sequence homology, allowing the identi cation of subdomains. The subdomain structure of some oxidases has been con rmed by X-ray crystallography (Zaitseva et al. 1996). Each domain is characterized by an eight-stranded Greek 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 “cupredoxin fold.” The phylogenetic relationship between the individual domains of multicopper oxidases and the blue copper proteins indicates that the multicopper oxidases evolved from the latter by domain duplication (Ryden and Hunt 1993; Murphy et al. 1997; Abolmaali et al. 1998). Extension and modi cation of domains resulted in the rearrangement of Cu-binding sites and enabled the formation of new types of Cu centers with catalytic activity (Abolmaali et al. 1998). The type I center in multicopper oxidases still serves its ancestral function in that it shuttles electrons from the electron donor to the catalytic center (also see below). Small modi cations in the protein environment of the Cu sites allowed the ne-tuning of their redox potentials to speci c redox partners (Messerschmidt 1998). This generated a family of oxidase enzymes with a large variety of redox potentials and substrate speci cities, potentially including multicopper oxidases that are able to oxidize transition metals such as Mn. The properties and postulated 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 spectroscopic characteristics, sequence similarity, and reactivity. Members of this family include plant and fungal laccase, plant and bacterial ascorbate oxidase, human ceruloplasmin, yeast Fet3p, and bacterial CopA. Recently, additional members have been isolated and partly characterized, as discussed below. In general, multicopper oxidases contain one type I Cu site, and a type II and type III center organized in a trinuclear cluster (Solomon et al. 1996). Ceruloplasmin is unique among the multicopper oxidases in that it contains two additional type I centers. All multicopper oxidases have absorption maxima at » 600 nm (type I) or 330 nm (type III) and show EPR signals characteristic of type I and type II sites. The amino acid sequence similarity in the regions containing Cu-binding ligands is substantial (Figure 6). Generally, two of these regions are situated near the C terminus and separated by 35–75 amino acid residues; the other two are near the N terminus and separated by 35–60 residues. Each of the N- and C-terminal regions contains a conserved HXH motif. The eight histidines within the four motifs are the conserved peptide ligands of the trinuclear center. The type II Cu is coordinated by two of these histidines, and each of the two Cu ions belonging to the type III site is coordinated by three histidines (Figure 6)

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FIGURE 6 Alignment of the (putative) copper binding regions of different multicopper oxidases. The binding regions A, B, C, and D correspond to those shown in Figure 4. The copper-binding residues are designated I, II, IIIa, or IIIb on the basis of the types of copper they can potentially bind. Abbreviations used and Genbank accession number: LacF(ungus), Trametes versicolor laccase (X84683; Jonsson et al. 1995); LacP(lant), Acer pseudoplatanus laccase (U12757; Sterjiades et al. 1992); CpAO, Cucurbita pepo medullosa ascorbate oxidase (J04494; Ohkawa et al. 1989); HsCp, Human ceruloplasmin (M13699; Koschinsky et al. 1986); Fet3, Saccharomyces cerevisiae Fet3p (L25090; Askwith et al. 1994). CopA, copper resistance protein Pseudomonas syringae (M19930; Mellano and Cooksey 1988); PcoA, copper resistance protein plasmid pRJ1004 Escherichia coli (X83541; Brown et al. 1995); CumA, Pseudomonas putida GB-1 (AF086638; Brouwers et al. 1999); MofA, Lepthothrix 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 histidine nitrogen and a water molecule. The conserved ligands of the type I Cu are located in 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 methionine, 10 amino acids distant from the cysteine residue. Others contain a noncoordinating leucine or phenylalanine at this position (cf. Figure 6). The cysteine residue is anked by two of the histidines coordinating the trinuclear cluster (Figure 6). This arrangement provides a functional proximity between the type I Cu and the trinuclear cluster. Type I Cu is currently thought to oxidize the substrate by four subsequent one-electron oxidations. The electrons are passed to the trinuclear cluster through the cysteine–histidine pathway (Lowery et al. 1993). The trinuclear cluster is the site of the four-electron reduction of oxygen to water, probably through a peroxide intermediate (Sundaram et al. 1997). Virtually all multicopper oxidases consist of three cupredoxin domains, whereas ceruloplasmin consists of six (the putative multicopper oxidase from Bacillus SG-1 also contains six subdomains; see above). Most multicopper oxidases oxidize organic substrates. At present, only ceruloplasmin and Fet3p are known to directly catalyze the oxidation of a metal ion, in this case, Fe2 + (see below). One fungal laccase has recently been shown to be able to directly use Mn2 + as a substrate (Höfer and Schlosser 1999; see also below). Some multicopper oxidase homologs such as CopA do not seem to be redox-active at all (see below). Multicopper oxidases occur as enzymes with low (K m in the millimolar range) or high (K m in the micromolar range) substrate speci city. Laccases generally fall into the former category and are not supposed to contain a substrate-binding pocket, in contrast to the multicopper oxidases with high substrate speci city, such as ascorbate oxidase and ceruloplasmin.


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Laccase Laccase is a polyphenol oxidase (p-diphenol:dioxyge n oxidoreductase), rst identi ed in the Japanese lacquer tree Rhus vernicifera at the end of the nineteenth century (Yoshida, 1883). Since then it has been detected in a variety of plants (O’Malley et al. 1993) and shown to be a ubiquitous fungal enzyme as well (Thurston 1994). Laccase activity has also been found in insects (Binnington and Barrett 1988) and in one bacterium, Azospirillum lipoferum (Givaudan et al. 1993). Recently, a laccase-like pluripotent polyphenol oxidase was identi ed in a marine Alteromonas species (Sanchez-Amat and Solano 1997). Laccases have received a great deal of attention in view of their application in industrial oxidative processes such 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 pdiphenols, methoxy-substituted phenols, and methoxy-substituted diamines, and substrate speci cities vary from one laccase to another (Thurston 1994). Laccases have been shown to catalyze the formation of Mn(III) chelates from Mn2 + , either through oxidized phenolic intermediates (Archibald and Roy 1992) or directly in the presence of the chelator sodium pyrophosphat e (Höfer and Schlosser 1999). Many plant and fungal species produce isoforms of laccase encoded by multigene families (Mansur et al. 1998; Ranocha et al. 1999). The functions of the different laccases are as yet unresolved and may depend on the organism or type of tissue in which they are expressed (Ranocha et al. 1999). Most notably, they are implicated in the biosynthesis (by plants) or degradation (by fungi) of lignin, a complex aromatic biopolymer. Plant laccases have been puri ed from several species, allowing biochemical characterization, immunolocalization, and measurements of in vitro activity. Laccase has been immunolocalized 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 of herbaceous and woody species (Bao et al. 1993; O’Malley et al. 1993; Liu et al. 1994; McDouglas et al. 1994; McDouglas and Morrison 1996). Several isolated plant laccases have been shown to catalyze the dehydrogenative polymerization of monolignols (lignin monomers) (Sterjiades et al. 1992; Bao et al. 1993). Laccase genes were preferentially expressed in differentiating xylem tissue of poplar (Ranocha et al. 1999). These data convincingly point to involvement of laccase in lignin formation, in contrast to previous opinions proposing an exclusive role for lignin peroxidases in lignin biosynthesis (Nakamura 1967; Harkin and Obst 1973). However, neither laccase nor peroxidase alone can account for the stereospeci city of the monolignol polymerization reaction. Sterjiades et al. (1993) hypothesized that laccases and peroxidases work in a coordinated manner in lignin formation, with laccases producing oligolignols, and peroxidases catalyzing the formation of lignin from oligolignols. In some species, moreover, e.g., the lacquer tree, laccase does not seem to be involved in ligni cation at all (Nakamura 1967). At present studies are underway to downregulate laccase genes in transgenic plants and to study the effect on lignin content and composition (Ranocha et al. 1999). These experiments may clarify the speci c role of different laccases in lignin biosynthesis. Some proposed functions of fungal laccases are pigment formation, lignin degradation, and detoxi cation of phenoxy radicals produced during deligni cation or of phenols produced by other organisms (Thurston 1994). The best established function is pigment formation. A laccase-negative mutant of Aspergillus nidulans produced white spores instead of the green spores formed by the wild type. Addition of partly puri ed laccase to the growth medium of the mutant restored the wild-type phenotype (Clutterbuck 1972). The bacterial Azospirillum lipoferum laccase also has a function in pigment formation (Givaudan et al. 1993). The pigments may protect the microbial cells from radiation damage or from attack by cell wall–degrading enzymes (Givaudan et al. 1993).

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Because laccases are ubiquitous in wood-rotting fungi, they are assigned a role in lignin degradation, along with hemoprotein peroxidases. They are assumed to take part in the oxidative cleavage of some of the numerous structures in the complex biopolymer. The best evidence in support of such a role is that a laccase-negative mutant of Sporotrichum pulverulentum was unable to degrade lignin. The lignin-degrading ability was restored in laccase-positive revertants (Ander and Eriksson 1976). Mn(III)-catalyzed degradation of lignin by puri ed laccase, in concert with manganese peroxidase, has been demonstrated in vitro (Sterjiades et al. 1993). As stated above, laccase can catalyze the formation of strongly oxidizing Mn(III) chelates (Archibald and Roy 1992; Höfer and Schlosser 1999). These Mn(III) chelates, like the Mn(III) chelates produced by manganese peroxidase, are able to oxidatively degrade lignin. However, many lignin-degrading fungal strains do not produce laccase (Thurston 1994). Perhaps a combination of oxidative enzymes, manganese peroxidase and laccase, or manganese peroxidase and lignin peroxidase, is the minimum requirement for lignin breakdown (de Jong et al. 1992). In conclusion, the functions of fungal laccases, like those of plant laccases, are as yet not well established. Ascorbate Oxidase Ascorbate oxidase, which catalyzes the oxidation of ascorbate to dehydroascorbate, is mainly found in higher plants, especially in members of the Cucurbitaceae (e.g., cucumber, squash, and melon; Ohkawa et al. 1989). It has been studied extensively biochemically, and various genes encoding ascorbate oxidases have been isolated and sequenced (Nakamura et al. 1968; Ohkawa et al. 1989, 1990; Esaka et al. 1992; Moser and Kanellis 1994). In some plants various isoforms are encoded by a multigene family. The enzymes are associated with the cell wall (Lin and Varner 1991; Esaka et al. 1992). Ascorbate oxidase is 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 enzyme structure and expression, its role in plant metabolism is still obscure. Ascorbate oxidase transcripts and activities are greatest in actively growing tissues (Ohkawa et al. 1989; Lin and Varner 1991; Esaka et al. 1992). Consequently, the enzyme has been suggested to play a role in cell elongation, possibly by cell wall loosening as a result of the interaction of dehydroascorbat e with cell wall components (Lin and Varner 1991). Alternatively, ascorbate oxidase may function in regulation of the cell cycle by controlling the concentration of cellular ascorbic acid [ascorbic acid indirectly promotes the progression from the G1 to the S phase in the cell cycle (Arrigoni 1994)]. Ascorbate oxidase may induce cell cycle arrest by lowering the ascorbic acid concentration, for instance, during early stages of fruit development when cells do not divide (Diallinas et al. 1997). Finally, ascorbate is one of the compounds involved in plant oxidative defense systems (Foyer et al. 1994), often activated under stress conditions. This may explain why ascorbate oxidase expression is repressed under 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 an organic substrate have thus far been characterized: ceruloplasmin, ubiquitous in vertebrates, and Fet3p from the yeast Saccharomyces cerevisiae. Both enzymes catalyze the oxidation of Fe2 + (K m < 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 is more closely related to laccase and ascorbate oxidase than to ceruloplasmin (see below).


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Fet3p is an integral membrane protein with an extracellular multicopper oxidase domain (Askwith et al. 1994; Stearmann et al. 1996). Spectroscopic analysis of isolated recombinant Fet3p indicates the presence of one type I Cu site and a trinuclear cluster, similar to, among other things, laccase (Hassett et al. 1998). Fet3p mediates Fe transport by converting ferrous iron into ferric iron, the substrate for the permease Ftr1p (Kaplan and O’Halloran 1996; Stearman et al. 1996). Ferrous iron is generated by cell surface ferrireductases, which solubilize extracellular ferric iron pools to make them available for transport. The involvement of the multicopper oxidase Fet3p in Fe metabolism is illustrated by the fact that mutations in Cu transport proteins such as the Fet3p Cu loader Ccc2p result in a de ciency of Fet3p activity and of high-af nity Fe transport (Dancis 1998). All genes involved in Cu loading of Fet3p have human homologs with high degrees of sequence conservation (see below). Ceruloplasmin is to date unique among the multicopper oxidases in that it contains three type I Cu sites next to a trinuclear cluster (Zaitseva et al. 1996). One of the type I sites appears to be permanently reduced (Machonkin et al. 1998) and thus is catalytically irrelevant. One of the remaining type I sites is connected through a cysteine–histidine pathway to the trinuclear cluster, similar to the type I sites in laccase and ascorbate oxidase. The function of the third type I site is still uncertain. Recently, Machonkin et al. (1998) suggested that the resting form of ceruloplasmin in plasma under aerobic conditions is a four-electron oxidized form consistent with its function in the four-electron reduction of O2 to H2 O. Although isolated ceruloplasmin is able to oxidize aromatic substrates (Solomon et al. 1996), it oxidizes Fe2 + with much higher af nity. Its main function is thought to be the mobilization of cellular iron by acting as a ferrooxidase. The ferric iron thus produced is bound to plasma transferrin and transported to body tissues. The link between iron metabolism and copper was established years ago in Cu-de cient swine, which had low plasma ceruloplasmin activity, developed anemia, and accumulated iron in several tissues (Lee et al. 1968). The human genetic disease aceruloplasminemia (absence of ceruloplasmin) results in defects in iron homeostasis, including iron accumulation in tissues (Harris et al. 1995). Defects in the Menkes/Wilson disease protein, a Cu transporter, result in low concentrations of active plasma ceruloplasmin and anemia, similar to the symptoms in Cu-de cient swine (Bull et al. 1993). The Menkes/Wilson disease protein is highly similar to the yeast Cu-transporter Ccc2p; substituted for defective Ccc2p, it can restore the Cu-loading and activity of Fet3p in ccc2p mutants (Payne and Gitlin 1998). This illustrates the evolutionary conservation of proteins responsible for homeostasis of transition metals (Askwith and Kaplan 1998). In contrast to the proposed role of ceruloplasmin in stimulation of iron egress from cells, recent studies with isolated cell cultures have indicated a function in cellular Fe uptake (Attieh et al. 1999). Alternatively, ceruloplasmin could be involved in Cu metabolism rather than in that of Fe, controlling the concentration of Cu and maintaining the redox balance in plasma (Leung 1998), or delivering Cu to Cu-dependent enzymes such as cytochrome c oxidase and superoxide dismutase (Marceau and Aspin 1973a, 1973b; Hsieh and Frieden 1975). Studies indicating involvement of ceruloplasmin in superoxide-dependen t oxidative modi cation of plasma lipoproteins (Mukhopadhyay and Fox 1998) have further complicated 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 recently been described and partially characterized. Bilirubin oxidase, sulochrin oxidase, and dihydrogeodin oxidase have been identi ed in various fungi, and phenoxazinone synthase has been found in several Streptomyces species (see Solomon et al. 1996 for an overview).

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The amino acid sequences of these proteins show the conserved Cu-binding domains present in all multicopper oxidases. Studies on overproduce d wild-type and mutant bilirubin oxidase have indicated intramolecular electron transport from the type I Cu to the trinuclear cluster via a cysteine–histidine pathway (Shimizu et al. 1999). Bilirubin oxidase and phenoxazinone oxidase show a small but important sequence similarity outside the Cu-binding domains to the 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 be redox-active. They appear to be involved in Cu resistance, for instance, CopA, a plasmidborne homolog of multicopper oxidases isolated from P. syringae pv tomato (Mellano and Cooksey 1988). The copA gene is part of an operon consisting of four genes, copA through copD. All four gene products are involved in Cu resistance but only CopA and CopB are essential. Mutation analysis indicates that CopC and CopD are required for full activity, but 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 ions entering the bacterial cells are thus accumulated in the periplasm. Cooksey (1993) proposed that four Cu atoms are bound to the type I, II, and III Cu sites and that additional Cu ions are bound in octapeptide motifs. Homologs of the cop gene cluster have been identi ed on the chromosome of Xanthomonas campestris pv juglandis (Lee et al. 1994) and on the Escherichia coli plasmid pRJ1004 (Brown et al. 1995). In the latter organism, resistance is based on reduced Cu uptake instead of Cu accumulation (Cervantes and Gutierrez-Corona 1994). Multicopper oxidases involved in Cu resistance appear to depend on their Cu2 + binding, not their oxidative potential. The possibility cannot be excluded, however, that they 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 various oxidizing systems differ in many respects. For instance, the process can be catalyzed by metabolically inert spores, by cellular outer membrane components, or by bacterial sheaths. Except for the intracellular oxidation of Mn2 + by Lactobacillus plantarum, which uses Mn2 + ions in scavinging superoxide radicals (Archibald and Fridovich 1981), bacterial Mn oxidation appears to be con ned to outer surface coverings. Because of diversity in oxidizing systems, it has been hard to formulate unifying concepts on the mechanisms and functions of the process. Only recently has a common theme been revealed: Proteins with sequence similarity to multicopper oxidases play a role in Mn2 + oxidation in a marine Gram-positive and two fresh-water Gram-negative species. These proteins, MnxG, MofA, and CumA, all contain the highly conserved Cu-binding ligands characteristic of multicopper oxidases. MnxG appears to consist of subdomains. The multicopper oxidases ceruloplasmin, laccase, and ascorbate oxidase contain subdomains with a speci c so-called cupredoxin fold. Both MofA and CumA can also be expected to contain cupredoxin subdomains. This can be veri ed by a computerized search for internal homology regions in the proteins. The amino acid sequences can be analyzed for their potential to adapt a cupredoxin fold. Cloning the respective genes allows overproduction and puri cation of the proteins. For instance, parts of MofA have already been produced and puri ed in substantial quantities after expression in E. coli (Brouwers 1999). Optimization of the overproduction systems will open the way for spectroscopic and eventually crystallographic characterization of these putative multicopper oxidases. The question arises as to whether MnxG, MofA, and CumA are directly or indirectly involved in Mn2 + oxidation. The genes encoding MnxG and CumA have been identi ed by mutagenesis and characterization of nonoxidizing mutants. This approach will also detect


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genes encoding indirectly involved products, for example, regulatory genes, genes encoding enzymes involved in transport, genes responsible for biosynthesis of potential cofactors, and so forth. MofA, on the other hand, has been identi ed by antibodies against an active Mn2 + -oxidizing factor, which supports a direct involvement of the multicopper oxidases in Mn2 + oxidation. If MnxG, MofA, and CumA are directly involved in metal oxidation, they can be compared with the metal-oxidizing multicopper oxidases ceruloplasmin and Fet3p, which oxidize Fe2 + with a K m comparable with that of bacterial Mn2 + oxidation. However, neither MnxG, MofA, nor CumA is able to oxidize Mn2 + when produced from an expression vector in E. coli (Tebo et al. 1997; Brouwers 1999). Spectroscopic analysis of the overproduce d proteins should indicate whether Cu ions are correctly incorporated in the protein structure. Analysis of Cu incorporation in Fet3p has shown that speci c genes of the Cu metabolism have to be operative for Fet3p to attain activity. Such genes may be absent or silent in E. coli. To circumvent potential dif culties in the production of active multicopper oxidases because of the absence of metal cofactors, expression of, for instance, CumA in closely related organisms, such as the nonoxidizing P. putida strains, may also be attempted. In principle, multicopper oxidases oxidize their substrates directly with the concomitant reduction of oxygen to water. Except for multicopper oxidases with low substrate speci city such as laccases, the oxidases are proposed to contain speci c substrate-binding pockets. Consequently, MnxG, MofA, and CumA may contain Mn2 + -binding sites. This can be con rmed by Mn2 + -binding studies, and the potential binding sites may be identi ed by site-directed mutagenesis. An alternative approach to assessing the role of the multicopper oxidases in Mn2 + oxidation is to localize the proteins in subcellular fractions with the use of speci c antibodies in wild-type and mutant cells. Production of such antisera has become feasible by the development of overproduction and puri cation protocols for recombinant proteins. For instance, if CumA represents the structural Mn2 + -oxidizing enzyme in P. putida GB-1, it should be localized at the outer membrane of wild-type cells. The protein may be found in the periplasm, plasma membrane, or cytosol of the transport mutants. Localization can also be performed with GFP (green uorescent protein) or other uorescent protein fusions. Antibodies may be used to attempt puri cation of the native multicopper oxidases by af nity chromatography. To date, puri cation by other biochemical methods has been very problematic. Studies on the Mn2 + -oxidizing factors as they are electrophoretically detected in extracts of Bacillus SG-1 spores, Pseudomonas putida cells, and Leptothrix discophora spent media suggest that these factors are not isolated as single proteins. They appear to reside in complexes originating from spore coats (Bacillus), sheath remnants (Leptothrix), or the outer membrane (Pseudomonas). Because these putative complexes display the oxidizing activity, the Mn2 + -oxidizing multicopper oxidases may depend on additional factors for Mn2 + oxidation. For instance, attached saccharides may assist in binding Mn2 + . Isolation of the native multicopper oxidases will allow characterization of potential nonprotein components. Because inhibition of cytochrome c synthesis in P. putida GB-1 and MnB-1 results in loss of oxidizing activity, and the mof operon of L. discophora appears to encode a protein with a potential heme-binding site, c-type hemes may play a role in Mn2 + oxidation. However, the genes of the cytochrome c operon have been shown to ful ll dual functions, and the determinants for the different functions appear to reside in different regions of the genes. For instance, the 30 end of ccmI was shown to be essential for Cu resistance and not for cytochrome c synthesis in P. uorescens 09906 (Yang et al. 1996). Different amino acid residues in the CcmC protein were found to function in pyoverdine production and cytochrome c biogenesis in P. uorescens ATCC 17400 (Gaballa et al. 1998). Thus, possibly one or more of the ccm gene products are involved in the Mn2 + -oxidizing process,

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for instance, in the metabolism of Cu on which the Mn2 + -oxidizing enzyme is supposed to depend. Studies of the effect of site-directed mutagenesis of the cytochrome c biogenesis genes on Mn2 + oxidation and on the production of CumA will resolve this question. Unfortunately, studies on the effects of speci c mutagenesis of the mof operon are not feasible, because no transformation system is available for L. discophora. Finally, Mn2 + may not be the primary substrate of the potential multicopper oxidases MnxG, MofA, and CumA. Instead, they might be primarily involved in the oxidative crosslinking of cell surface structures such as spore coat proteins or sheath components, with the ability to oxidize Mn2 + being a (bene cial) side effect. If so, these oxidases may be compared with laccases, which have been proposed to function, among other processes, in the oxidative polymerization of lignin monomers. However, unpublished results in our laboratory showed that neither the spent medium of L. discophora SS-1 nor cells of P. putida GB-1 were able to directly oxidize syringaldazine, a model laccase substrate. Determining the substrate speci city of the multicopper oxidases MnxG, MofA, and CumA is of utmost importance for identifying their physiologic role. This underlines the necessity of overproduction systems and puri cation protocols for active proteins.

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