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Structure and Molecular Evolution of Multicopper Blue Proteins

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Structure and Molecular Evolution of Multicopper Blue Proteins Article in press - uncorrected proof BioMol Concepts, Vol. 1 (2010), pp. 31–40 • Copyright ᮊ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BMC.2010.004 Short Conceptual Overview Structure and molecular evolution of multicopper blue proteins Hirofumi Komori1,2 and Yoshiki Higuchi1,2,* containing enzymes participate in reactions involving 1 Department of Life Science, Graduate School of Life molecular oxygen. During the evolution of copper-containing Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, proteins, the number of domains in a subunit and the pattern Ako-gun, Hyogo 678-1297, Japan of subunit assembly have varied from protein to protein, 2 RIKEN SPring-8 Center, 1-1-1 Koto, Mikazuki-cho, together with point mutations. The variation appears to pro- Sayo-gun, Hyogo 679-5248, Japan mote the functional evolution of various copper-binding sites for an efficient catalysis of complex redox processes. In the * Corresponding author course of evolution, unique copper sites were created for e-mail: [email protected] oxygen binding and electron storage. Type I copper, called blue copper, has unique spectro- scopic properties and was originally found in electron trans- Abstract fer proteins (2, 3). It has unusually high redox potential and assists electron transfer systems in photosynthesis and nitrate The multicopper blue protein family, which contains cupre- respiration (1, 4). The cupredoxin-like fold containing Type doxin-like domains as a structural unit, is one of the most I copper is found as a structural unit in numerous proteins diverse groups of proteins. This protein family is divided into and is a typical motif for the multicopper blue proteins two functionally different types of enzymes: multicopper (MCBPs). The MCBP family is one of the most important oxidase and nitrite reductase. Multicopper oxidase catalyzes copper-containing protein groups, which contains a diverse the oxidation of the substrate and then reduces dioxygen. The group of proteins containing 2–6 copper ions with anywhere structures of many multicopper oxidases are already known, from 300 to more than 1000 amino acid residues in a single and until recently they were classified into two main groups: peptide chain. They have a multidomain structure and are the three- and six-domain types. Both function as monomers divided into two types of enzymes: multicopper oxidase and have three spectroscopically different copper sites: Types (MCO) and nitrite reductase (NIR). I (blue), II, and III (tri-nuclear). Nitrite reductase is a closely MCO uses the distinctive redox ability of copper to cata- related protein that contains Types I and II (mono-nuclear) lyze the oxidation of a wide range of substrates, followed by coppers but reduces nitrite instead of dioxygen. Nitrite reduc- the reduction of dioxygen (O ) to water (H O) (Figure 1) (5). tase, which consists of two domains, forms a homotrimer. 2 2 MCO contains three different types of copper ions. All types Multicopper oxidase and nitrite reductase share similar struc- of copper are involved in the transfer of electrons from the tural architectures and also contain Type I copper. Therefore, substrate to dioxygen, the final electron acceptor. MCO has it is proposed that they have a common ancestor protein. cupredoxin-like domains as structural units and has been Recently, some two-domain type multicopper oxidases have classified into two groups according to the number of been found and their crystal structures have been determined. domains: three-domain type (3dMCO) and six-domain type They have a trimeric quaternary structure and contain an (6dMCO), while they possess very similar optical spectral active site at the molecular interface such as nitrite reductase. properties related to the copper ions. These results support previous hypotheses and provide an NIR is highly related to MCO but no longer reduces the insight into the molecular evolution of multicopper blue dioxygen, instead functioning to reduce nitrite (NO -) (Figure proteins. 2 1). Therefore, it is thought to be another distinct branch of Keywords: ascorbate reductase; blue copper; MCO (6–8). ceruloplasmin; laccase; multicopper oxidase; nitrite Since the first crystal structure of MCO was obtained in reductase; Type I copper. 1989, many crystal structures of 3dMCOs, 6dMCOs, and NIRs have been determined (8–10). They share similar struc- tural architectures, but the oligomeric state of NIRs is com- Introduction pletely different from MCOs. Therefore, it was widely believed that they have a common ancestor protein, which Copper is essential for life, through its role in various pro- consists of consecutive cupredoxin-like domains (8, 11). teins (1). Among the bioelements, copper is thought to be a As the genome projects have given us an increasing modern metal that is available mainly after the photosyn- amount of sequence data, new homologs of MCOs have been thetic generation of an oxidizing environment. Many copper- identified. In 2003, Nakamura et al. suggested a new class 2010/6 Article in press - uncorrected proof 32 H. Komori and Y. Higuchi Figure 2 Structure of a cupredoxin fold, which has an eight- stranded Greek key b-barrel fold. Type I coppers are shown in blue. It has a distorted tetrahedral coordination system coordinated by three strong ligands (one cys- teine and two histidines) and one weaker ligand, a methionine. The Figure 1 Schematic presentation of the catalytic mechanism of figures were prepared by the PyMOL program (http:// multicopper oxidase (A) and nitrite reductase (B). pymol.sourceforge.net), using the coordinate from PDB file 1PLC (Popar plastocyanin). of two-domain type MCO (2dMCO) (12). On the basis of Three-domain type MCO (3dMCO) sequence alignments, several types of 2dMCOs have been predicted and are classified according to the location of the Type I copper-binding sites. At the same time, the novel 3dMCO, representing the main group of MCOs, consists of types of MCOs, which have much smaller molecular weight three cupredoxin-like domains. There are various types of than those of the typical 3dMCOs have been characterized 3dMCO, including laccases and several oxidases with spe- biochemically (13–15). They are thought to lack the second cific substrates such as ascorbate, copper, iron or bilirubin. domain and are considered 2dMCOs. Laccases catalyzes the oxidation of a variety of aromatic Recently, three crystal structures of 2dMCOs have been compounds including diphenols. They have been found in a determined. All structures of 2dMCOs show the same tri- wide variety of plants, fungi, bacteria, and insects and have meric architecture as that of NIR. Furthermore, 2dMCOs and hence been studied extensively (16). Ascorbate reductase NIRs have their active sites at a similar molecular interface. (AO), which exhibits a high specificity toward L-ascorbate, These results confirm the evolutionary relationship between is one of the best characterized MCOs and the crystal struc- MCOs and NIRs. In this study, we describe the molecular ture was solved 20 years ago (9). Many crystallographic results are available for 3dMCOs (laccases from fungus evolution of MCBPs on the basis of the three-dimensional (16–24), CueO (25), CotA (26), Fet3p (27), and phenoxa- structures of 2dMCOs. zinone synthase (28)). They contain single peptide chains of around 500 amino acid residues with four copper ions in three distinct sites. They all have the same structural archi- tecture with three sequentially arranged cupredoxin-like Multicopper oxidase domains (Domains 1, 2, and 3) (Figure 3A). Each of them has eight conserved b-strands, constituting the core structure MCO is a Type I (blue) copper-containing enzyme, which is of each cupredoxin-like domain, which are connected by sev- involved in the oxidation of substrate, followed by the reduc- en structurally unconserved regions (8). These unconserved tion of dioxygen (Figure 1) (5). MCOs are the only enzymes regions are likely to be involved in the modulation of the known to catalyze the four-electron reduction of dioxygen to substrate recognition and molecular stability in 3dMCOs. water, except for cytochrome c (CYTC) oxidase, the terminal enzyme in the respiratory system. They all consist of cupre- Three types of coppers doxin-like domains, which have eight stranded Greek key b- barrel folds (Figure 2). The cupredoxin fold was originally Coppers in MCOs are classified into three types (I, II, and found in small copper proteins, such as plastocyanin and III) on the basis of optical and electromagnetic resonance azurin (2, 3). They contain Type I copper, exhibiting a bright (EPR) spectroscopic features. The spectroscopic features of blue color; they generally have high redox potential and a Type I copper are basically the same as those of electron function as an electron transfer protein. Until recently, the transfer proteins. It has an absorption peak around 600 nm multicopper proteins were divided into two subfamilies, and a narrow hyperfine coupling in EPR spectroscopy. This 3dMCO and 6dMCO, according to the number of cupre- copper has a distorted tetrahedral coordination system coor- doxin-like domains. dinated by three strong ligands (one cysteine and two histi- Article in press - uncorrected proof Multicopper blue proteins 33 Figure 3 Overall structure of multicopper blue proteins, 3dMCO (AO) (A), 6dMCO (CP) (B), NIR (C), 2dMCO_wBx (SLAC) (D), and 2dMCO_wCx (mgLAC) (E). The first (Class IV) and second (Class V) domains are shown in pink and light green, respectively. 3dMCO and 6dMCO are single chain proteins. NIR and 2dMCOs are trimers of two consecutive domains. Type I coppers are indicated by blue circles. The Type II and Type III coppers in MCOs are shown in green and cyan circles, respectively. The Type II coppers in NIR are shown in cyan circles. The figures were prepared by the PyMOL program, using coordinates from PDB files 2BW4 (NIR from Achromobacter cycloclastes), 1AOZ (AO from Cucurbita pepo var. melopepo), and 2J5W (CP from human).
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