Multi-copper oxidases

Ambient temperature catalysis of O2 reduction at low overpotentials is a challenge in development of conventional proton exchange membrane fuel cells (pol5mer electrolyte membrane fuel cells, PEMFCs) [Ralph and Hogarth, 2002]. In this chapter, we discuss two classes of enz5mes that catalyze the complete reduction of O2 to H2O multi-copper oxidases and heme iron-containing quinol oxidases. 

Of direct interest for biofuel cell applications are the reported reduction of O2 by multi-copper oxidases on carbon nanotube electrodes [Yan et al., 2006 Zheng et al., 2006] and the oxidation of H2 by hydrogenase covalently bound to carbon nanotubes [Alonso-Lomillo et al., 2007]. The hydrogenase/nanotube anode is extremely stable (>1 month), and shows 33-fold enhanced enzyme coverage compared with similarly treated graphite of the corresponding geometric surface area. A. vinosum... 

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

A. Messerschmidt, Multi-Copper Oxidases, World Scientific, Singapore, 1997. 

The salient features of A. faecalis pseudoazurin are that (1) it has a Cu-Met bond length shorter than that of either plastocyanin or azurin (see Table III) (2) it has only one NH - S bond, as does plastocyanin and (3) its overall architecture resembles plastocyanin (see Fig. 4), with an extended carboxy terminus folded into two a helices [a preliminary sequence comparison suggested that the folding would resemble plastocyanin (Adman, 1985)]. It retains the exposed hydrophobic face found in azurin and plastocyanin. Just how it interacts with nitrite reductase is still a subject of investigation. It is intriguing that the carboxy-terminal portion folds up onto the face of the molecule where the unique portions of other blue proteins are the flap in azurin, and, as we see below in the multi-copper oxidase, entire domains. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

A new putative member of the blue multi-copper oxidases has been isolated using the Escherichia coli yacK gene. Six copper ions per polypeptide chain were determined and assigned to two type 1 copper centers and further one type 2 and one type 3 copper. Phenoloxidase and ferroxidase properties were ascertained98 A new copper containing nitrite reductase was purified from a halophilic archaeon and the ligands to type 1 and type 2 coppers in the sequence were... 

T. Sakurai and S. Suzuki, in Multi-Copper Oxidases , ed. A. Messerschmidt, World Scientific, Singapore, 1997, p. 225-250. 

One of the major goals of studying active sites in copper proteins has therefore been to understand the spectroscopic features associated with the active site. This has led to a classification of three general types of copper protein active sites based on their unique spectral features Blue copper, normal copper and coupled binuclear copper. An additional class of copper proteins, the multi-copper oxidases, contains a combination of these three types of copper active sites. A reasonably firm understanding of the optical and EPR spectra of a number of copper proteins has now been achieved1,2K This article presents an overview of these electronic spectral features and their relationship to geometric and electronic structure. 

For a two-pulse (90° - t - 180°), or primary echo experiment, the integrated intensity of the spin echo, which occurs at time t after the 180° pulse, is measured as a fimction of increasing t from the probe s dead-time ( 100 ns) to a time where the echo amplitude has decayed to a few percent of its initial amplitude (2-8 ps for most powder samples). A two-pulse ESE decay envelope for the type-1 Cu(II) site of a multi-copper oxidase, Fet3p, is shown in Figure 1(a). The data show an overall decay characterized by a phase memory time, Tm or T, of < 1.0 ps. Superimposed on this decay are echo modulations that arise ft om hyperfine coupling to the N nuclei of two histidyl imidazole ligands and the protons of the snrronnding matrix. 

Figure 5 The iron reductase and high affinity uptake system in yeast. Frel,2P, iron reductase proteins Fet3P, multi-copper oxidase FtrP, high affinity transport protein (after Eide, 1998).

Brouwers G. J., de Vrind J. P. M., Corstjens P., Cornelis P., Baysse C., and DeJong E. (1999) cumA, a gene encoding a multi-copper oxidase, is involved in oxidation in... 

Francis C. A. and Tebo B. M. (2001) cumA multi-copper oxidase genes from diverse Mn(ll)-oxidizing and non-Mn(ll)-oxidizing Pseudomonas strains. Appl. Environ. Microbiol. 67, 4272-4278. 

O. Farver and I. Pecht, ElectronTransfer Reactions in Multi-Copper Oxidases, A. Messerschmidt, Ed., World Scientific Publications, Singapore 1997, pp. 355-389. 

L. Avigliano A. Finazzi-Agro, Biological Function and Enzyme Kinetics of Ascorbate Oxidase. In Multi-Copper Oxidases ... 

Lindley, P. Multi-copper oxidases. In Handbook on Metalloproteins Bertini, I. Sigel, A. Sigel, H., Eds. Marcel Dekker New York, NY, 2001 pp 763-811. 

This review is concerned with the chemical and physical properties of proteins and enzymes containing three distinct and unique forms of Cu The "blue center or, in the nomenclature proposed by Vdnng rd, Type 1 Cu2+ the colorless or Type 2 Cu2+, common to all multi-copper oxidases which reduce molecular oxygen to two molecules of water and the Cu associated with the 330 nm absorption band, again common to the oxidases. The purposes of the review are to assemble chemical and physical data related to the indicated types of Cu binding sites, to offer some interpretations (and occasionally re-interpretations) of experimental results concerned with structure-function relationships, and to generalize some of the information available as it concerns the structures of these unique Cu-co-ordination complexes. Special emphasis will be placed on the kinetic and mechanistic work which has been carried out on the multi-copper oxidases while the physiological roles of the various protein systems will not be of particular importance. 

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