The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E0 =455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the kcat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

The multicopper oxidases (MCOs) constitute a family of enzymes that present broad substrate specificity, oxidizing numerous aromatic phenols and amines. The one-electron oxidation of these substrates occurs concomitantly with a four-electron reduction of molecular oxygen to water. The redox reactions catalyzed by these enzymes depend on the presence of three copper sites, designated Cu types, 1, 2 and 3; a mononuclear T1 copper centre that is the primary acceptor for electrons; and a trinuclear centre comprising one T2 and two T3 copper ions that is involved in dioxygen reduction to water [1,2].The laccases constitute a large subfamily of MCOs and have been implicated in various biological activities related to lignolysis, pigment formation, detoxification and pathogenesis [3]. Laccases have a great potential in various biotechnological processes mainly owing to their high relative nonspecific oxidation capacity, the lack of a requirement for cofactors and the use of readily available oxygen as an electron acceptor. A few MCO members are able to oxidize lower valence metal ions, such as Cu The gene, Aquifex aeolicus AAC07157.1, encoding a multicopper oxidase (McoA) and localized in the genome as part of a putative copper-resistance determinant, has been cloned, over-expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. The purified enzyme shows spectroscopic and biochemical characteristics typical of the well-characterized multicopper oxidase family of enzymes. McoA presents higher specificity (k cat ⁄ K m ) for cuprous and ferrous ions than for aromatic substrates and is therefore designated as a metallo-oxidase. Addition of copper is required for maximal catalytic efficiency. A comparative model structure of McoA has been constructed and a striking structural feature is the presence of a methionine-rich region (residues 321-363), reminiscent of those found in copper homeostasis proteins. The kinetic properties of a mutant enzyme, McoADP321-V363, deleted in the methionine-rich region, provide evidence for the key role of this region in the modulation of the catalytic mechanism. McoA has an optimal temperature of 75°C and presents remarkable heat stability at 80 and 90°C, with activity lasting for up to 9 and 5 h, respectively. McoA probably contributes to copper and iron homeostasis in A. aeolicus.Abbreviations ABTS, 2,2¢-azinobis-(3-ethylbenzo-6-thiazolinesulfonic acid); IPTG, isopropyl thio-b-D-galactoside; MCO, multicopper oxidase; McoA, multicopper oxidase from Aquifex aeolicus; SGZ, syringaldazine.

The multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV–visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the multicopper oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site‐directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 °C, a half‐life of inactivation of ∼ 6 h at 80 °C, and temperature values at the midpoint from 97 to 112 °C. McoP is an efficient metallo‐oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min−1, respectively, at 40 °C. It is noteworthy that McoP follows a ping‐pong mechanism, with three‐fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of multicopper oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.

The aim of this study was to characterize the immune system profile in the uterine cervix of 17 human papillomavirus (HPV)-infected women, compared with 17 whom were coinfected with HIV-1. Five histologically normal cervices in immunocompetent women were used as controls. HPV infection was associated with a marked increase in cells expressing interleukin (IL)-6, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha). Coinfection by HPV and HIV-1 led to decreased expression of IL-6, TNF-alpha, and IFN-gamma. However, coinfection led to increased numbers of cells expressing IL-4, IL-10, and IL-8. Compared with the histologically normal cervices, increased numbers of macrophages (CD68, RFD7) and T lymphocytes (CD4, CD8) were seen in HPV-infected cervices; coinfection with HIV-1 was associated with a higher number of CD8 cells and lower number of CD68 cells. HPV DNA localized exclusively to the dysplastic squamous cells, whereas HIV-1 RNA was detected mainly in CD68-positive stromal cells. In conclusion, this study shows differential expression of various cytokines and classes of inflammatory cells, relative to HIV-1 infection and HPV coinfection, which may relate to the risk of transmission of HIV-1 and increased risk of cervical cancer in these women.