Insight into the multicopper oxidases stability

Dissertation presented to obtain the PhD degree in Biochemistry

This dissertation portrays recent development on the knowledge of the stability determinants and of functional characteristics of multicopper oxidases (MCO). Multicopper oxidases are a family of enzymes that includes laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2), ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.3) and ceruloplasmin (Fe2+ oxygen oxidoreductase, EC 1.16.3.1). MCO are characterized by having four copper ions that are classified into three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The classical T1 copper site comprises two histidine residues and a cysteine residue arranged in a distorted trigonal geometry around the copper ion with bonding distances approx. 2.0 Å (1 Å=0.1 nm); a weaker fourth methionine ligand completes the tetrahedral geometry. The copper–cysteine linkage is characterized by an intense S(π)→Cu(dx2−y2) CT (charge transfer) absorption band at approximately 600 nm, and a narrow parallel hyperfine splitting A\\ = (43–90)×10−4 cm−1 in the electron paramagnetic resonance (EPR) spectrum. The function of the T1 copper site is to shuttle electrons from substrates to the trinuclear copper centre where molecular oxygen is reduced to two molecules of water during the complete four-electron catalytic cycle. The trinuclear center contains a T2 copper coordinated by two histidine residues and one water molecule, lacks strong absorption bands and exhibits a large parallel hyperfine splitting in the EPR spectrum (A\\ = (150–201)×10−4 cm−1). The T2 copper site is in close proximity to two T3 copper ions, which are each coordinated by three histidine residues and typically coupled, for example, through a dioxygen molecule. The T3 or coupled binuclear copper site is characterized by an intense absorption band at 330 nm originating from the bridging ligand and by the absence of an EPR signal due to the antiferromagnetically coupling of the copper ions.(...)

Apoio financeiro da FCT e do FSE no âmbito do Quadro Comunitário de Apoio, BD nº SFRH/BD/31444/2006