RESUMO
A multicopper oxidase, CueO was doubly mutated at its type I copper ligand, Cys500 and an acidic amino acid residue located in the proton transfer pathway, Glu506, to Ser and Ala, respectively. Cys500Ser/Glu506Ala was mainly in a novel resting form to afford the absorption band at ca. 400 nm and an EPR signal with a highly anisotropic character derived from type III copper. However, Cys500Ser/Glu506Ala gave the same reaction intermediate (peroxide intermediate) as that from Cys500Ser and Cys500Ser/Glu506Gln.
Assuntos
Cobre/química , Proteínas de Escherichia coli/química , Mutação de Sentido Incorreto , Oxirredutases/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/metabolismo , Ligação ProteicaRESUMO
Glu506 involved in the hydrogen bond network leading from solvent waters to the trinuclear copper center in a multicopper oxidase, CueO plays a crucial role to transport protons in the four-electron reduction of dioxygen to water. We performed X-ray crystal structure analyses of the Glu506Ala and Glu506Ile mutants, showing the formation of a compensatory proton transport pathway with only water molecules and a disruption of the hydrogen bond network due to the bulky side chain, respectively. We discuss the efficiency of proton transport through the hydrogen bond network based on the present results and our previous modification of the proton transport pathway by the Glu506 to Gln mutation, which have allowed us to trap and characterize the reaction intermediates.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/química , Escherichia coli/genética , Oxirredutases/química , Oxirredutases/genética , Oxigênio/metabolismo , Prótons , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/metabolismo , Conformação ProteicaRESUMO
CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.