ABSTRACT
Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.
La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.
Subject(s)
Animals , Cattle , Electron Transport Complex IV/metabolism , Cytochromes c/metabolism , Heme/chemistry , Hydrogen/metabolism , Oxidation-Reduction , Paracoccus denitrificans/enzymology , Protons , Rhodobacter sphaeroides/enzymology , Amino Acid Sequence , Thermus thermophilus/enzymology , Escherichia coli/enzymologyABSTRACT
Reported models of denitrification rates integrate in an unique parameter the pH-dependent inhibition by HNO2 and the pH effect on the bacterial metabolic activity; furthermore, they do not quantify separately the pH effect on the nitrate and on the nitrite reduction rates. The goal of this work was to quantify both effects on the kinetics of nitrate and nitrite reduction to improve the models predictive value. Assays were performed at a pH range of 6.5-9.0 in batch reactors at 37ºC with an activated sludge. At the studied pH range and at below the HNO2 inhibitory concentration (0.004 mg L-1), the maximum nitrate reduction rate diminished 23 percent and 50 percent by decreasing or increasing, respectively, one pH unit from 8.0. The maximum nitrite reduction at pH 8.0 diminished 15 percent at pH 7.0 and 40 percent at pH 9.0. At HNO2 concentrations over the inhibitory concentration, except at pH > 8.0, the maximum nitrate reduction rate diminished 50 percent upon decreasing the pH from 8.0 to 7.0 or increasing it from 8.0 to 9.0. Inclusion of the pH effect in the reported models improved their predictive value; average deviations from the experimental data were reduced from 53 percent to 10.7 percent or 33.8 percent to 10.5 percent for nitrite and nitrate reduction rates, respectively.