Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach / Investigación de las especifidades de la unión de substrato para la citocromo-c-oxidasa: un enfoque asistido por computadora

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

La deficiencia de la citocromo-c-oxidasa (COX) se halla asociada con un número de enfermedades, pero los detalles del mecanismo de acción - especialmente la interacción con su substrato, el ferrocitocromo c - no está aún claro. Se sabe que la transferencia de electrones del ferrocitocromo c a la COX, es facilitada por la formación de los complejos enzima-substrato (ES), los cuales son estabilizados por puentes intermoleculares de sal. No obstante, la identidad de los residuos que participan en los puentes sigue sin estar clara. Recurriendo a las estructuras publicadas de dos proteínas, se emplearon simulaciones por computadora a fin de obtener un modelo de sus interacciones, en un intento por identificar los residuos que toman parte en los puentes de sal. El proceso de simulación fue guiado principalmente por estudios de reticulación, que proponen que el Lys-13 del citocromo c está pareado con el Asp-18 de la COX. El complejo enzima-sustrato inicial creado mediante la manipulación asistida por computadora de las dos estructuras, exhibía tres puentes de sal. Tras aplicar los procedimientos de minimización de la energía, el número de puentes de sal aumentó a siete y hubo veinticuatro enlaces intermoleculares de hidrógeno. Los puentes de sal emanaron de: Glu-119 y Asp-221 de la subunidad I; Glu-114, Asp-115 y Asp-158 de la subunidad II y Asp-73 y Glu-78 de la subunidad VIb. Estos fueron pareados con Lys-87, 8, 25, 27, 13, 22 y 100 respectivamente del citocromo c. Estos resultados sugieren que las subunidades I, II y VIb juegan un papel directo en la unión del substrato. Los resultados también sugieren que los enlaces de hidrógeno contribuyen significativamente a la estabilidad del complejo-ES.

Molecular phylogenic location of the Plagiorchis muris (Digenea, Plagiorchiidae) based on sequences of partial 28S D1 rDNA and mitochondrial cytochrome C oxidase subunit I

To determine the molecular phylogenic location of Plagiorchis muris, 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome C oxidase subunit I (mtCOI) were sequenced and compared with other trematodes in the family Plagiorchiidae. The 28S D1 tree of P. muris was found to be closely related to those of P. elegans and other Plagiorchis species. And, the mtCOI tree also showed that P. muris is in a separate clade with genus Glypthelmins. These results support a phylogenic relationship between members of the Plagiorchiidae, as suggested by morphologic features.

Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.

Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand.

The mitochondrial cytochrome c oxidase subunit I (CO I) gene and the second internal transcribed spacer region (ITS II) gene of Opisthorchis viverrini were compared among O. viverrini from various areas in northeast Thailand. The nucleotide sequences of partial CO I gene (417 bp) of O. viverrini differed among O. viverrini originated from Ubon Ratana, Leongpleuy, Ban Phai, Maha Sarakham, and Chatturat. These intraspecific variations were classified into 5 patterns but no area-specific pattem was observed. Amino acid sequence deduced from the nucleotide sequences of these genes was identical. Nucleotide sequences of a region of the O. viverrini ITS II gene (296 bp) from different areas were identical. However, they were different from those of Clonorchis sinensis, Haplorchis taichui, H. pumilio, Fasciola gigantica, Echinostoma malayanum and Centrocestus sp..