RESUMEN
We report twelve novel mutations in patients with isolated sulfite oxidase deficiency. The mutations are in SUOX, the gene that encodes the molybdohemoprotein sulfite oxidase. These include two frameshift mutations, a four-basepair deletion (562del4) and a single-basepair insertion (113insC), both resulting in premature termination. Nonsense mutations predicting Y343X and Q364X substitutions were identified in a homozygous state in three patients, the latter in two sibs. The remaining eight are missense mutations generating single amino acid substitutions. From the position of the substituted residues, seven of these mutations are considered to be causative of the enzyme deficiency: I201L, R211Q, G305S, R309H, K322R, Q339R, and W393R. The eighth, a C>T transition, predicts an R319C substitution, which could affect the binding of the molybdenum cofactor and thus severely reduce sulfite oxidase activity. This mutation, however, is downstream of a frameshift mutation and is therefore not the causative mutation in this individual.
Asunto(s)
Errores Innatos del Metabolismo de los Metales/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , Humanos , Errores Innatos del Metabolismo de los Metales/enzimología , Errores Innatos del Metabolismo de los Metales/patología , Datos de Secuencia Molecular , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/deficienciaRESUMEN
The molybdenum site of the Arginine 160 --> Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine Oepsilon to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.
Asunto(s)
Molibdeno/química , Compuestos Organometálicos/química , Sulfito-Oxidasa/química , Sulfito-Oxidasa/metabolismo , Algoritmos , Arginina/química , Arginina/genética , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Glutamina/análogos & derivados , Glutamina/genética , Concentración de Iones de Hidrógeno , Mutación , Conformación Proteica , Análisis Espectral , Sulfito-Oxidasa/genética , Rayos XRESUMEN
The redox chemistry of the molybdenum site of the C207S mutant of recombinant human sulfite oxidase has been studied via potentiometric titrations employing both electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS) as probes of the active site structure. In earlier EXAFS studies, oxidized Cys207Ser enzyme has been shown to possess a novel tri-oxo active site, in which Ser207 does not appear to be a ligand to Mo [George, G. N.; Garrett, R. M.; Prince, R. C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 8588-8592]. Redox titrations show that the active site is modified under reducing conditions to a mono-oxo Mo(IV) species, probably with Ser207 ligated to the metal. The Mo(IV) species can be reoxidized to a mono-oxo Mo(V) species still coordinated to Ser207, which in turn can be further reoxidized to yield the initial tri-oxo Mo(VI) structure with loss of Ser207 ligation.