The active site structure and catalytic mechanism of arsenite oxidase

Arsenite oxidase is thought to be an ancient enzyme, originating before the divergence of the Archaea and the Bacteria. We have investigated the nature of the molybdenum active site of the arsenite oxidase from the Alphaproteobacterium Rhizobium sp. str. NT-26 using a combination of X-ray absorption spectroscopy and computational chemistry. Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. We propose that this is an entatic state imposed by the protein on the active site through relative orientation of the two molybdopterin cofactors, in a variant of the Rây-Dutt twist of classical coordination chemistry, which we call the pterin twist hypothesis. We discuss the implications of this hypothesis for other putatively ancient molybdopterin-based enzymes

A new family of periplasmic-binding proteins that sense arsenic oxyanions

Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that's role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate

Arsenite oxidase as a novel biosensor for arsenite

Contamination of groundwater with the toxic soluble arsenic species, arsenite (AsIII) and arsenate (AsV) has led to an epidemic of arsenic poisoning effecting over 100 million people worldwide. The World Health Organisation (WHO) recommended maximum contaminant level (MCL) for arsenic in water is 0.13 μM (10 μg L−1). Accurate quantification of arsenic below the MCL usually requires highly sensitive laboratory based techniques, the practical uses of which are limited within the effected populations, principally due to cost. Biosensors are a potentially powerful technology for overcoming this problem. Amperometric biosensors couple the analytical sensitivity of electrochemistry with the selectivity of enzyme substrate interactions. The bioenergetic metalloenzyme AsIII oxidase (Aio) catalyses the oxidation of AsIII to AsV in a number of physiologically diverse microorganisms including the Rhizobium sp. str. NT-26. To develop a biosensor for AsIII it was first necessary to optimise the expression and purification of the biological recognition element, a recombinant NT-26 Aio in Escherichia coli str. DH5α. with final a yield of ca. 1.1 mg per L of culture. The recombinant NT-26 Aio was characterised using biophysical techniques to confirm the correct insertion of the enzyme cofactors during heterologous expression in E. coli. The reduction midpoint potentials of the 3Fe-4S (270 mV) and the Rieske 2Fe-2S (225 mV) clusters were confirmed by redox titration. The thermostability of the recombinant Aio was ≤ 64.5 °C. The oxidised structure of the Mo at the active site was confirmed to have a di-oxo (Mo = O2) coordination. The kinetics and pH dependence of AsIII oxidation were investigated using various artificial and physiological electron acceptors. Electrochemical studies were performed to develop a system for AsIII concentration determination, using the biological recognition element Aio. The electron transfer mediator ferrocene methanol was found to produce the greatest currents during catalytic voltammetry experiments at pH 8.0. A chronoamperometric detection system incorporating the electron transfer mediators ferrocene methanol and potassium ferricyanide was able to resolve AsIII concentrations of 0.07 – 0.53 μM (5 – 40 μg L−1), below the WHO MCL for arsenic, suggesting such a system would be capable of determining the safe levels of arsenic in drinking water

Molybdenum-Containing Enzymes

An overview of modern methods used in the preparation and characterization of molybdenum-containing enzymes is presented, with an emphasis on those methods that have been developed over the past decade to address specific difficulties frequently encountered in studies of these enzymes