Abstract
Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s−1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.
Highlights
Arsenic, in the inorganic forms arsenite (+III) and arsenate (+ V), is toxic to most organisms [1]
Aio is a member of the dimethyl sulfoxide reductase (DMSOR) superfamily of molybdoenzymes, which all contain two equivalents of an organic pyranopterin cofactor coordinated to the molybdenum, usually present as the dinucleotide of guanine and termed MGD
There are four electron transfer events in the proposed electron pathway for arsenite oxidation catalysed by Aio: arsenite to the molybdenum centre, from the molybdenum centre to the 3Fe-4S cluster, from the 3Fe-4S cluster to the Rieske 2Fe-2S cluster and from the Rieske 2Fe-2S cluster to the terminal electron acceptor [13]
Summary
In the inorganic forms arsenite (+III) and arsenate (+ V), is toxic to most organisms [1]. The physiological electron acceptor for Aio has been shown to be c-type cytochromes or azurin [4,5,6,7]. The molybdenum centre of Aio exhibits highly cooperative two electron transfer, with the intermediate Mo(V) oxidation state not typically observed upon reduction of Mo(VI) to Mo(IV) [9] until recently in a mutant with altered hydrogen bonding to the MGD [10]. Heterologous expression of the NT-26 Aio in Escherichia coli has facilitated a more detailed study of the mechanisms of electron transfer [7,8,10,14] and its development as a biosensor for arsenite [15]. Using salt is not physiologically relevant as the Aio is a periplasmic enzyme which means that the pH and salt concentration would be in equilibrium with the environment and NT-26 was isolated from a low-salt environment [18]
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