The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster

Thomas P Warelow,Barbara Schoepp-Cothenet,Joanne M Santini,Ganesh N Sivalingam,Ulrike Kappler,Nicole Bruselat,Muse Oke,Konstantinos Thalassinos,James H Naismith,Jan U Dahl,Silke Leimkühler,Claudio M Soares

doi:10.1371/journal.pone.0072535

Abstract

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.

Highlights

Aerobic arsenite oxidation in the Alphaproteobacterium Rhizobium sp
Aerobic arsenite oxidation is catalysed by arsenite oxidase (Aio) [2] which is thought to be an ancient bioenergetic enzyme that was present in the last universal common ancestor prior to the divergence of the Bacteria and Archaea [3,4]
The aioB and aioA genes were cloned without the aioB Tat leader sequence into pPROEX-HTb (Invitrogen) under the control of the IPTG inducible trc promoter which allows for expression in the E. coli cytoplasm

Summary

Introduction

Aerobic arsenite oxidation in the Alphaproteobacterium Rhizobium sp. NT-26 is an energy-generating process where the electron donor, arsenite, is oxidized to the less toxic arsenate and this is coupled to the reduction of oxygen to water [1]. NT-26 can oxidize arsenite either autotrophically with carbon dioxide as the sole carbon source or heterotrophically, where yeast extract is used as the source of carbon [1]. Aerobic arsenite oxidation is catalysed by arsenite oxidase (Aio) [2] which is thought to be an ancient bioenergetic enzyme that was present in the last universal common ancestor prior to the divergence of the Bacteria and Archaea [3,4].

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Conclusion