title: The respiratory arsenite oxidase: structure and the role of residues surrounding the rieske cluster.
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.
authors.affiliation: Institute of Structural and Molecular Biology, University College London, London, United Kingdom.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Warelow
authors.forename: Muse
authors.initials: M
authors.lastname: Oke
authors.forename: Barbara
authors.initials: B
authors.lastname: Schoepp-Cothenet
authors.forename: Jan U
authors.initials: JU
authors.lastname: Dahl
authors.forename: Nicole
authors.initials: N
authors.lastname: Bruselat
authors.forename: Ganesh N
authors.initials: GN
authors.lastname: Sivalingam
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Thalassinos
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kappler
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Santini
mesh_terms: D000332:Aerobiosis; D042481:Alcaligenes faecalis / Q000201:enzymology; D020561:Alphaproteobacteria / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008982:Molybdenum / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D008982:Molybdenum; D010088:Oxidoreductases; C078179:arsenite oxidase; D014450:Electron Transport Complex III
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