Alcaligenes faecalis Arsenite Oxidase A new molybdenum protein structure from beamline 9-2 By Paul Ellis
What we knew Induced in A. faecalis by the presence of arsenite (AsO2-). Oxidizes arsenite to the less-toxic arsenate (AsO43-): AsIIIO2- + 2H2O AsVO43- + 4H+ + 2e- DMSO reductase family: HiPIP: 300-400 mV reduction potential Rieske: 200-300 mV reduction potential AsVO43-/AsIIIO2-: +60 mV CO2/HCOOH: -200 mV DMSO/DMS: +160 mV TMAO/TMA: NO3-/NO2-: DMSO reductase from R. sphaeroides Mo(VI/V): +144 mV Mo(V/IV): +160 mV Mo Mo Mo 4Fe/4S 4Fe/4S HiPIP or 3Fe/4S Rieske 2Fe/2S DMSO Reductase, TMAO Reductase Formate Dehydrogenase Arsenite Oxidase
Arsenite oxidase crystals Cell solvent content P1 triclinic 59% P21 monoclinic 57% P2 monoclinic 56% C2 monoclinic 71% I222 orthorhombic -5% P4 tetragonal 33% P4x212 tetragonal 18% R32 trigonal 58%
Mercury derivatives of the P1 form
Phased anomalous map from SHARP Hg Mo Fe Fe
Beamline 9-2
MAD data collection from a P21 crystal low energy = 6900 eV maximum f’’ minimum f’ high energy = 12650 eV
Locating the metal atoms in the P21 cell superpose
Picking the correct solution Incorrect Correct
Backbone autotraced by wARP
Sequencing Sequence based on electron density and hydrogen-bonding patterns ASP=ASN GLU=GLN 95% of residues assigned Matches known N-terminal sequence of the long chain
Final model
Acknowledgements Department of Molecular Biochemistry, Ohio State University Russ Hille Thomas Conrads Structural Molecular Biology, SSRL Peter Kuhn Mike Soltis Aina Cohen Tim McPhillips Henry Bellamy Department of Energy: Office of Basic Energy Sciences Office of Biological and Environmental Research National Institutes of Health, National Center for Research Resources, Biomedical Technology Program
Summary Phased using data from two crystal forms. Sequenced approximately 95% of residues based on density and hydrogen bonding. Protein consists of a long chain containing the Mo site and a 3Fe/4S cluster and a short chain containing a Rieske-type 2Fe/2S site. Long chain is homologous to other members of the DMSO reductase family, but there is no protein sidechain bound to Mo. Short chain has no counterpart among known Mo protein structures but is similar to the Rieske Fe/S protein domains of: cytochrome bc1 cytochrome b6f naphthalene 1,2‑dioxygenase.