Engineering a novel iron-sulfur cluster into the catalytic subunit of Escherichia coli dimethyl-sulfoxide reductase.
Dimethyl-sulfoxide reductase (DmsABC) is a complex [Fe-S] molybdoenzyme that contains four [4Fe-4S] clusters visible by electron paramagnetic resonance (EPR) spectroscopy. The enzyme contains four ferredoxin-like Cys groups in the electron transfer subunit, DmsB, and an additional group of Cys residues in the catalytic subunit, DmsA. Mutagenesis of the second Cys, Cys-38, in the DmsA group to either Ser or Ala promotes assembly of a fifth [Fe-S] cluster into the mutant enzyme. The EPR spectra, the temperature dependences, and the microwave power dependences demonstrate that the new clusters are [3Fe-4S] clusters. The [3Fe-4S] clusters in both of the C38S and C38A mutant enzymes are relatively unstable in redox titrations and have midpoint potentials of approximately 178 and 140 mV. Mutagenesis of the DmsA Cys group to resemble a sequence capable of binding an [4Fe-4S] cluster did not change the cluster type but reduced the amount of the cluster present in this mutant enzyme. This report demonstrates that all four EPR detectable [Fe-S] clusters in the wild-type enzyme are ligated by DmsB. Wild-type DmsA does not ligate an [Fe-S] cluster that is visible by EPR spectroscopy.
Trieber
C A
CA
Department of Biochemistry and the Medical Research Council Group in the Molecular Biology of Membranes, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Rothery
R A
RA
Weiner
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008872:Microwaves; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry*; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase