Abstract
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.
Highlights
Escherichia coli grows anaerobically using Me2SO as respiratory oxidant by expressing an electron transfer chain terminating with Me2SO reductase, DmsABC1 [1]
Ferredoxins that contain [4Fe-4S] clusters usually ligate these clusters by Cys groups consisting of four Cys residues spaced such that the first two Cys residues are separated by two amino acids, while the spacing between the second, third, and fourth Cys residues is somewhat variable
The C38S and C42S mutants were blocked in using electrons from the quinol pool to reduce substrate, the [4Fe-4S] clusters responded to the redox state of the quinol pool. In this manuscript we show that the DmsA Cys group does not coordinate an electron paramagnetic resonance (EPR) visible [Fe-S] cluster, but when the second Cys of this group is mutated, a [3Fe-4S] cluster assembles into the mutant enzyme
Summary
Bacterial Strains and Plasmids—E. coli strain F36 is a mutant of E. coli HB101(supE44 hsdS20 (rBϪmBϪ) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1), which is impaired in molybdenum cofactor insertion into DmsABC [4]. The plasmids used are the vector pBR322 (Pharmacia Biotech Inc.), pDMS160, which contains the dmsABC operon cloned into pBR322 [31], and pC38S and pC38A, which are derivatives of pDMS160-containing point mutations in the dmsA gene [32]. PTZ18R (Pharmacia) was used as a cloning vector and to sequence fragments containing mutant DNA. Harvesting of Cells and Preparation of Membrane Fractions—For whole cell EPR samples, cells were harvested, washed, and resuspended in degassed 100 mM MOPS, pH 7.0, 5 mM EDTA buffer containing 20 mM succinate [31]. Glycerol-fumarate grown cells were harvested, washed, and resuspended in 50 mM, MOPS, pH 7.0, 5 mM EDTA. EPR Spectroscopy—Samples were prepared as described by Cammack and Weiner [4] from either whole cells or washed membranes.
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