Acetylene hydratase of Pelobacter acetylenicus: Molecular and spectroscopic properties of the tungsten iron-sulfur enzyme

Rainer U. Meckenstock, Robert Krieger, Scott Ensign, Peter M.H. Kroneck, Bernhard Schink

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62 Scopus citations

Abstract

Acetylene hydratase of Pelobacter acetylenicus is a tungsten iron- sulfur protein involved in the fermentation of acetylene to ethanol and acetate. Expression of the enzyme was increased 10-fold by feeding a 50-L batch culture continuously with 104 Pa acetylene at pH 6.8-7.0. Acetylene hydratase was purified to homogeneity by a three-step procedure in either the absence or presence of dioxygen. The enzyme was a monomer with a molecular mass of 73 kDa (SDS/PAGE) or 83 kDa (matrix-assisted laser-desorption ionization MS) and contained 0.5 ± 0.1 W (inductively coupled plasma/MS) and 1.3 ± 0.1 molybdopterin-guanine dinucleotide per mol. Selenium was absent. EPR spectra (enzyme as isolated, under air) showed a signal typical of a [3Fe-4S] cluster with g(av) = 2.01, at 10 K. In enzyme prepared under N2/H2, this signal was absent and reaction with dithionite led to a rhombic signal with g(z) = 2.048, g(y) = 1.939 and g(x) = 1.920 indicative of a low- potential ferredoxin-type [4Fe-4S] cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared with g(x) = 2.007, gy = 2.019 and g(z) = 2.048 (g(av) = 2.022), which disappeared after further oxidation. The signal was still visible at 150 K and was tentatively assigned to a W(V) center. The iron-sulfur center of acetylene hydratase (prepared under N2/H2) gave a midpoint redox potential of -410 ± 20 mV in a spectrophotometric titration with dithionite. Enzyme activity depended on the redox potential of the solution, with 50% of maximum activity at -340 ± 20 mV. The presence of a pterin-guanine dinucleotide cofactor differentiates acetylene hydratase from the aldehyde ferredoxin oxidoreductase-type enzymes which have a pterin mononucleotide cofactor.

Original languageEnglish
Pages (from-to)176-182
Number of pages7
JournalEuropean Journal of Biochemistry
Volume264
Issue number1
DOIs
StatePublished - 15 Aug 1999
Externally publishedYes

Keywords

  • Acetylene hydratase
  • Iron-sulfur cluster
  • Molybdopterin
  • Pelobacter acetylenicus
  • Tungsten enzymes

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