Acetylene hydratase of Pelobacter acetylenicus. Molecular and spectroscopic properties of the tungsten iron-sulfur enzyme.
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 gav = 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 gz = 2.048, gy = 1.939 and gx = 1.920 indicative of a low-potential ferredoxin-type [4Fe-4S] cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared with gx = 2.007, gy = 2.019 and gz = 2.048 (gav = 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.
Meckenstock
R U
RU
Fakultät für Biologie, Universität Konstanz, Germany. Rainer.Meckenstock@uni.konstanz.de
Krieger
R
R
Ensign
S
S
Kroneck
P M
PM
Schink
B
B
D001421:Bacteria, Anaerobic / Q000201:enzymology*; D020134:Catalytic Domain; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry; D008970:Molecular Weight; D000090006:Molybdenum Cofactors; D010084:Oxidation-Reduction; D011621:Pteridines / Q000737:chemistry; D014414:Tungsten / Q000737:chemistry*
D003067:Coenzymes; D006836:Hydro-Lyases; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D014414:Tungsten; C022261:molybdopterin; C096200:acetylene hydratase