Acetylene hydratase of Pelobacter acetylenicus

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 10 4 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 N 2 /H 2 , 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, g y = 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 N 2 /H 2 ) 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.