On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5.
Hydrogenase, purified to an average specific activity of 328 mumol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule. Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H2O yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy. Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (g-values at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins. These oxidations and reductions appeared to occur within the turnover time of the enzyme. Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance. 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster. These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands.
D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000032:analysis; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000032:analysis
D013455:Sulfur; D007501:Iron; D010088:Oxidoreductases