RESUMEN
The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy. At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near -400 mV vs. SHE. Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster. Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule. Another signal (g = 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200 degrees K and is not attributed to any iron-sulfur cluster. The possible origin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site.
Asunto(s)
Aldehído Oxidorreductasas , Clostridium/enzimología , Formiato Deshidrogenasas , Tungsteno/análisis , Aldehído Oxidorreductasas/análisis , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Formiato Deshidrogenasas/análisis , Oxidación-Reducción , TemperaturaRESUMEN
Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Formiato Deshidrogenasas/metabolismo , Nitrato Reductasas/metabolismo , Azotobacter/enzimología , Clostridium/enzimología , Activación Enzimática , Neurospora crassa/enzimología , Nitrogenasa/metabolismoRESUMEN
Formate dehydrogenase (FDH) (EC 1.2.1.43) from C. thermoaceticum has been purified in two forms. One contains tungsten (W), and the other is enriched in molybdenum (Mo). The W-FDH is clearly active, while the Mo results are ambiguous with enzymatic activities generally lower in the Mo-enriched samples. Spectroscopic studies (EPR, absorption, and CD) on W-FDH and Mo-FDH demonstrate that no signal correlates to the group VI metal active site in the dithionite-reduced enzyme. This lack of a W(V) EPR signal is in contrast to the results observed for tungsten-substituted sulfite oxidase which is inactive.