Spectroscopic and Kinetic Properties of the Molybdenum-Containing, NAD+-Dependent Formate Dehydrogenase from Ralstonia eutropha. [Molecular Biophysics]

November 9th, 2015 by Niks, D., Duvvuru, J., Escalona, M., Hille, R.

We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD+-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the EPR signal of the molybdenum center in its MoV state and demonstrated the direct transfer of the substrate Ca hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power and level of enzyme reduction, we are able to clearly identify the EPR signals for four of the iron-sulfur clusters of the enzyme, and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron-sulfur centers, permitting assignment of this signal to a specific iron-sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a Mo=S group of the molybdneum center.
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