Abstract
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 electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα 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 electron paramagnetic resonance 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 molybdenum-sulfur group of the molybdenum center.
Highlights
(ϳ21% identity) and expected strong structural homology to a corresponding subunit of the matrix- or cytosol-exposed portion of NADH dehydrogenase, and the spatial layout of the several redox-active centers is highly conserved
This domain is present in Nqo3 as well, the molybdenum center itself and one of the more C-terminal iron/sulfur clusters have been lost by the NADH dehydrogenases over the course of evolution [1, 3, 6]
Kinetics of the R. eutropha FdsABG Formate Dehydrogenase—The steady-state kinetics reported here demonstrate that our enzyme preparation has the characteristics previously reported and in particular has approximately the same high specific activity
Summary
Organisms and Growth Conditions—R. eutropha strains H16 (ATCC 17699) and HF210 were grown as previously described [1], except that the final molybdenum concentration was increased to 0.15 mM. The pellet containing FdsABG was dissolved in 80 mM K-PO4, 20 mM KNO3, pH 8.0 (saturated with 15% ammonium sulfate), at 0.4 ml/g cells to maintain a relatively small. One pellet was redissolved in 0.5 ml of HIC-A buffer, loaded onto a 5 ml butylSepharose HP column (GE Healthcare), washed with 0.5 column volume HIC-A buffer, and eluted with a linear gradient of 0.5– 0 M ammonium sulfate over 4 column volumes. During simulations of MoV-containing spectra, g values for the iron/sulfur clusters were allowed to vary by no more than Ϯ 0.001, whereas anisotropic line broadening (simulated as HStrain in the EasySpin software package) varied by no more than Ϯ 10 MHz. Iron/sulfur linewidths used in the molybdenum-containing spectra were initially estimated from experimental spectra that contained the various iron/sulfur components at the temperatures where the molybdenum-containing spectra were obtained. Because of the broad nature of the iron/sulfur centers, the exclusion of electron-electron coupling for the Fe/S3 did not have a discernible effect on either the accuracy of the simulation or the resultant parameters
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