Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha

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.

Keywords: electron paramagnetic resonance (EPR); enzyme catalysis; enzyme kinetics; enzyme mechanism; iron/sulfur protein; molybdenum.

FIGURE 1.

Reductive titration of FdsABG. A, oxidized (blue) and sodium dithionite-reduced (black) spectra. B, change in absorbance as a function of reduction. The inset plots the relative absorbance at 550 nm versus relative absorbance at 450 nm, with the diagonal (reflecting strict proportionality in the absorbance change at the two wavelengths) indicated. Details of titration are described under “Experimental Procedures.”

FIGURE 2.

A, pH dependence of k_cat for FdsABG. The reactions were performed at 30 °C in a buffer containing 75 mm of malate, K₂HPO₄, Tris, and glycine, brought to pH 5–10. The fit to the data (solid line) yielded two pK_a values of 5.6 and 9.3, respectively. B, Lineweaver-Burk plots for the reaction of FdsABG with formate at the following [NAD⁺]: 1.62 mm (closed circles), 0.54 mm (open circles), 0.18 mm (closed triangles), and 0.06 mm (open triangles). C, Lineweaver-Burk plots for the reaction of FdsABG with NAD⁺ at the following [formate]: 3.24 mm (closed circles), 1.08 mm (open circles), 0.36 mm (closed triangles), and 0.12 mm (open triangles). D, secondary plots of 1/y intercept taken from B versus 1/[NAD⁺]. E, secondary plots of 1/y intercept taken from C. versus 1/[formate]. The reactions in B and C were performed in 75 mm K-PO₄, pH 7.7, at 30 °C. Linear regression analysis of plots D and E yielded a K_m^NAD+ = 130 μm and K_m^formate = 310 μm, respectively, and an average k_cat of 201 s⁻¹.

FIGURE 3.

Pre-steady-state kinetics for the reduction of FdsABG with formate or [²H]formate. A, rapid scanning stopped flow traces for the reaction of 6 μm of FdsABG with 5 mm formate performed at 10 °C in 75 mm K-PO₄, pH 7.7. B, a representative kinetic trace of the reaction of FdsABG with 0.8 mm sodium formate monitored at 450 nm. The trace is best represented by three phases with k_obs = 124, 14, and 2.5 s⁻¹, with ΔA = 0.011, 0.017, 0.006, respectively. C, plots of k_fast (black circles) and k_intermediate (red squares) versus formate concentrations or k_fast (black triangles) versus [²H]formate concentrations. All reactions were performed at 10 °C in 75 mm K-PO₄, pH 7.7, with 2.5 μm FdsABG. Hyperbolic fits (solid lines) yielded k_fast = 140 s⁻¹, K_d^formate = 82 μm, k_intermediate = 19 s⁻¹, and K_d^formate = 230 μm for reaction with formate and k_fast = 66 s⁻¹, and K_d^[2H]formate = 193 μm for reaction with [²H]formate.

FIGURE 4.

EPR of molybdenum center of FdsABG collected at 150 K. A, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of FdsABG collected with modulation amplitude = 2 Gauss and microwave power = 4 mW. The sample was prepared under anaerobic conditions by reduction of 100 μm of FdsABG in 75 mm K-PO₄, pH 7.5, in the presence of 10 mm KNO₃, with 5 mm buffered sodium dithionite. The Mo^V component represents ∼33% of total spin density. B, simulation of the Fe/S1 contribution to the spectrum in A. C, simulation of the Mo^V contribution to the spectrum in A. The hyperfine splitting caused by the 25% naturally occurring ^95,97Mo (I = 5/2) is shown in the 5-fold expanded spectrum above. D, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of deuterated FdsABG collected with modulation amplitude = 2 Gauss and microwave power = 4 mW. The sample was prepared under anaerobic conditions by reduction of 100 μm of FdsABG in 75 mm K-PO₄, pD 7.1, in the presence of 10 mm KNO₃, with 0.4 mm sodium formate. The Mo^V component represents ∼38% of total spin density. E, simulation of the Fe/S1 contribution to the spectrum in D. F, simulation of the Mo^V contribution to the spectrum in D. The hyperfine splitting caused by the 25% naturally occurring ^95,97Mo (I = 5/2) is shown in the 3-fold expanded spectrum above. Simulation parameters are summarized in Table 1. sim, simulation.

FIGURE 5.

Expanded view of the EPR of molybdenum center of FdsABG from Fig. 4. Superposition of the composite Mo^V-Fe/S EPR spectra from Fig. 4A (black) with Fig. 4D (red). The line diagram above indicates the location of the principal g tensors (red vertical lines) and the approximate location of the ¹H hyperfine splitting (I = 1/2) in the spectrum from Fig. 4A (black vertical lines).

FIGURE 6.

A, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of FdsABG collected at 150 K with modulation amplitude = 3 Gauss and microwave power = 4 mW. The sample was prepared under anaerobic conditions by reduction of 50 μm of FdsABG in 200 mm Tris-HCl, pH 8.5, in the presence of 10 mm KNO₃, with 0.25 mm sodium formate on ice and mixing for 5 s before freezing. The Mo^V component represents ∼10% of total spin density. B, simulation of the Fe/S1 contribution to the spectrum in A. C, simulation of the Mo^V contribution to the spectrum in A. D, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of FdsABG collected at 150 K with modulation amplitude = 2 Gauss and microwave power = 4 mW. The sample in A above was thawed in a room temperature water bath and incubated for an additional 55 s before refreezing. The Mo^V component represents ∼19% of total spin density. E, simulation of the Fe/S1 contribution to the spectrum in D. F, simulation of the Mo^V contribution to the spectrum in D. sim, simulation.

FIGURE 7.

EPR of Fe/S centers of FdsABG. A, Fe/S1 EPR spectrum (black) and simulation (red) of FdsABG collected with modulation amplitude = 4 Gauss and microwave power = 4 mW at 100 K. The sample was prepared by thawing out the sample from Fig. 6D under aerobic conditions in a room temperature water bath and incubating for an additional 1 min before refreezing. Locations of g₁, g₂, and g₃ are indicated. B, iron/sulfur EPR spectrum (black) and simulation (red) of FdsABG collected at 60 K with modulation amplitude = 4 Gauss and microwave power = 4 mW. The sample was prepared by incubation of 100 μm of FdsABG in 75 mm K-PO₄, pH 7.7, in the absence of 10 mm KNO₃ for >6 h. (aerobically at 4 °C), followed by reduction under anaerobic conditions with 5 mm buffered sodium dithionite and further incubation for 1 h before freezing. Locations of g₁, g₂, and g₃ corresponding to the Fe/S2 component of the spectrum are indicated. C, Fe/S EPR spectrum (black) and simulation (red) of FdsABG sample from B collected at 20 K at with modulation amplitude = 3 Gauss and microwave power = 0.2 mW. Locations of g₁, g₂, and g₃ corresponding to the Fe/S3 component of the spectrum are indicated. D, iron/sulfur EPR spectrum (black) and simulation (red) of FdsABG sample from B collected at 20 K at with modulation amplitude = 10 Gauss and microwave power = 100 mW. Locations of g₁, g₂, and g₃ corresponding to the Fe/S4 component of the spectrum are indicated. E, spectrum from D enlarged to show the broad peaks corresponding to Fe/S4. Simulation parameters are summarized in Table 1.

FIGURE 8.

Simulations of EPR spectra for Fe/S1–Fe/S4 from parameters summarized in Table 1. The iron/sulfur clusters are classified by the increasing value of their g₁ tensor.

FIGURE 9.

EPR of deuterated molybdenum center of FdsABG collected at 20–60 K. A, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of FdsABG sample as described in Fig. 4D collected with modulation amplitude = 2 Gauss and microwave power = 0.2 mW at 60 K. The Mo^V component represents ∼27% of total spin density. B, simulation of the Fe/S1 and Fe/S2 contributions to the spectrum in A. C, simulation of the Mo^V contribution to the spectrum in A. D, Mo^V-Fe/S EPR spectrum (black) and simulation (red) of FdsABG sample from A. above collected with modulation amplitude = 2 Gauss and microwave power = 0.02 mW at 20 K. The Mo^V component represents ∼16% of total spin density. E, simulation of the Fe/S1–Fe/S3 contributions to the spectrum in D. F, simulation of the Mo^V contribution to the spectrum in D. The simulation parameters are summarized in Table 1. sim, simulation.

FIGURE 10.

The active site of the FdhF formate dehydrogenase from E. coli (PDB code 1FDO). The molybdenum is at the center of the figure, coordinated by the two equivalents of pyranopterin cytidine dinucleotide (PCD), a terminal sulfido (represented as a coordinated water in the original PDB file), and Sec-140. The substrate binding site is delineated by two loops consisting of ¹³⁷ARVUHGP¹⁴³ and ³²⁸GVNPLRGQNNVQG³⁴⁰. Arg-333 has been proposed to interact with the negatively charged substrate. In these two stretches, only two residues differ in the FdsA subunit of the R. eutropha formate dehydrogenase: Sec-140, which is a Cys that coordinates the molybdenum in FdsA, and Val-338, which is a Leu in FdsA.

FIGURE 11.

A proposed hydride transfer mechanism for the formate dehydrogenases. Beginning with the ionized substrate, the second carbon-oxygen double bond forms displacing hydride, which attacks the molybdenum-sulfur group, bringing about the formal two-electron reduction of the metal with concomitant transfer of the Cα-H to the sulfur.

FIGURE 12.

A structural model for the R. eutropha formate dehydrogenase. The molybdenum center is at left, and the FMN is at right, with the several iron/sulfur clusters intervening. The model is based on homologies to the T. thermophilus NADH dehydrogenase (PDB code 3IAM) and the E. coli formate dehydrogenase (PDB code 1AA6), with the position of the additional [4Fe-4S] cluster present in FdsA but absent in Nqo3 indicated by the red circle.