Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator

Abstract

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD⁺-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe₄S₄ in FdsB and Fe₂S₂ in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH^•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH^-, initially formed via NADH-mediated reduction, to the Fe₂S₂ cluster. This Fe₂S₂ cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD⁺ binding in proper NADH dehydrogenases.

Keywords: FdsABG; carbon assimilation; electron transfer; enzyme kinetics; enzyme structure; flavin mononucleotide (FMN); formate dehydrogenase; nicotinamide adenine dinucleotide (NADH); protein crystallization.

Figure 1.

EPR spectra of the iron-sulfur clusters of the FdsBG complex. A, observed iron-sulfur EPR spectrum (black trace) and simulated composite spectrum (red trace) of the dithionite-reduced FdsBG complex, collected at 9 K with modulation amplitude of 8 gauss and microwave power of 2 microwatts. The sample was prepared by incubation of 125 μm of FdsBG complex in 100 mm KPO₄, pH 7.0, with 2 mm buffered sodium dithionite under anaerobic conditions for 1 h at room temperature prior to freezing. B, the individual component spectra resulting from the simulation of the composite spectrum in A: the spectrum corresponding to the previously assigned Fe/S₁ (blue dashed trace (6)) and an additional Fe/S₅ signal (red dashed trace) resolved only in the FdsBG complex. C, superposition and graphical alignment of the spectrum presented in A (red trace) with the spectrum of dithionite-reduced FdsABG collected at 20 K (blue trace, expanded to match the amplitude of the g₁ feature in the Fe/S₁ component of the FdsBG spectrum; reprinted with permission (6)). Dashed lines mark the location of g₁ and g₃ features of the Fe/S₃ component of the FdsABG holoenzyme spectrum.

Figure 2.

Arrangement of the two FdsBG complexes of the asymmetric unit and electron density map of representative regions. A, placement of the two FdsBG complexes of the asymmetric unit within the crystallographic unit cell. The view is down the ∼179° rotation axis between the two FdsBG complexes of the asymmetric unit. Crystallographic axes are in blue, with the b-axis being the crystallographic 2-fold. The angle between the ∼179° rotation axis and the crystallographic 2-fold (i.e. b-axis) is ∼92°. B–E, the (2F_o − F_c)-electron difference map for different regions of the final model. B, the α-helical region between residues 234^B and 244^B (superscript B indicates that residues belong to FdsB) contoured at 1.0 σ and carve radius of 2.0 Å; C, the FMN bound to FdsB contoured at 1.0 σ and carve radius of 1.5 Å; D, the Fe₂S₂ cluster of FdsG contoured at 1.0 and 6 σ and carve radius of 2.5 Å; E, the Fe₄S₄ cluster of FdsB contoured at 1.0 and 6 σ and carve radius of 2.0 Å. The electron difference map contoured at 1.0 and 6.0 σ are shown as light blue and blue meshes, respectively.

Figure 3.

Domain structure of FdsB and FdsG and their sequence and structure alignment with NuoE and NuoF. A, primary structures of FdsB, Nqo1 from T. thermophilus, and NuoF from A. aeolicus. The domains are indicated as boxed regions. The FdsB-specific N-terminal thioredoxin-like domain (Txr-like), N-terminal region of Nqo1, Rossmann-like fold, ubiquitin-like domain, and four-helical bundle (4-HB) are shown in brown, light green, white, green, and beige. B, primary structures of FdsG, Nqo2, and NuoE. The N-terminal four-helical bundle and the thioredoxin-like domain (Txr-like) are shown in brown and light green. C and D, structures of FdsB and FdsG subunits with domains color-coded according to A and B. E, superposition of FdsB (beige) and NuoF (light green) subunits, of FdsG (brown) and NuoE (green) subunits, and of FdsBG (beige and brown) and NuoEF complexes (light green and green).

Figure 4.

Structural comparison of the FdsBG and NuoEF complexes. A and B, effect of the structural difference in the 183^B–190^B loop of the Rossmann-like domain on the positioning of the Fe₂S₂ cluster (A), of the ubiquitin and four-helical bundle domains, and of the Fe₄S₄ cluster (B). The Rossmann-like, ubiquitin, and four-helical bundle domains of FdsB are displayed in white, green, and brown, and the C-terminal domain of FdsG is shown in gold. The corresponding domains of NuoF and NuoE are shown in gray, light green, beige, and pale yellow. C, difference in the C- and N-terminal domain arrangement of FdsG (brown and green) and NuoE (beige and light green). D, hinging of FdsG's C-terminal domain toward the Rossmann-like domain of FdsB compared with NuoE's C-terminal domain. E and F, overall arrangement of the Fe₂S₂ cluster, Fe₄S₄ cluster, and FMN in FdsBG (E) and NuoEF (F).

Figure 5.

FMN-binding site. The binding site of the flavin ring (A) and of the ribityl-phosphate moiety (B and C) of FMN are displayed. The Fe₄S₄ cluster of FdsB is rendered in a space-fill representation, whereas FMN, residues of the binding site, and coordinating water molecules are rendered in a ball-and-stick representation. FMN, selected residues of FdsB, and water molecules are shown in gold, beige, and red, respectively.

Figure 6.

Reductive titration of FdsBG complex at pH 7.0. A, oxidized (red) and sodium dithionite-reduced (blue) spectra. B, change in absorbance as a function of reduction. The inset plots the relative absorbance change at 550 nm (y axis) against relative absorbance change at 450 nm (x axis) with the diagonal reflecting strict proportionality in the absorbance change between two wavelengths. The titration was performed at room temperature in 100 mm KPO₄, pH 7.0, under anaerobic conditions.

Figure 7.

NADH binding site. A, the unbiased (F_o − F_c)- and (2F_o − F_c)-electron difference maps for FdsBG crystals soaked with NADH. The final models for FMN and the adenosine diphosphate of NADH are displayed in ball-and-stick (gold and cyan). The (F_o − F_c)-electron difference map contoured at 3.0 σ with a carve radius of 1.8 Å and the (2F_o − F_c)-electron difference map contoured at 1.0 and 3.0 σ with a carve radius of 1.8 Å are shown in pale green, light blue, and blue meshes, respectively. B, steric overlap between the Asp-184^B–Glu-185^B peptide bond with FMN in crystals of FdsBG complex soaked with NADH. The position of the Asp-184^B–Glu-185^B peptide bond shown was determined for FdsBG complex without FMN bound. C, comparison of the NADH/NAD⁺ positions relative to the FMN of the NADH-bound FdsBG and the NAD⁺-bound NuoEF complex. Shown are the FMN and NAD⁺ from the NuoEF complex (pale cyan and light yellow; PDB entry 6HLI) and the NADH of the FdsBG complex (cyan). D, binding site of adenosine diphosphate of NADH. Displayed are FMN, NADH, and the adenosine-binding site on FdsB in gold, blue, and beige. Residues Val-295^B–Ala-297^B and Lys-292^B are omitted from this view for clarity.

Figure 8.

Single-wavelength pre-steady-state kinetics for the reduction of FdsBG with NADH. Shown is a plot of k_obs (black circles) versus NADH concentrations. Hyperbolic fits (solid line) yielded a k_red of 680 s⁻¹ and K_d of 0.19 mm. Each point is the average of 3–5 measurements, and the error bars are the S.D. of these measurements. The inset shows a typical trace for the reaction of 9 μm FdsBG complex with 14 μm NADH monitored at 450 nm. All reactions were performed at 5 °C in 100 mm KPO₄, pH 7.0, under anaerobic conditions.

Figure 9.

Rapid-reaction kinetics for the reaction of FdsBG with NADH. A, selected traces for the reaction of 10 μm FdsBG complex with 5 μm NADH at 5 °C in 100 mm KPO₄, pH 7.0, performed under anaerobic conditions and monitored with a photodiode detector. The oxidized spectrum was obtained by diluting FdsBG complex with buffer in the stopped-flow instrument. The inset shows a time course extracted at 450 nm. B, difference spectra from the data presented in A.

Scheme 1.

Proposed electron transfer in FdsBG upon NADH reduction.

Figure 10.

EPR of the neutral flavin semiquinone, FMNH^• of FdsBG. A, sample was prepared by rapid freeze-quench of a reaction of 40 μm enzyme with 0.8 mm NADH at 0 °C (quenching time ∼40 ms). The EPR spectrum was collected at 150 K with modulation amplitude of 8 gauss and microwave power of 0.4 milliwatts. B, sample was prepared by mixing 160 μm FdsBG with 60 μm NADH at room temperature for 10 s prior to freezing. The EPR spectrum was collected at 9 K with modulation amplitude of 8 gauss and microwave power of 2 microwatts. All samples were prepared in 100 mm KPO₄, pH 7.0, under anaerobic conditions.

Figure 11.

Sequence and structure comparisons of FdsBG, NuoEF, and HoxF. A, comparison of the domain structure of FdsB, FdsG, NuoE, NuoF, and HoxF. B, sequence alignment of the FdsB, NuoF, and HoxF regions for which an oxidation state–dependent peptide flip has been reported in NuoEF. C, structure comparison of FdsBG in the presence and absence of NADH. D, structure of NuoEF by itself (PDB entry 6HL2) and in oxidized form with bound NAD⁺ (PDB entry 6HL3). E, structure of HoxFUHY in reduced form (PDB entry 5XFA) and oxidized form (PDB entry 5XF9). Shown is the main chain in the region of interest for each protein. The “reduced” and “oxidized” form of each protein is shown in beige and brown, respectively. Also indicated (−FMN) is the absence of FMN in the NADH-bound FdsBG and H₂-reduced HoxFUHY structures.