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CO Binding to the FeV Cofactor of CO‐Reducing Vanadium Nitrogenase at Atomic Resolution

Abstract Nitrogenases reduce N2, the most abundant element in Earth's atmosphere that is otherwise resistant to chemical conversions due to its stable triple bond. Vanadium nitrogenase stands out in that it additionally processes carbon monoxide, a known inhibitor of the reduction of all substrates other than H+. The reduction of CO leads to the formation of hydrocarbon products, holding the potential for biotechnological applications in analogy to the industrial Fischer–Tropsch process. Here we report the most highly resolved structure of vanadium nitrogenase to date at 1.0 Å resolution, with CO bound to the active site cofactor after catalytic turnover. CO bridges iron ions Fe2 and Fe6, replacing sulfide S2B, in a binding mode that is in line with previous reports on the CO complex of molybdenum nitrogenase. We discuss the structural consequences of continued turnover when CO is removed, which involve the replacement of CO possibly by OH−, the movement of Q176D and K361D, the return of sulfide and the emergence of two additional water molecules that are absent in the CO‐bound state.


Isolation of protein
The component proteins of the vanadium-dependent nitrogenase were isolated from Azotobacter vinelandii according to established procedures. [1] Molybdenum-depleted cultures of A. vinelandii were grown in vanadium-containing growth medium. Protein isolation was carried out under strict exclusion of dioxygen, in a chamber containing an N2/H2 atmosphere (95:5) or using modified Schlenk techniques with N2 gas. The isolation protocol consisted of two consecutive anion exchange chromatography columns followed by size exclusion chromatography. Pure protein was stored in a buffer containing 100 mM NaCl, 2.5 mM sodium dithionite and 50 mM Tris/HCl buffer at pH 7.4.

Turnover assays with carbon monoxide
CO turnover assays were carried out with an ATP regeneration system. 5 mg•mL-1 VnfDKG was combined with 0.5 mg•mL-1 VnfH, yielding an approximate 3:1 ratio of the two nitrogenase components for low electron flux turnover. Buffer solutions contained 20 mM Tris/HCl buffer at pH 7.4, 10 mM MgCl2, 15 mM phosphocreatine, 0.125 mg•mL-1 phosphocreatine kinase and 25 mM sodium dithionite. Mixtures were sealed in a Wheaton vial and the headspace was replaced with 100 % carbon monoxide. The gas phase was equilibrated with the solution by incubation for 3 min at 30 °C under agitation at 320 rpm in a water bath shaker. CO turnover was initiated by the addition of ATP to a final concentration of 2.5 mM. The samples were given ample time (~ 2 h) for full depletion of ATP under continuous agitation. Carbon monoxide was eventually removed from the headspace prior to crystallization. To obtain the CO-free structure, the CO-turnover sample was concentrated (100 kDa MWCO, Sartorius) and desalted on a G-25 column (GE Healthcare) under gravitational flow. The turnover assay was then repeated with the same component ratio and buffers, but with argon replacing carbon monoxide in the headspace.

Crystallization
Crystallization of VnfDKG was carried out based on previously published conditions in an anoxic chamber under N2/H2 atmosphere (95:5) and was further optimized. [2] Sitting drop vapor diffusion-based crystallization trials were conducted in 24-well crystallization plates (Cryschem). ZnCl2 was added to a final concentration of 0.1 mM in turnover samples prior to crystallization. Drops consisted of a mixture of 2 µL of protein solution at a concentration of 10 mg•mL-1 and 2 µL of reservoir solution. Reservoir solutions contained 100 mM Tris/HCl at pH 7.5, 30 mM MgCl2, 20 % (v/v) of ethylene glycol, 7-12 % (w/v) of polyethylene glycol 8000 and 1 mM sodium dithionite. After the first crystals were obtained from initial crystallization plates, 0.5 µL of a diluted microcrystal suspension were added to each drop of subsequent CO-turnover and crystallization trials. This microseeding procedure was applied repeatedly in order to optimize the amount and shape of crystals formed within each drop. Crystals appeared within a day and grew continuously for 2 weeks. After 2 weeks, crystals were harvested with a nylon loop and flash-frozen in liquid nitrogen.

Data Collection, Structure Solution and Refinement
Diffraction data were collected at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland). High-resolution data of the CObound state was collected at a wavelength of 0.85 Å using an Eiger 16M detector (Dectris) at beam line X06SA, while the high-resolution data set of the CO-removed state was collected at a wavelength of 0.91 Å using a Pilatus 2M-F detector (Dectris) at beam line X06DA. To obtain the anomalous scattering contribution of sulfur, crystals of both states were measured at the Pilatus 2M-F detector at a wavelength of 2.0 Å (6200 eV), and in order to maximize the multiplicity of the data, 9 datasets were collected at increasing chi angles, in steps of 5°, by using the PRIGo multi-axis goniometer. [3] XDS was used for indexing and integration, [4] and Aimless from the CCP4 suite for merging and scaling of the data. [5] An existing structure of VnfDKG (PDB-entry: 6FEA) served as a starting model, and further adjustments were made using Coot. [6] This included changes at the C-terminus of VnfD, since a Zn2+ ion at the surface that was found in the previous turnover structure was missing. The high-resolution electron density map also allowed to identify and assign Tris and ethylene glycol molecules in the crystal that were added to the model. For the CO-bound state, the prior ligand at the Fe2-Fe6 of the VFe cofactor did not sufficiently explain the observed density map peak and was replaced by CO, which led to a clear improvement in the Fo-Fc difference map. Adjustments to Q176D and K361D were made. The structure was refined with REFMAC5 [7] using anisotropic temperature factors and a ligand dictionary file from the previous published high resolution crystal structure. [8] All figures were generated with PyMOL. [9]

Results and Discussion
The high level of detail provided by the present crystallographic analysis at a resolution of 1.0 Å for the CO adduct of FeV cofactor provides a wealth of direct information regarding the nature and properties of the individual atoms in the structure. Most importantly, the structure shows individual single maxima for each atom, thus fulfilling the criterion for true atomic resolution in crystallography. The present analyses of the CO-bound and turnover states are the most highly resolved that are available for vanadium nitrogenase today and will be instrumental as a future reference for theoretical approaches. Furthermore, the quality of the electron density map is sufficient to show consistent differences in the electron density maxima for carbon vs. nitrogen vs. oxygen atoms. In the well-defined core region of the enzyme, this allows for the distinction of light atoms (Fig. S1). Notably, as in our previous analysis of the turnover state of V-nitrogenase, [8] we refrain from assigning the nature of the light atom in the CO-removed structure of the enzyme solely based on electron density, even at 1.05 Å resolution (Fig. 4). As seen from anomalous difference maps, we typically find a small fraction of resting state even in turnover state structures that result from inhomogeneous conformations across the entire crystal. A final assignment will ideally include data from separate experiments, in particular EPR spectroscopy.  No residual peaks in the map indicate the presence of residual sulfur at either the S2B belt position or in the holding site, indicating that the CO-bound state is very near 100 % occupancy. The diffraction data underlying this map was collected at an X-ray energy of 6200 eV (see Table S1) to 2.0 Å resolution, with an overall multiplicity of 28 for the crystal belonging to space group P1. The electron density map is contoured at the 5 σ level. B) Plot of anomalous scattering coefficients f " for sulfur, vanadium and iron, displaying the K-edge positions for all elements and highlighting their relative anomalous scattering contributions at 6200 eV, where the data set for panel (A) was collected.
At the high resolution of the present analysis, further details with respect to the conformational changes related to CO binding to FeV cofactor became apparent. The rearrangement of residue Q176D form the CO-bound to the turnover state hinges on an H-bonding interaction of the amide nitrogen Nε2 with the α-carboxylate of the homocitrate ligand to vanadium. While the length of this bond only shortens from 2.89 Å in the in-conformation to 2.84 Å in the CO-bound out-conformation, the side chain moves towards the organic ligand and displaces residue K361D that is H-bonded to the α-carboxylate of homocitrate in the in-conformation, but relocates to a weaker (3.02 Å) hydrogen bond to the δ-carboxylate in the out-conformation of residue Q176D (Fig. S2). Concomitant with this movement, both the amide of Q176D and the amine of K361D displace a coordinated water molecule towards a pool of solvent molecules that characteristically fill a cavity adjacent to homocitrate within the protein matrix (Fig. S4). It is tempting to consider that these water molecules may really be product NH3/NH4+, whichdue to an identical number of electronscould not be distinguished in an electron density map. This arrangement may serve to eject product concomitant with the formation of the inward-facing conformation of the turnover state of the enzyme that can only be attained if a single light atom is bound at Fe2 and Fe6. Notably, K361D is replaced by a glutamate residue in Mo nitrogenase, but in the absence of a turnover state structure its effect on the solvent structure around FeMo cofactor remains to be determined. We note that Hu and Ribbe recently reported a MoFe protein structure obtained from protein isolated in the absence of reductant that they interpret to reveal multiple N2 binding sites at FeMo cofactor. [10] Although this would represent a substrate-bound state of this nitrogenase variant, the diatomic N2like CO in the present workwould not accommodate the glutamine residue in the inward-facing conformation and is thus not a turnover/intermediate state that would inform on side chain flexibility. Figure S4. The reorientation of residue K361D in response to the conformational change of Q176D. A) In the CO-bound structure (as well as the resting state of FeV cofactor with sulfide S2B in place at Fe2 and Fe6), K361 forms a hydrogen bond to the δ-carboxylate of the homocitrate ligand, whose α-carboxylate interacts with the amide nitrogen of Q176 in the out-conformation. B) In the turnover state obtained after the removal of CO by continuous turnover in the absence of the gas, Q176 is in the in-conformation, forming a much tighter hydrogen-bonding network that involves H180 and the bound light atom at the cluster. Residue K361 also changes its conformation to now interact with the α-carboxylate of homocitrate, creating space for coordinated water (or NH4+) molecules in close proximity to the cluster.