The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase†

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, aiso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.


S2
(DJ2330) were constructed by transforming DJ2253 with plasmids that carry deletions and Km R insertions in cloned segments of either nifB (pDB218) or nifE (pDB259).
Cell growth, and protein purification. Azotobacter vinelandii DJ strains expressing the Vdependent nitrogenase system were grown at 30°C in a 150-liter custom built fermenter (W. B. Moore, Inc., Easton, PA) in modified Burk medium containing 2 µM V2O5 as the vanadium source and 10 mM urea as a nitrogen source. Parameters for growth and cell harvesting were the same as described previously. 4 The Strep-tagged VFe proteins were purified according to a published procedure using a Strep-Tactin (IBA Lifesciences, Gӧttingen, Germany) column. 4 Fe protein specific for the V-dependent system was purified from strains DJ1258 or DJ2330 using a procedure similar to that for purification of Fe protein from the Mo-dependent system as described before. 5,6 The identity of purified proteins was determined by mass spectrometry as previously described. 7 In vitro incubation of VFe StrnifE protein. Crude extract from DJ2330 (100 g wet cells) was incubated with 10 mM sodium dithionite (DT), 50 µM V2O5, 2.0 mM α-ketoglutaric acid and MgATP regenerating mix, that consisted of 2.5 mM ATP, 30 mM creatine phosphate, 10 mM MgCl2 and 0.1 mg/mL of creatine phosphokinase. The reaction mix was incubated at room temperature for 4h under continuous stirring under an argon atmosphere. The reaction mixture was then filtered through a 0.45 µm membrane and loaded on 2 tandem strep-tactin columns (5 mL each). Nonspecifically bound proteins were removed from the column by washing with 30 mL of Buffer A (50 mM Tris, 500 mM NaCl, 20% glycerol, 2 mM DT, pH 8.0). Elution was performed with 50 mM biotin in buffer A.
Protein activity assays. Substrate reduction assays were conducted in 9.4-mL septum-sealed serum vials with 1 mL of an assay buffer containing a MgATP regeneration system (5.0 mM ATP, 6.7 mM MgCl2, 30 mM phosphocreatine, 0.2 mg/mL creatine phosphokinase, and 1.0 mg/mL BSA) and 10-12 mM DT in 100 mM MOPS at pH 7.3. The reaction vials were degassed under vacuum and the headspace was refilled with Ar for proton reduction or with N2 for N2 reduction and adjusted to 1 atm. The VFe Str protein was then added to the reaction buffer to a final concentration of 0.1 mg/mL. Each reaction vial was preincubated in a 30 C water bath for 2 min before the addition of Fe protein with a molar ratio to VFe Str protein of ≥ 20 to start the reaction at the same temperature. The reactions lasted for 8-10 min as specified with a shaking rate of ca. 140 rpm before the reactions were quenched by addition of 500 µL of 400 mM EDTA at pH 8.0. The products (H2 and NH3) were quantified according to methods described before. 8 The detailed information about assay conditions and protein concentrations are listed in the corresponding table captions and figure legends.
EPR sample preparation. The resting state of VFe Str proteins were prepared in 100 mM MOPS buffer, pH 7.3, with 150 mM NaCl and 20 mM DT, and the resting state Fe protein was prepared in turnover buffer described described below. Turnover samples prepared for EPR analyses included 200 mM MOPS buffer at pH 7.3 with a MgATP regeneration system (20 mM MgATP, 20 mM phosphocreatine, 1 mg/mL bovine serum albumin, and 0.4 mg/mL creatine phosphokinase from rabbit muscle) and 20 mM DT. VFe Str protein was first added to the designated final concentration and the reaction initiated by addition of Fe protein to the designated concentration. After the reaction was incubated at room temperature for about 20-25 s, an aliquot of 300 µL of the reaction mixture was rapidly transferred into a 4-mm quartz EPR tube and frozen in a pentane slurry before being stored in liquid nitrogen for EPR measurement. The protein concentrations of the samples can be found in the corresponding figure legends.
The samples for EPR study of the redox properties of VFe Str protein were made in 100 mM MOPS at pH 7.3 with 150 mM NaCl as the following: (1) a resting state sample of VFe Str protein with 20 mM DT; (2) two methylene blue (MB, Em = +11 mV vs SHE) 9 oxidized samples were prepared in parallel in the same buffer with a final concentration of 612 μM MB and VFe Str protein and no DT . After these two reactions were incubated for 15 min at room temperature, one sample was immediately frozen in an EPR tube whereas DT was added to the other sample, giving a DT concentration of 20 mM, and then frozen. All three samples contained a final VFe Str protein concentration of 50 μM.
To confirm the observed intermediate hyperfine splitting is associated with a catalytic intermediate, a pair of EPR samples under Ar turnover condition were made. One Ar turnover EPR sample was made as mentioned above with a final VFe Str protein concentration of 10 μM and Fe protein concentration of 100 μM. The sample was freeze quenched after incubation for 25 sec at room temperature. The other sample was made with the same amount of the proteins and incubation time (25 sec) at room temperature before the addition of a degassed and dithionitereduced 400 mM EDTA stock solution to bring the final protein concentrations to the same as those in the first sample and with a final EDTA concentration of 75 mM. After 300 s relaxation at room temperature,to allow for relaxation, the sample was frozen in a pentane slurry before being stored in liquid nitrogen for EPR measurement. Figure S5 shows the signal from the intermediate disappears during the relaxation period.
EPR spectroscopy. Continuous-wave (CW) X-band EPR spectra were recorded using a Bruker ESP-300 spectrometer with an EMX PremiumX microwave bridge and an EMX PLUS standard resonator in perpendicular mode, equipped with an Oxford Instruments ESR900 continuous helium flow cryostat using VC40 flow controller for helium gas. Spectra were recorded at the following conditions: temperature, 12 K or otherwise stated in the figure legends; microwave frequency, ~9.38 GHz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 8.14 G; time constant, 20.48 ms. Each spectrum is the sum of five or ten scans as specified for each data set as in the figure legends, and is presented in this work after subtracting the cavity background signal recorded with an EPR tube with frozen 100 mM MOPS buffer at pH 7.3 with 150 mM NaCl. EPR simulations were performed with the EasySpin program operating in Matlab 5.2.83 Spin quantification of EPR signals. Spin quantification of S = ½ EPR signals observed for VFe protein preparations was carried out under non-saturating conditions with Cu 2+ -EDTA as standards according to an established procedure. 10 The concentration of a stock Cu 2+ -EDTA solution (21.766 mM) in 100 mM MOPS buffer with 150 mM NaCl at pH 7.3 were calibrated S4 with an extinction coefficient of 0.094 mM -1 •cm -1 at 730 nm. 11,12 The Cu 2+ -EDTA standard samples were made by sequential dilutions to final concentrations of 50 and 100 μM. EPR measurements of the samples were collected at the same microwave power, modulation amplitude, temperature, and number of scans. The baselines were corrected, when necessary, before the final integrations were done using Bruker WinEPR processing software. The g-value differences were taken into account for the final quantitative analysis. Table S1. Specific activities of VFe proteins from the EPR samples listed in Figure 4