Hu et al. 10.1073/pnas.0602647103. |
Table 3. Designations of NifEN' proteins in this work
Designation | Preparation |
NifENcomplete | NifEN was incubated with molybdate, homocitrate, MgATP and Fe protein and re-purified as described in Material and Methods. |
nifB NifENcomplete | Same as NifENcomplete except that NifEN was replaced by nifB NifEN. |
NifENminus Mo/homocitrate | Same as NifENcomplete except that molybdate and homocitrate were omitted. |
NifENminus homocitrate | Same as NifENcomplete except that homocitrate was omitted. |
NifENminus Mo | Same as NifENcomplete except that molybdate was omitted. |
NifENminus MgATP | Same as NifENcomplete except that MgATP was omitted. |
NifENminus Fe protein | Same as NifENcomplete except that Fe protein was omitted. |
NifENapo Fe protein | Same as NifENcomplete except that Fe protein was replaced by apo Fe protein. |
NifENA157S Fe protein | Same as NifENcomplete except that Fe protein was replaced by A157S Fe protein. |
NifENM156C Fe protein | Same as NifENcomplete except that Fe protein was replaced by M156C Fe protein. |
NifENA157G Fe protein | Same as NifENcomplete except that Fe protein was replaced by A157G Fe protein. |
NifENMgADP | Same as NifENcomplete except that MgATP was replaced by MgADP. |
NifENATPgS | Same as NifENcomplete except that MgATP was replaced by ATPgS. |
NifENAMPPNP | Same as NifENcomplete except that MgATP was replaced by AMPPNP. |
NifEN' | General term for all processed, repurified NifEN proteins listed in this table. |
Table 4. Fe K-edge extended x-ray absorption fine structure (EXAFS) fit results
[7Fe-9S] Fit | [Mo-7Fe-9S] (FeMoco) Fit | |||||||||||
NifEN precursor | NifENcomplete precursor | NifENcomplete precursor | MoFe protein FeMoco | |||||||||
Fit Parameter | N | R , Å | s2 , Å2 | N | R , Å | s2 , Å2 | N | R , Å | s2 , Å2 | N | R , Å | s2 , Å2 |
Scatterer | ||||||||||||
Fe-S | 3.1 | 2.28 | 0.0035 | 3.1 | 2.26 | 0.0031 | 3.1 | 2.26 | 0.0031 | 3.1 | 2.24 | 0.0048 |
Fe-Fe | 3.1 | 2.65 | 0.0052 | 3.1 | 2.63 | 0.0044 | 3.4 | 2.64 | 0.0053 | 3.4 | 2.60 | 0.0055 |
Fe-Fe | 0.3 | 2.89 | 0.0023 | 0.3 | 2.82 | 0.0008 | ― | ― | ― | ― | ― | ― |
Fe-Mo | ― | ― | ― | ― | ― | ― | 0.4 | 2.71 | 0.0016 | 0.4 | 2.73 | 0.0010 |
Long Fe-Fe | 0.9 | 3.68 | 0.0010 | 1.7 | 3.70 | 0.0045 | 1.7 | 3.70 | 0.0044 | 1.7 | 3.68 | 0.0034 |
Long Fe-Fe | 0.9 | 3.80 | 0.0026 | ― | ― | ― | ― | ― | ― | ― | ― | ― |
DE0, eV | -10.6 | -10.6 | -8.2 | -9.8 | ||||||||
F | 0.222 | 0.357 | 0.355 | 0.264 | ||||||||
All fits were calculated over a k-range of 2-16 Å-1 as described in Supporting Text. The coordination number, N; interatomic distance, R; mean-square thermal and static deviation in R, s2; and the shift in the threshold energy from 7130 eV, DE0, variables have respective uncertainties of ± 0.02 Å, ± 0.0001 Å2, ± 20 %, and ± 0.2 eV. The goodness of fit, F = [S k6(c exptl - ccalcd)2/ Sk6cexptl2]1/2. ―, not included in fit.
Table 5. Mo K-edge extended x-ray absorption fine structure (EXAFS) fit results
MoFe protein | NifENcomplete | ||||||||
3-component fit | 4-component fit | ||||||||
Fit parameter | N | R , Å | s2 , Å2 | N | R , Å | s2 , Å2 | N | R , Å | s2 , Å2 |
Scatterer | |||||||||
Mo-O/N* | ― | ― | ― | ― | ― | ― | 1.0 | 2.00 | 0.0039 |
Mo-O/N* | 3.0 | 2.22 | 0.0027 | 3.0 | 2.13 | 0.0128 | 2.0 | 2.17 | 0.0036 |
Mo-S | 3.0 | 2.37 | 0.0021 | 2.0 | 2.37 | 0.0018 | 2.0 | 2.37 | 0.0016 |
Mo-Fe | 3.0 | 2.69 | 0.0029 | 2.0 | 2.70 | 0.0042 | 2.0 | 2.70 | 0.0041 |
DE0, eV | -7.3 | -9.8 | -10.2 | ||||||
F | 0.145 | 0.235 | 0.228 | ||||||
All fits were calculated over a k-range of 4-16 Å-1 as described in the Supporting Text. The coordination number, N; interatomic distance, R; mean-square thermal and static deviation in R, s2; and the shift in the threshold energy from 20025 eV, DE0, variables have respective uncertainties of ± 0.02 Å, ± 0.0001 Å2, ± 20 %, and ± 0.2 eV. The goodness of fit, F = [S k6(c exptl - ccalcd)2/ Sk6cexptl2]1/2. ―, not included in fit. *Scatterers differing by Z ±1 are not distinguishable in an EXAFS analysis. The ordering O/N indicates that an O atom was used to model the scattered wave in the calculation of phase and amplitude functions.
Supporting Text
Cell Growth and Protein Purification
. All Azotobacter vinelandii strains were grown in 180-liter batches in a 200-liter New Brunswick fermentor on Burke's minimal medium supplemented with 2 mM ammonium acetate. The growth rate was measured by cell density at 436 nm by using a Spectronic 20 Genesys Spectrophotometer (Spectronic, Westbury, NY). After ammonia consumption, the cells were derepressed for 3 h followed by harvesting using a flow-through centrifugal harvester (Cepa, Lahr/Schwarzwald, Germany). The cell paste was washed with 50 mM Tris·HCl (pH 8.0). Published methods were used for the purification of all Fe proteins (1), wild-type MoFe protein (2), His-tagged DnifB MoFe protein (3), His-tagged NifEN, and His-tagged DnifB NifEN (4, 5).EPR Spectroscopy.
All EPR samples were prepared in an Ar-filled Vacuum Atmospheres dry box with <4 ppm O2. All dithionite-reduced samples were in 25 mM Tris·HCl (pH 8.0), 10% glycerol and 2 mM Na2S2O4. Indigo disulfonate (IDS)-oxidized samples were prepared as described (1). Samples were either used as they were or were concentrated in a Centricon-30 (Amicon) in anaerobic centrifuge tubes outside of the dry box. All EPR spectra were recorded using a Bruker ESP 300 Ez spectrophotometer, interfaced with an Oxford Instruments ESR-9002 liquid He continuous flow cryostat. All spectra were recorded at 13 K using a microwave power of 50 mW, a gain of 5 × 104, a modulation frequency of 100 kHz, and a modulation amplitude of 5 G. A microwave frequency of 9.43 GHz was used to record 10 scans for each sample.Preparation of Apo Fe protein.
Apo Fe protein was prepared by chelating the [4Fe-4S] cluster from the Fe protein with the iron chelator bathophenanthroline disulfonate. The chelation reaction contained, in a 10 ml total volume, 25 mM Tris·HCl (pH 8.0), 20 mM ATP, 40 mM MgCl2, 20 mM bathophenanthroline disulfonate, and 100 mg wild-type Fe protein. The reaction mixture was incubated for 15 min and the Fe protein was subsequently passed over a 2.5 × 100-cm Ultrogel AcA34 (ICF) gel filtration column in 25 mM Tris·HCl (pH 8.0). The resulting Fe protein was completely Fe deficient (or apo) based on Fe analysis.FeMoco Maturation Assay.
Assays designed to covert NifEN-bound FeMoco precursor to mature FeMoco contained, in a 0.8 ml total volume, 25 mM Tris·HCl (pH 8.0), 20 mM Na2S2O4, 0.5 mg of purified FeMoco-deficient DnifB MoFe protein from A. vinelandii strain DJ1143 (6), 1.4 mg of Fe protein, 0.3 mM homocitrate, 0.3 mM Na2MoO4, 0.8 mM ATP, 1.6 mM MgCl2, 10 mM creatine phosphate, and 8 units of creatine phosphokinase. FeMoco maturation was initiated by the addition of 2 mg isolated FeMoco precursor containing-NifEN (4) to the mixture above. Such reaction mixtures were incubated at 30°C for 30 min, stopped by the addition of 40 nmol (NH4)2MoS4 (4), and the enzymatic activities were then determined as described (2, 7, 8). The reaction products H2 and C2H4 were analyzed as described (7), whereas ammonium was determined by using a high performance liquid chromatography fluorescence method (9). Homocitrate lactone (Sigma) containing an undefined mixture of stereochemical configurations was converted to the free acid as described (4).X-Ray Absorption Spectroscopy (XAS) Data Collection
. The NifEN' XAS samples were prepared in an Ar-filled Vacuum Atmospheres dry box with <4 ppm O2 and concentrated in a Centricon-30 (Amicon) concentrator in anaerobic centrifuge tubes outside the dry box to final concentrations of ~50 mg/ml (4 mM in Fe, 0.3 mM in Mo) for NifENcomplete, and 53 mg/ml (2 mM in Fe) for DnifB NifENcomplete. Fe K-edge samples in Tris×HCl (pH 8.0), 250 mM imidazole, 500 mM NaCl, 2 mM Na2S2O4 and 50% glycerol were loaded into 70 ml, 1-mm pathlength Lucite cells with Kapton tape windows and flash frozen in a pentane/liquid N2 slush. The Mo K-edge NifENcomplete sample was similarly prepared, but with 33% glycerol, and was loaded into a 250-ml, 15-mm pathlength Nylon cell before flash freezing as above. Na2MoO4, 8 mM in 33% glycerol/water, was measured in a similar Nylon cell. The preparation of Mo K-edge samples of MoFe protein (10) and Fe K-edge samples of MoFe protein, DnifB MoFe protein, NifEN, and DnifB NifEN (11) has been described.XAS data were measured at the Stanford Synchrotron Radiation Laboratory (SSRL) under 3 GeV, 80-100 mA beam conditions using beam line 9-3 with a Si(220) double-crystal monochromator and two Rh-coated mirrors: one flat premonochromator mirror for harmonic rejection and vertical collimation and one toroidal postmonochromator mirror for focusing. The samples were maintained at 10 K during data collection in an Oxford Instruments CF1208 continuous-flow liquid He cryostat. Data were collected as Fe or Mo Ka fluorescence using a Canberra 30-element solid-state Ge detector. Radiation from elastic/inelastic scattering and Kb fluorescence were minimized at the detector by placing a set of Soller slits with either a Mn (for Fe) or Zr (for Mo) filter between the cryostat and the detector. The x-ray energy at the Fe K-edge was calibrated to the inflection point at 7111.2 eV of a standard Fe foil measured concurrent with the samples. At the Mo-edge, the x-ray energy was similarly calibrated to 20003.9 eV. Changes in the edge position over time, which would indicate photoreduction of an oxidized metal site, were not observed for any sample.
XAS Data Analysis.
The analysis of Mo K-edge data from MoFe protein (10) and Fe K-edge data from MoFe protein, DnifB MoFe protein, NifEN, and DnifB NifEN (11) has been described previously. Following a similar procedure, Fe and Mo K-edge data from NifEN' and MoFe protein were processed using the program XFIT (12) to (i) fit a background absorption curve to the data in the pre-edge region, with a slope matching that of the post-edge region, using a polynomial function and weighted control-points; (ii) fit a four-segment polynomial spline over the EXAFS region; and (iii) normalize the spectra to have an edge-jump of 1.0 between the background and spline curves at 7130 eV (for Fe) or 20025 eV (for Mo). The MoFe protein-bound FeMoco data, NifEN-bound FeMoco precursor data, and NifENcomplete precursor data were obtained by subtraction of the appropriate DnifB protein from MoFe protein, NifEN, or NifENcomplete in an 8/7:15/7 ratio. The EXAFS data were subtracted separately from the edge data.Fe K-edge EXAFS data from MoFe protein-bound FeMoco, the NifEN-bound precursor, and the NifENcomplete precursor over the k-range 2-16 Å-1 were fit by using the nonlinear least squares fitting program, opt, from the EXAFSPAK program suite (13). During the fitting process, the variables for interatomic distance (R) and mean-square thermal and static deviation in R (s2) were allowed to vary for all scattering components. The shift in the threshold energy (DE0) was also varied for each fit, but constrained to be the same for all components. The amplitude reduction factor (S02) was fixed to a value of 1.0 for all fits. Coordination numbers (N) were fixed to values established by the input models of either FeMoco or a Mo-free [7Fe-9S] homolog of FeMoco. The ab initio theoretical phase and amplitude functions used in the fitting program were generated by feff (version 7.0) (14, 15) from input models based on the crystallographic coordinates of FeMoco in the MoFe protein (16).
Mo K-edge EXAFS data from MoFe protein or NifENcomplete over the k-range 4-16 Å-1 were similarly fit with phase and amplitude functions generated by feff (version 7.0) (14, 15) from a FeMoco model structure (16) using the opt program (13). For MoFe protein, N were fixed to values established by the FeMoco input model. The determination, by EXAFS analysis, of N values for unknown site is complicated by the high degree of correlation between N and s2, which provides a measure of thermal and static disorder, in a fit (17). To overcome this difficulty in fitting NifENcomplete, N values were iteratively tested over a range of appropriate initial R values, while R, s2, and single DE0 value were allowed to float (18, 19). The N values for NifENcomplete are thus estimates and may be artificially low due to contributions from static disorder.
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