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www.sciencemag.org/cgi/content/full/334/6058/940/DC1
Supporting Online Material for
Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor Thomas Spatzal, Müge Aksoyoglu, Limei Zhang, Susana L. A. Andrade, Erik Schleicher,
Stefan Weber, Douglas C. Rees, Oliver Einsle*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 18 November 2011, Science 334, 940 (2011)
DOI: 10.1126/science.1214025
This PDF file includes:
Materials and Methods Figs. S1 and S2 Table S1 References
SUPPLEMENTARY ONLINE MATERIAL
T. Spatzal, M. Aksoyoglu, L. Zhang, S.L.A. Andrade, E. Schleicher, S. Weber, D.C. Rees & O. Einsle
Evidence for Interstitial Carbon in Nitrogenase FeMo Cofactor
MATERIALS AND METHODS
Cell growth and protein purification. Azotobacter vinelandii (Lipman, 1903) was grown in modified
Burke’s medium (pH 8.2) in a 70% air, 30% N2 atmosphere. Pre-culture medium (100 mL) contained
10 mM NH4Cl as the sole nitrogen source. Main cultures (500 mL) were complemented with 1.3 mM
NH4Cl resulting in de-repression of nitrogenase gene expression upon ammonium depletion. The
ammonium concentration of the medium was monitored via the Berthelot reaction. Cells were
harvested by centrifugation at an optical density (OD600nm) of 4.0 and the pellet was washed with 50
mM Tris/HCl buffer at pH 7.4. All subsequent steps were carried out under strict exclusion of
dioxygen. Buffers were degassed and supplemented with 5 mM Na2S2O4 at pH 7.5. Cells were
ruptured in a French Pressure cell (Aminco) at 1000 bar under Ar atmosphere. The lysate was
centrifuged at 100,000×g for 1 h and the supernatant was loaded onto a HiTrap Q anion exchange
column (GE Healthcare) pre-equilibrated with 50 mM Tris/HCl buffer at pH 7.4. MoFe protein was
eluted with a linear NaCl gradient at 340 mM. After heat-treatment at 323K for 1 min, precipitate was
removed by centrifugation (15,000×g for 15 min) and the supernatant was loaded onto a size exclusion
column (S200, 26/60, GE Healthcare) equilibrated with 200 mM NaCl, 50 mM Tris/HCl buffer at pH
7.4. Pure MoFe protein was concentrated to 80 mg·mL–1 using a Vivaspin concentrator (100,000 kDa
MWCO, Sartorius) under 5 bar N2 pressure. Nitrogenase activity was assayed by monitoring acetylene
reduction as described previously.
Isotope labeling. 13C labeled MoFe protein was purified from cells grown on Burke’s medium with
pure 13C-D-glucose as the sole carbon source. Pre-cultures were 12C-depleted over four consecutive
generations, resulting in >99% labeling efficiency in the main culture. 15N labeled MoFe was obtained
by using 15NH4Cl as sole nitrogen source. Cells of the 15N cultivation were harvested as soon as the
whole-cell acetylene reduction activity reached 0.1% of its maximum to ensure that 14N incorporation
due to intrinsic nitrogen fixation activity was minimized.
Crystallization and Data Collection. MoFe protein was crystallized by sitting drop vapor diffusion at
291 K, in an anaerobic chamber at less than 5 ppm O2. 4 µL protein solution at 80 mg·mL–1 were
mixed with the same volume of reservoir solution containing 0.55 M NaCl, 16.5% (v/v) PEG 6000,
12.5% (v/v) MPD, 1.5% (v/v) Xylitol, 0.2M imidazole/malate buffer at pH 8.0, 0.55 mM spermine,
and 0.1 mM Zwittergent 3-14 (Hampton Research). Seeding was used to optimize the crystal shape.
Crystals were transferred to harvesting buffers with stepwise increase of MPD concentrations up to
19% (v/v), and flash-frozen in liquid nitrogen. Diffraction data were collected on beam line X06SA at
the Swiss Light Source (Paul-Scherrer-Institut, Villigen, CH) using a Dectris Pilatus 6M detector.
Structure Solution, Refinement and Analysis of Electron Densities. Data were indexed and
integrated using HKL2000 (5) and XPREP (Bruker). Phase information was obtained by molecular
replacement using the 1.16 Å resolution structure as a search model (PDB-ID 1M1N). Rebuilding was
done in COOT (6) and the structure was refined using REFMAC5 (Table 1) (7). Electron density
analyses were carried out using MAPMAN (8) and proprietary software.
EPR Spectroscopy / ESEEM. 12C/14N-MoFe, 13C/14N-MoFe and 12C/15N-MoFe proteins were
concentrated to 120, 60 and 90 mg·mL–1, respectively. All samples contained 5 mM Na2S2O4. X-band
three pulsed (3P) ESEEM spectra were recorded using a commercial pulse EPR spectrometer (Bruker
E580, Bruker BioSpin GmbH, Rheinstetten, Germany) in conjunction with a dielectric-ring resonator
Bruker ER 4118X-MD5-EN), which was immersed in a helium-gas flow cryostat (Oxford CF-935).
The temperature was regulated to ± 0.1 K by a temperature controller (Oxford ITC-4). For 3P ESEEM,
a microwave pulse-sequence π/2–τ–π/2–T–π/2 using 16 ns π/2 pulses was used. The separation times,
τ and T between the microwave pulses were set to 130 or 180 ns, and 400 ns, respectively. All
measurements were performed at 3.6 K and were recorded at a magnetic field position of 342.5 mT
(g = 2.0346).
SUPPORTING FIGURES
Figure S1: Effects of higher resolution data. A) The resolution-dependent electron density artifacts in
the center of the FeMo cofactor are introduced by the cluster geometry (5). They disappear only at
resolutions higher than 0.6 Å, yielding an unbiased value (dotted red line). At 1.16 Å resolution these
artifacts lead to a slight overestimation of the actual electron density, while this effect is negligible at
1.0 Å resolution. B) Integrated average electron density vs. radius of the integration sphere for the 1.16
Å resolution structure of nitrogenase MoFe protein (PDB 1M1N) (5). Compared to the higher
resolution data (Fig. 1B), the central atoms are less well-defined and are best interpreted as N.
Figure S2: Plots of ρ0 vs. Biso for data to 1.0 Å (left) and 1.16 Å resolution (right) (2). The ellipsoids
depict the 99% level (~ 2.5 σ) for a Gaussian fit of the data for individual C- (black), N- (blue) and O-
(red) atoms. The figure is analogous to Fig. 1C, but here the negative slope represents the
anticorrelation between ρ0 and Biso. Atoms with large B-factors (>20 Å2 for the 1.0 Å data and >30 Å2
for the 1.16 Å data) were excluded from the calculation.
SUPPORTING TABLES
Table 1: Data collection and refinement statistics. Values in brackets represent the highest resolution
shell.
Wavelength (Å) 0.8000 Resolution range (Å) 47.5 – 1.0 (1.09 – 1.00) Unique reflections 1,112,854 (252,121) Completeness (%) 99.1 (98.3) Multiplicity 6.4 (5.5) Space group P21
Unit cell parameters
a, b, c 81.2, 130.7, 107.2 α, β, γ 90.0, 110.7, 90.0
Rmerge 0.100 (0.687) Rp.i.m. 0.042 (0.311)
I / σ(I) 8.6 (1.5)
Rcryst 0.128 (0.351) Rfree 0.146 (0.349) r.m.s.d. bond lengths (Å) 0.011 r.m.s.d. bond angles (˚) 1.541 Average B-factor (Å2) 8.1 Diffraction precision index (Å) 0.014
SUPPORTING REFERENCES
5. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307 (1996).
6. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Acta Crystallogr. D. 66, 486 (2010).
7. G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Cryst. D53, 240 (1997).
8. G. Kleywegt, T. A. Jones. Daresbury Study Weekend Proceedings. (1992).
References and Notes
1. D. C. Rees et al., Structural basis of biological nitrogen fixation. Philos. Trans. R. Soc. Lond. A 363, 971 (2005). doi:10.1098/rsta.2004.1539
2. O. Einsle et al., Nitrogenase MoFe-protein at 1.16 A resolution: A central ligand in the FeMo-cofactor. Science 297, 1696 (2002). doi:10.1126/science.1073877 Medline
3. Material and methods are available as supporting material on Science Online.
4. D. Lukoyanov et al., Testing if the interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C: ENDOR, ESEEM, and DFT studies of the S = 3/2 resting state in multiple environments. Inorg. Chem. 46, 11437 (2007). doi:10.1021/ic7018814 Medline
5. Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307 (1996). doi:10.1016/S0076-6879(97)76066-X
6. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486 (2010). doi:10.1107/S0907444910007493 Medline
7. G. N. Murshudov, A. A. Vagin, E. J. Dodson, Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240 (1997). doi:10.1107/S0907444996012255 Medline
8. G. Kleywegt, T. A. Jones, in Molecular Replacement, E. J. Dodson, S. Gover, W. Wolf, Eds. (CCP4 Daresbury Laboratory Study Weekend Proceedings, Warrington, UK, 1992).