108 N Ni if fB B- -c co o, , a a K Ke ey y F Fe eM Mo o- -c co o P Pr re ec cu ur rs so or r, , i is s a a F Fe e 6 6 S S 9 9 C Co or re e w wi it th h a an n I In nt te er rs st ti it ti ia al l L Li ig gh ht t A At to om m L-cysteine Molybdenum Fe NifS NifU NifB NifX NifQ NifH NifH NafY NafY NifV ATP ATP Fe-S Core FeMo-co Proposed FeMo-co biosynthetic complex Active NifDK Inactive apo-NifDK SAM R- homocitrate NifEN homocitrate Cys-275 His-442 Mo X Fe S Chemical Science Fig. 1. Left: the structure of FeMo-co as determined by X-ray crystallography on the Mo-N 2 ase enzyme (Protein Data Bank: 1M1N). Right: Proposed scheme for FeMo-co biosynthesis showing the proposed roles of NifB, NifX, NifH, NafY, and the MoFe protein (NifDK) N 2 ase [1]. All life depends on the input of the element nitrogen into the biosphere by biological nitrogen fixation (the reduction of dinitrogen to ammonia, N 2 2NH 3 ) performed only by diazotropic microorganisms. These microorganisms achieve N 2 fixation using a family of metalloenzymes, called nitrogenases (N 2 ases). Biological N 2 fixation is responsible for about half of the protein available for human consumption. The other half is produced using natural gas in fertilizer factories by the Haber-Bosch process. A better understanding of N 2 ases may have an impact on our ability to transition to a more sustainable energy economy. The active site of Mo-N 2 ase, which is contained within the so-called MoFe protein (one of the component proteins in Mo-N 2 ase), is a remarkable [7Fe-9S-Mo-X-homocitrate] cluster, called the iron-molybdenum cofactor (FeMo-co). FeMo-co is regarded as one of the most complex Fe-S clusters found in biology (Fig. 1). The exact identity of the interstitial light atom X is unknown; it can be assigned to C, N, or O. Knowledge of the FeMo-co biosynthetic assembly process, and the nature of X, is critical to understanding the N 2 ase catalytic mechanism. Figure 1 shows the current understanding of the biosynthetic pathways for Mo-N 2 ase [1]. A key metabolic intermediate in the biosynthesis of FeMo-co is NifB-co, a low-molecular weight Fe-S cluster. In addition to being a precursor of FeMo-co, NifB-co is also hypothesized to be the precursor of FeV-co and FeFe-co, suggesting that NifB-co forms the core portion that is common among all three N 2 ase active site cofactors. Recently, we used two synchrotron radiation techniques, namely K-edge extended X-ray absorption fine structure (EXAFS) and nuclear resonance vibrational spectroscopy (NRVS) to probe the structural and dynamics of the Fe sites of NifB-co bound to the small protein NifX (NifX:NifB-co) ( BL09XU ) [2]. EXAFS is a well-developed technique [3], capable of solving the local structure (typically within 5 Å) of the probed atom (in our case, Fe). NRVS is a novel vibrational spectroscopy [4]. The NRVS experiment involves scanning an extremely monochromatic X-ray beam ( ∆ E ~ 1 meV) through a nuclear resonance. Apart from the 'zero phonon' (recoil-free) Mössbauer resonance, there are additional transitions that correspond to nuclear excitation plus excitation or de- excitation of vibrational modes. Figure 2 shows the EXAFS and NRVS spectra of NifX:NifB-co and the simulations. Spectra for FeMo-co are also shown to help calibrate and interpret the NifX:NifB-co results. The EXAFS simulation of NifX:NifB-co reveals a set of S ligand at ~2.3 Å and a set of Fe next nearest neighbors at ~2.6 Å. A set of Fe-Fe interactions at 3.7 Å is also clear, which has so far been observed only in Fe-S centers for FeMo-co. The above features can be viewed as a strong indicator for the presence of the Fe 6 S 9 core in NifB-co, similar with the trigonal prism core in a matured FeMo- co. Three different structural models (Fig. 3) have been used in the EXAFS simulation, the best fit was obtained by using the 6Fe model. Furthermore, a search profile in the simulation confirms a Fe-X interaction with ~1 atom at 2.06 Å, consistent with the same interaction in a maturated FeMo-co. This is the first time this interaction has been reported in NifB-co, and is highly significant for the understanding Yisong Guo a , Simon J. George b , Luis M. Rubio c and Stephen P. Cramer a,b, * a Dept. of Applied Science, University of California, USA b Physical Biosciences Division, Lawrence Berkeley National Lab., USA c Dept. of Plant and Microbial Biology, University of California, USA *E-mail: spjcramer@ucdavis.edu References [1] L.M. Rubio and P.W. Ludden: Ann. Rev. Microbiol. 62 (2008) 93. [2] S.J. George, R.Y. Igarashi, Y. Xiao, J.A. Hernandez, M. Demuez, D. Zhao, Y. Yoda, P.W. Ludden, L.M. Rubio and S.P. Cramer: J. Am. Chem. Soc. 130 (2008) 5673. [3] J.J. Rehr and R.C. Albers: Rev. Mod. Phys. 72 (2000) 621. [4] J.T. Sage et al. : J. Phys.: Condensed Matter 13 (2001) 7707. [5] Y. Xiao et al. : J. Am. Chem. Soc. 128 (2006) 7608. 109 Fig. 3. Proposed models for NifB-co as described in the text. (a) 6Fe (b) 7Fe (c) 8Fe. Color code: Fe (brown), sulfur (yellow), interstitial atom X (red). Fig. 2. Left: Fe K -edge Fourier transformed EXAFS of (a) NifX:NifB-co (---) with the 6Fe model fit (---), and (b) data (---) and fit (---) of NafY:FeMo-co. Right: (a) NRVS of NifX:NifB- co (---) and the 6Fe model fit (---); (b) NRVS of NafY:FeMo-co (---) and NMF:FeMo-co (---). of FeMo-co maturation and the N 2 ase catalytic mechanism. In our simulation, X is simulated as N atom, C, and O also can obtain similar results. In addition, measurements in the Mo K-edge region conclusively demonstrate the absence of Mo in NifB-co. The NRVS spectrum of NifX:NifB-co shows similar spectral features as the NRVS of NafY:FeMoco (FeMo-co bound to the small protein NafY) and NMF:FeMoco (FeMo-co isolated in N-methyl- formamide (NMF)) (Fig. 2). Features above 250 cm -1 are primarily due to Fe-S stretching modes. The most striking feature is the intensity observed between 180 and 200 cm -1 in the NifX:NifB-co NRVS. This feature is considered to arise from the breathing modes of the Fe 6 S 9 core of FeMo-co in the presence of the interstitial light atom X [5], indicating that the interstitial light atom has already been incorporated into the NifB-co cluster. A simulation using the models in Fig. 3 confirms this assignment, while a model without this interstitial atom results in the absence of this 180-200 cm -1 band. Taken together, the EXAFS and NRVS data show that NifB-co, the key FeMo-co biosynthetic intermediate, is most likely to consist of a Fe 6 S 9 cage (Fig. 3). The presence of an interstitial atom in NifB-co is confirmed for the first time. This finding is significant because it rules out the possibility that the interstitial atom is incorporated in a later stage of the FeMo-co biosynthetic pathway - it implies that the function of the NifB enzyme is to perform the reaction of synthesizing this unique structure. This study demonstrates that EXAFS and NRVS are a powerful combination to reveal the structural information in the iron-containing metalloproteins. 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 1 2 3 4 5 6 7 R (Å) Transform Magnitude Transform Magnitude Energy (cm -1 ) PVDOS (cm) 0 0.02 0.015 0.01 0.005 0 0.02 0.015 0.01 0.005 0 100 200 300 400 500 PVDOS (cm) Data Fit (a) (b) (b) (a) (a) 6Fe model (b) 7Fe model (c) 8Fe model Fe X S Fe X S Fe X S
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