Structural insights into a -bound - precursor

Mary C. Corbett*, Yilin Hu†, Aaron W. Fay†, Markus W. Ribbe†‡, Britt Hedman‡§, and Keith O. Hodgson*‡§

*Department of Chemistry, Stanford University, Stanford, CA 94305; †Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; and §Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015

Edited by Helmut Beinert, University of Wisconsin, Madison, WI, and approved November 29, 2005 (received for review September 11, 2005) The iron-molybdenum cofactor (FeMoco) of the MoFe cluster from NifB, is transferred to the NifEN protein complex, protein is a highly complex metallocluster that provides the cata- where it is further processed and potentially molybdenum and lytically essential site for biological nitrogen fixation. FeMoco is homocitrate are added. The iron protein (NifH) is somehow assembled outside the MoFe protein in a stepwise process requir- involved in this process, and, although its exact role is unknown, ing several components, including NifB-co, an iron- and sulfur- it is thought to effect a change in NifEN that facilitates modi- containing FeMoco precursor, and NifEN, an intermediary assembly fication of the FeMoco precursor. Comparison with the chemical protein on which NifB-co is presumably converted to FeMoco. synthesis of P-cluster topologs indicates that an intermediate in Through the comparison of strains express- FeMoco might consist of two bridged [4Fe–4S] ing the NifEN protein in the presence or absence of the nifB gene, clusters (14, 18), however, there was previously no structural the structure of a NifEN-bound FeMoco precursor has been ana- evidence to support this theory or any proposed mechanism. lyzed by x-ray absorption spectroscopy. The results provide phys- Furthermore, the lack of a synthetic route to a FeMoco analog ical evidence to support a mechanism for FeMoco biosynthesis. The has limited the scope of the comparison between the synthetic NifEN-bound precursor is found to be a molybdenum-free analog and biochemical assembly reactions. The x-ray absorption spec- of FeMoco and not one of the more commonly suggested cluster troscopy (XAS) and extended x-ray absorption fine structure types based on a standard [4Fe–4S] architecture. A facile scheme by (EXAFS) analyses of a FeMoco precursor cluster bound to which FeMoco and alternative, non-molybdenum-containing ni- NifEN presented herein provide physical insight into how the trogenase cofactors are constructed from this common precursor is cofactor assembly reaction may proceed. Unexpectedly, the presented that has important implications for the biosynthesis and results indicate that a molybdebnum-free, iron- and sulfur- biomimetic chemical synthesis of FeMoco. containing cluster with a core structure similar to FeMoco is present on NifEN at an early stage in the biosynthetic pathway. biosynthesis ͉ extended x-ray absorption fine structure ͉ nitrogenase ͉ x-ray absorption spectroscopy Results and Discussion A FeMoco precursor bound to NifEN (expressed in a ⌬nifHD- itrogenase has served as a focal point in the field of KTY strain of A. vinelandii) was previously identified through ⌬ because of the complexity of its EPR comparison with a NifEN protein expressed in a nifB N ⌬ associated metalloclusters and because of its important role in background ( nifB NifEN) (19). NifEN is a heterotetramer that catalyzing biological , the essential process by shares significant sequence homology with the MoFe protein, which atmospheric nitrogen is converted into a bioavailable form including several of the residues surrounding both the P-cluster (see reviews in refs. 1–5). Minimally, nitrogenase enzyme sys- and FeMoco binding sites (20). Two different types of iron– tems consist of a catalytic component and a specific reductase, sulfur clusters, neither of which contain molybdenum, have been which, in the standard system, are referred to as the MoFe identified on NifEN: a permanent cluster and a transient ¶ protein and the Fe protein. The MoFe protein contains two FeMoco precursor (19, 21). The permanent clusters, alone, are ⌬ unique, high-nuclearity metal clusters: the [8Fe–7S] P-cluster, present on nifB NifEN, which has an iron content of 8 mol of ϭ ͞ which functions in electron transfer, and the iron-molybdenum iron per mol of protein and a signature S 1 2 EPR signal that cofactor (FeMoco), which is the site of substrate reduction. indicates the presence of two [4Fe–4S]-like clusters (19, 22). FeMoco consists of fused [Mo–3Fe–3S] and [4Fe–3S] subcu- NifEN has an iron content of 16 mol of iron per mol of protein ϭ ͞ banes that are bridged by three additional sulfides and share a and displays an additional, distinct S 1 2 EPR signal (19). The extra iron on NifEN is believed to comprise a FeMoco precursor central ␮6-light atom of unknown identity (see Fig. 1) (6–8). At the cluster termini, FeMoco is externally coordinated by two because NifEN can activate apo-MoFe protein upon addition of ⌬ protein residues and an organic homocitrate entity. Some or- Fe protein, MgATP, homocitrate, and molybdate, whereas nifB ganisms can express additional genetically distinct nitrogenase NifEN cannot (19). systems having catalytic components with either an iron– vanadium cofactor (FeVco) or an iron-only cofactor (FeFeco) Conflict of interest statement: No conflicts declared. (9, 10), however, the molybdenum-based system is the most efficient and best-characterized. This paper was submitted directly (Track II) to the PNAS office. Based largely on the genetic analysis of Azotobacter vinelandii, Abbreviations: FeMoco, iron-molybdenum cofactor; FeFeco, iron-only cofactor; FeVco, iron-vanadium cofactor; XAS, x-ray absorption spectroscopy; EXAFS, extended x-ray ab- it its known that FeMoco is first assembled and then inserted into sorption fine structure. the MoFe protein in a complicated process requiring several ‡To whom correspondence may be addressed. E-mail: [email protected], hedman@ssrl. nitrogen fixation gene (nif) products (see reviews in refs. 11–15). slac.stanford.edu, or [email protected]. The pathway for FeMoco assembly is separate from that of ¶The concentration of molybdenum in the purified MoFe protein in this study was previ- FeVco and FeFeco, yet all three cofactors may originate from a ously determined to be 1.8 Ϯ 0.1 mol of molybdenum per mol of protein (19); ⌬nifB MoFe common precursor formed by the NifB protein (9). This protein, protein, NifEN, and ⌬nifB NifEN were found to have Ͻ0.1 mol of molybdenum per mol of protein (19, 39). The iron concentrations of the purified in this study, expressed which is required for all of the nitrogenase systems, has been as mol of iron per mol of protein, were determined to be 29.5 Ϯ 2.0, 18.1 Ϯ 1.9, 16.1 Ϯ 2.4, shown to contain an unusual iron–sulfur cluster and to be the and 8.5 Ϯ 1.0 for MoFe protein, ⌬nifB MoFe protein, NifEN, and ⌬nifB NifEN, respectively source of the iron that eventually comprises FeMoco in the (19, 39). MoFe protein (16, 17). For FeMoco synthesis, the precursor © 2006 by The National Academy of Sciences of the USA

1238–1243 ͉ PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507853103 Downloaded by guest on September 26, 2021 Fig. 1. Structure of FeMoco in the MoFe protein and structural models consistent with the XAS analysis of the NifEN-bound FeMoco precursor [adapted from the crystallographic coordinates of the MoFe protein (6)]. The atoms are molybdenum (purple), iron (green), sulfur (orange), oxygen (red), nitrogen (blue), (gray), and X (black), which may be carbon, nitrogen, or oxygen. In the 8Fe and 7Fe models for the NifEN-bound FeMoco precursor, sulfur is shown as the ligand to one of the terminal iron atoms because the Cys residue that ligates FeMoco in the MoFe protein is conserved in the NifE Fig. 2. Iron K-edge EXAFS of MoFe protein (purple), ⌬nifB MoFe protein primary sequence (20). The ligand at the opposite terminal iron atom is not (green), and MoFe protein-bound FeMoco (data in dotted black with best fit given because it could be any number of light-atom ligands, including Asn, in orange) (A) and NifEN (gray), ⌬nifB NifEN (blue), and the NifEN-bound which replaces the FeMoco His ligand in the NifE sequence (20). FeMoco precursor (data in dotted black with best fit in red) (B) with associated Fourier transforms of MoFe protein-bound FeMoco (data in dotted black with best fit in orange) (C) and the NifEN-bound FeMoco precursor (data in dotted The presence of two different iron-containing clusters on black with best fit in red) (D). The MoFe protein-bound FeMoco data were NifEN presents a problem for effective XAS analysis of the obtained by subtraction of ⌬nifB MoFe protein EXAFS from wild-type MoFe FeMoco precursor because both types of clusters contribute to protein EXAFS in a ratio of 8͞7:15͞7. The NifEN-bound precursor data were an iron K-edge data set. To structurally characterize the pre- obtained by subtraction of ⌬nifB NifEN EXAFS from NifEN EXAFS in a 1:2 ratio. cursor on NifEN without interference from the permanent Fourier transforms for all spectra are shown in Fig. 4. clusters, a method was devised to remove the signal common to both NifEN and ⌬nifB NifEN by subtracting ⌬nifB NifEN XAS short-range iron–iron distances, 3.3 at 2.66 Å and 0.5 at 2.88 Å, data from NifEN XAS data after scaling both data sets based on and a total of 1.6 long-range iron–iron distances split between the respective iron contents of the two proteins. This method was 3.68 Å and 3.80 Å (see Table 1). The strength of the short- and tested by first using it to analyze MoFe protein, which is well long-range iron–iron components suggests that the NifEN- characterized by x-ray crystallography and similarly has two bound precursor is a high-nuclearity cluster. To find the struc- iron-containing clusters. ture that best matches these fit parameters, structural models A data set representing MoFe protein-bound FeMoco was generated by subtracting the iron K-edge XAS data of A. were derived from all of the current known high-nuclearity vinelandii ⌬nifB MoFe protein, which contains P-clusters but not iron–sulfur and mixed metal–sulfur cluster types (see refs. 18 and FeMoco (23), from that of wild-type A. vinelandii MoFe protein 25 for examples). Few of these models, including the bridged ina8͞7:15͞7 ratio (Fig. 2A; see also Fig. 3A). The EXAFS- bicubane structures commonly suggested as FeMoco precursors derived distances for the average iron environment in MoFe (14, 18, 25), had features consistent with the high number and protein-bound FeMoco obtained by this method (see Table 1) relatively short lengths of the long-range iron–iron scattering are consistent with those derived from both crystallography components found in the EXAFS analysis of the NifEN-bound of wild-type MoFe protein (Fe–S at 2.24 Ϯ 0.03 Å, Fe–Fe at precursor (see Table 1). The long-range iron–iron scattering Ϸ 2.63 Ϯ 0.03 Å, Fe–Mo at 2.70 Ϯ 0.03 Å, and Fe–Fe at 3.70 Ϯ 0.03 components comprise an intense peak at 3.5 Å in the Fourier Å) (6) and iron K-edge EXAFS of isolated FeMoco (24). The transform of the NifEN-bound precursor EXAFS data (Fig. 2D) correspondence between the results reported here for MoFe similar to one found in the Fourier transform for MoFe protein- protein-bound FeMoco with those reported previously for bound FeMoco (Fig. 2C) due to intercubane scattering between FeMoco by other methods indicates the suitability of the sub- the six iron atoms at the FeMoco core. This peak is absent from traction method to the analysis of the NifEN-bound FeMoco the ⌬nifB NifEN data, consistent with the presence of normal precursor cluster. Note that because the EXAFS technique is not [4Fe–4S] clusters having no long-range order, and from the sensitive enough to detect a small amount of scattering from a ⌬nifB MoFe protein data because the cross-cluster iron–iron light atom against a background of heavy atom scatterers, the scattering in the P-cluster is less ordered and occurs at a longer

EXAFS results cannot confirm the presence or absence of a distance with a lower coordination number than in FeMoco (see BIOCHEMISTRY central light atom in MoFe protein-bound FeMoco, nor would Fig. 4). Since its initial observation in the EXAFS Fourier it be possible to detect a similar atom in the NifEN-bound transform of isolated FeMoco, this peak has been regarded as a precursor should it exist. signature feature representative of the unique long-range order EXAFS spectra of NifEN, ⌬nifB NifEN, and the NifEN-bound in the FeMoco structure (24, 26–28). The occurrence of this precursor (prepared by a 1:2 subtraction of the former from the signature peak in the data for the NifEN-bound precursor latter) are shown in Fig. 2B; the Fourier transforms of these together with the EXAFS-fit results imply that the precursor has spectra are shown in Fig. 2D and Fig. 4, which is published as the same long-range order found in FeMoco. supporting information on the PNAS web site. The NifEN- A FeMoco-like structure having six, seven, or eight iron atoms bound precursor EXAFS data are best fit with a total of 3.8 could reproduce these long-range features, however, the inten-

Corbett et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1239 Downloaded by guest on September 26, 2021 Table 1. Final EXAFS fit results over a k range of 2–16 Å؊1 NifEN-bound precursor

MoFe protein-bound FeMoco 8Fe model 7Fe model

Fit parameter NR,A˚ ␴2,A˚ 2 NR,A˚ ␴2,A˚ 2 NR,A˚ ␴2,A˚ 2

Scatterer Fe–S 3.1 2.24 0.0048 3.1 2.28 0.0036 3.1 2.28 0.0035 Short Fe–Fe 3.4 2.60 0.0055 3.3 2.66 0.0058 3.1 2.65 0.0052 Short Fe–Fe — — — 0.5 2.88 0.0039 0.3 2.89 0.0023 Fe–Mo 0.4 2.73 0.0010 — — — — — — Long Fe–Fe 1.7 3.68 0.0034 0.8 3.68 0.0008 0.9 3.68 0.0010 Long Fe–Fe — — — 0.8 3.80 0.0023 0.9 3.80 0.0026

⌬E0,eV Ϫ9.8 Ϫ9.0 Ϫ10.6 F 0.264 0.210 0.222

Additional fit results are given in Table 3, which is published as supporting information on the PNAS web site. The variables are coordination number, N; interatomic distance, R; mean-square thermal and static deviation in R, ␴2; and the shift in the threshold energy 2 2 from 7,130 eV, ⌬E0. The estimated uncertainties in R, ␴ , and N are Ϯ0.02 Å, Ϯ0.0001 Å , and Ϯ20%, respectively. The goodness of fit 6 2 6 2 1͞2 is described by F, where F ϭ [⌺k (␹exptl Ϫ ␹calcd) ͞⌺k ␹exptl ] . The MoFe protein-bound FeMoco data were obtained by subtraction of ⌬nifB MoFe protein EXAFS from wild-type MoFe protein EXAFS in a ratio of 8͞7:15͞7. The NifEN-bound precursor data were obtained by subtraction of ⌬nifB NifEN EXAFS from NifEN EXAFS in the ratio of 1:2 for the 8Fe model or 8͞7:15͞7 for the 7Fe model. —, not included in fit.

sity of the short-range iron–iron scattering precludes a six-iron pre-edge spectra of MoFe protein-bound FeMoco and the cluster. A seven-iron cluster (Fig. 1, 7Fe model) is consistent NifEN-bound precursor consist of at least two well spaced peaks with the data, but fits based on this model (see Table 1) are worse centered at 7,112.1 and 7,113.6 eV (Fig. 3 and Table 2). These than those based on a model having eight iron atoms (Fig. 1, 8Fe spectra differ from those of typical protein-bound iron–sulfur model). Although the 7Fe model cannot be excluded on this clusters with tetrahedral iron site geometries, such as those of the basis, the 8Fe model is considered to be the more likely structure P-cluster-containing ⌬nifB MoFe protein and the [4Fe–4S]- because it is a better match both to the EXAFS fit results and containing ⌬nifB NifEN protein (Fig. 3 and Table 2), which previous biochemical studies (19). Overall, the fit parameters consist of a single broad peak or multiple closely spaced peaks suggest that the NifEN-bound precursor is structurally similar to centered at Ϸ7,112.2 eV (35). According to the crystal structure FeMoco but with a terminal iron atom in place of molybdenum of the MoFe protein (6), the predominate geometry of the iron and slightly elongated interatomic distances (see Fig. 1, 8Fe sites in FeMoco is intermediate between tetrahedral and trigonal model). Homology modeling and sequence alignments suggest pyramidal. This unconventional geometry is probably the source that this type of cluster would be easily accommodated in the of the observed spectral differences between MoFe protein- NifEN polypeptide environment, which is predicted to be very bound FeMoco and the more regular clusters of the ⌬nifB open with less-bulky amino acids than those found in the MoFe protein environment (29). The lone residue in this region of NifE, Cys-E250, is homologous to Cys-␣275 in the MoFe protein primary sequence and is therefore believed to serve as the ligand to the terminal iron atom of the NifEN-bound precursor in a manner similar to that observed between Cys- ␣275 and FeMoco in the MoFe protein (20, 29). The EXAFS-derived 8Fe model for the NifEN-bound FeMoco precursor is structurally homologous to the cluster proposed as the catalytic cofactor in the alternative iron-only nitrogenase (FeFeco) (9, 10) and is a good match to both the EXAFS results from this protein (27) and theoretically calcu- lated distances for FeFeco (30–32).ʈ In accordance with the observation of longer distances in the NifEN-bound precursor compared with MoFe protein-bound FeMoco, density func- tional theory calculations predict that FeFeco is less stable than its molybdenum-containing counterpart and therefore should have a larger cluster volume (32, 33). Further support for the assignment of the 8Fe or 7Fe model as the NifEN-bound precursor is provided by analysis of the x-ray Fig. 3. Normalized iron K-edge pre-edge spectra (Upper) and smoothed absorption pre-edge region. This region of the x-ray absorption second derivatives (Lower) of MoFe protein (dotted purple), ⌬nifB MoFe spectrum has been shown to be extremely sensitive to the protein (dashed green), and MoFe protein-bound FeMoco (solid orange) (A) geometry at an average iron site in a molecule, allowing for the and of NifEN (dotted gray), ⌬nifB NifEN (dashed blue), and the NifEN-bound differentiation of four-, five-, and six-coordinate complexes or FeMoco precursor (solid red) (B). The MoFe protein-bound FeMoco data were tetrahedral and square-planar geometries, for example (34). The obtained by subtraction of normalized ⌬nifB MoFe protein edge data from normalized wild-type MoFe protein edge data in a ratio of 8͞7:15͞7. The NifEN-bound precursor data were obtained by subtraction of normalized ⌬ ʈThe Mo¨ssbauer spectrum of the Fe-only nitrogenase has been interpreted to indicate that nifB NifEN edge data from normalized NifEN edge data in a 1:2 ratio. Full a central atom must be present in FeFeco, although there is no structural evidence of its edge spectra are shown in Fig. 5, which is published as supporting information occurrence (46). on the PNAS web site.

1240 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507853103 Corbett et al. Downloaded by guest on September 26, 2021 Table 2. Pre-edge fit results Peak 1 Peak 2

Data set Energy, eV Intensity Energy, eV Intensity Total intensity

⌬nifB MoFe protein 7,112.18 (0.03) 10.4 (0.8) — — 10.4 (0.8) ⌬nifB NifEN protein 7,112.25 (0.02) 9.2 (0.9) — — 9.2 (0.9) MoFe protein-bound FeMoco 7,112.11 (0.01) 6.7 (0.5) 7,113.61 (0.01) 11.5 (1.6) 18.2 (1.1) NifEN-bound precursor 7,112.06 (0.01) 7.6 (0.5) 7,113.56 (0.04) 6.4 (0.5) 14.0 (0.3)

Values are reported as averages from all fits to a data set with standard deviations in parentheses. Peak intensity is defined as the peak area (the product of peak amplitude and half-width) multiplied by 100 (29). MoFe protein-bound FeMoco data were obtained by subtraction of normalized ⌬nifB MoFe protein edge data from normalized wild-type MoFe protein edge data in a ratio of 8͞7:15͞7. NifEN-bound FeMoco precursor data were obtained by subtraction of normalized ⌬nifB NifEN edge data from normalized NifEN edge data in a 1:2 ratio. —, Two distinct peaks cannot be fit to the spectra of the ⌬nifB proteins at the resolution of this experiment (Ϸ1.2 eV).

proteins. Although the complexity of the clusters in these Transformation to FeVco would occur by simple insertion of proteins prohibits a detailed pre-edge analysis, the greater vanadium instead of molybdenum, whereas the cluster could correspondence between the pre-edge features of the NifEN- proceed as-is for homocitrate attachment and insertion into the bound precursor and MoFe protein-bound FeMoco, compared Fe-only protein. with that between the NifEN-bound precursor and the ⌬nifB For over 25 years, significant efforts have been focused on proteins, suggests that the average iron site geometry in the chemically synthesizing structural analogs of the nitrogenase precursor is more similar to the FeMoco geometry than that of clusters (18, 25). Despite recent advances leading to successful a typical iron–sulfur cluster. Importantly, all of the structural P-cluster topologs, no FeMoco-like clusters have been synthe- models considered except those based on a FeMoco core (see sized (18). The evidence presented here of a molybdenum-free Fig. 1) have tetrahedral iron site geometries and are therefore FeMoco analog in the FeMoco biogenesis pathway may prove inconsistent with the pre-edge analysis of the NifEN-bound instrumental in the development of a strategy for the chemical FeMoco precursor. synthesis of FeMoco and future opportunities for a detailed The assignment of a molybdenum-free FeMoco-like cluster analysis of this important yet enigmatic cluster. (see Fig. 1, 8Fe and 7Fe models) as the NifEN-bound FeMoco precursor is strongly supported by XAS results that are consis- Materials and Methods tent with an iron- and sulfur-containing cluster having at least Unless otherwise noted, all chemicals and reagents were ob- seven iron atoms, the same long-range order as FeMoco, and a tained from Fisher, Baxter Scientific Products (McGaw Park, similar average iron site geometry as FeMoco. This precursor is IL), or Sigma. formed in a strain of A. vinelandii lacking the structural genes for both the MoFe and Fe proteins and therefore represents the Cell Growth and Protein Purification. All A. vinelandii strains were state of FeMoco assembly up to the point where intervention of grown in 180-liter batches in a 200-liter New Brunswick fermen- the Fe protein (NifH) is required. Because the FeMoco archi- tor on Burke’s minimal medium supplemented with 2 mM tecture is already in place at this point, no further rearrangement ammonium acetate. The growth rate was measured by cell of the cluster is necessary. Thus the role of the Fe protein in density at 436 nm with a Spectronic (Westbury, NY) 20 Genesys FeMoco assembly cannot be to trigger a change in cluster Spectrophotometer. After consumption, the cells were structure, as has been previously suggested (14); instead, it is derepressed for 3 h and harvested with a flow-through centrif- more likely that the Fe protein is involved in another suggested ugal harvester (Cepa, Lahr, Germany). The cell paste was role, such as mediating heterometal insertion or homocitrate washed with 50 mM Tris⅐HCl, pH 8.0. Published methods were attachment (13). Furthermore, the occurrence of a FeMoco-like used for the purification of wild-type A. vinelandii MoFe protein cluster at this stage in the biosynthetic pathway indicates a (38) and the His-tagged MoFe proteins expressed by A. vine- considerable role for NifB in the formation of this distinctive landii DJ1143 (⌬nifB MoFe protein) (39), DJ1041 (NifEN structure and potentially a new synthetic route to bridged protein) (19, 22), and YM9A (⌬nifB NifEN protein) (19). All metalloclusters that relies on radical chemistry at the S- protein samples were prepared in a Vacuum Atmospheres adenosylmethonine domain of NifB (36). (Hawthorne, CA) Ar-filled dry box with Ͻ4 ppm O2 and Although it has long been known that NifEN acts as a scaffold concentrated in a Centricon-30 (Amicon) concentrator in an- by which potentially multiple FeMoco processing steps take aerobic centrifuge tubes outside the dry box to final concentra- place, the exact nature, number, and sequence of these steps tions of 53 mg͞ml MoFe protein (6 mM in Fe), 33 mg͞ml ⌬nifB have remained obscure (11–15). The results presented here MoFe protein (2 mM in Fe), 80 mg͞ml NifEN protein (5 mM in indicate that the first isolatable FeMoco precursor on NifEN is Fe), and 79 mg͞ml ⌬nifB NifEN protein (3 mM in Fe). Samples a molybdenum-free analog of FeMoco. The most likely trajec- for XAS analysis in Tris⅐HCl, pH 8.0͞250 mM imidazole͞500 tory for FeMoco assembly from this state, which is best repre- mM NaCl͞2 mM dithionite͞50% glycerol were loaded into 70-␮l sented by the 8Fe model (Fig. 1), would be loss of the non- Lucite cells with Kapton tape windows and flash-frozen in a ͞

cysteine-ligated terminal iron atom followed by incorporation of pentane liquid nitrogen slush. BIOCHEMISTRY molybdenum in its place. These types of reactions are well documented in synthetic and protein systems where site- XAS Data Collection. XAS data were measured at the Stanford differentiated [4Fe–4S] clusters commonly lose iron upon oxi- Synchrotron Radiation Laboratory under 3-GeV, 80- to 100-mA dation to form stable [3Fe–4S] clusters, which can, in turn, be beam conditions using beam line 9-3 with a Si(220) double- reconstituted upon reduction with either Fe(II) or other thio- crystal monochromator and two rhodium-coated mirrors: one philic metal ions (37). Consistent with the common requirement flat premonochromator mirror for harmonic rejection and ver- for NifB in all of the nitrogenase systems (9), the precursor tical collimation and one toroidal postmonochromator mirror structure proposed here for FeMoco could reasonably act as a for focusing. The samples were maintained at 10 K during data precursor for the three different nitrogenase cofactor types. collection in an Oxford Instruments CF1208 continuous-flow

Corbett et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1241 Downloaded by guest on September 26, 2021 liquid helium cryostat. Data were collected as iron K␣ fluores- initio theoretical phase and amplitude functions used in the cence using a Canberra 30-element solid-state germanium de- fitting program were generated by FEFF 7.0 (42, 43). Theoretical tector. Radiation from elastic͞inelastic scattering and iron K␤ functions were calculated separately for the MoFe protein- fluorescence was minimized at the detector by placing a set of bound FeMoco and NifEN-bound precursor data sets from input Soller slits with a manganese filter between the cryostat and the models based on the crystallographic coordinates of FeMoco in detector. The x-ray energy was calibrated to the inflection point the MoFe protein for the former or the same coordinates at 7,111.2 eV of a standard iron foil measured concurrent with modified to replace molybdenum with iron for the latter (6). The the samples. Changes in the edge position over time, which would use of theoretical functions calculated from the coordinates of indicate photoreduction of an oxidized metal site, were not other high-nuclearity clusters, such as [Fe8S8(SL)4] (44), observed for any sample. [(Fe4S4)2(␮2-S)2(SL)4] (45), and the P-cluster (6), did not affect the distances determined by the EXAFS fitting process for the XAS Data Analysis. The program XFIT (40) was used to process the NifEN-bound precursor. raw data from MoFe protein, ⌬nifB MoFe protein, NifEN, and The energies and intensities of the peaks in the pre-edge ⌬nifB NifEN. First, a polynomial background absorption curve spectra were quantified by using the methodology of Westre et was fit to the pre-edge region and extended over the post-edge al. (34) and the least-squares fitting program EDG࿝FIT, which is with weighted control points. Next, a four-segment polynomial part of the EXAFSPAK program suite (41). The pre-edge features spline was fit over the EXAFS region. The spectra were then were modeled as peaks composed of Lorentzian and Gaussian normalized to have an edge jump of 1.0 between the background functions in a fixed 1:1 ratio for which the energy, amplitude, and and spline curves at 7,130 eV. The MoFe protein-bound FeMoco half-width were allowed to vary. The background absorption was data were obtained by subtraction of ⌬nifB MoFe protein data empirically modeled as a single peak composed of Lorentzian from wild-type MoFe protein data in the ratio of 8͞7:15͞7. and Gaussian functions for which the mixing ratio was allowed Unless otherwise noted, the NifEN-bound FeMoco precursor to vary along with the other peak parameters. In all cases, the data were obtained by subtraction of ⌬nifB NifEN data from data were fit over three energy ranges: 7,108–7,116 eV, 7,108– NifEN data in a 1:2 ratio. The edge and EXAFS regions were 7,117 eV, and 7,108–7,118 eV using multiple background func- subtracted separately. tions. The fits were constrained to match a smoothed second EXAFS data from MoFe protein-bound FeMoco and the derivative of the data. NifEN-bound precursor over a k range of 2–16 ÅϪ1 were fit by using the nonlinear least squares fitting program, OPT, from the We thank Prof. R. H. Holm (Harvard University, Cambridge, MA) for EXAFSPAK program suite (41). During the fitting process, the insightful discussions and Prof. D. Dean (Virginia Polytechnic Institute, variables for interatomic distance (R) and mean-square thermal Blacksburg) for kindly providing A. vinelandii strain DJ1041. This work and static deviation in R (␴2) were allowed to vary for all was supported by National Institutes of Health Grants RR-01209 (to K.O.H.) and GM-67626 (to M.W.R.). M.C.C. was supported by scattering components. The shift in the threshold energy (⌬E0) was also varied for each fit but constrained to be the same for all an Evelyn Liang McBain Fellowship. XAS data were measured at components. The amplitude reduction factor (S 2) was fixed to the Stanford Synchrotron Radiation Laboratory, which is supported by 0 the U.S. Department of Energy, Office of Basic Energy Sciences. The a value of 1.0 for all fits. Coordination numbers (N) were fixed Stanford Synchrotron Radiation Laboratory Structural Molecular Biol- to values established by the input models. When compound ogy Program is supported by the National Institutes of Health, National scatterers of a single type were used, the sum was fixed to the Center for Research Resources, Biomedical Technology Program and by model value while different ratios of the two components were the U.S. Department of Energy, Office of Biological and Environmental iteratively tested to find the best empirical fit to the data. The ab Research.

1. Howard, J. B. & Rees, D. C. (1996) Chem. Rev. 96, 2965–2982. 19. Hu, Y., Fay, A. W. & Ribbe, M. W. (2005) Proc. Natl. Acad. Sci. USA 102, 2. Burgess, B. K. & Lowe, D. J. (1996) Chem. Rev. 96, 2983–3011. 3236–3241. 3. Smith, B. E. (1999) Adv. Inorg. Chem. 47, 159–218. 20. Brigle, K. E., Weiss, M. C., Newton, W. E. & Dean, D. R. (1987) J. Bacteriol. 4. Igarashi, R. Y. & Seefeldt, L. C. (2003) Crit. Rev. Biochem. Mol. Biol. 38, 169, 1547–1553. 351–384. 21. Roll, J. T., Shah, V. K., Dean, D. R. & Roberts, G. P. (1995) J. Biol. Chem. 270, 5. Rees, D. C., Tezcan, F. A., Haynes, C. A., Walton, M. Y., Andrade, S., Einsle, 4432–4437. O. & Howard, J. B. (2005) Philos. Trans. R. Soc. London A 363, 971–984. 22. Goodwin, P. J., Agar, J. N., Roll, J. T., Roberts, G. P., Johnson, M. K. & Dean, 6. Einsle, O., Tezcan, F. A., Andrade, S. L. A., Schmid, B., Yoshida, M., Howard, D. R. (1998) Biochemistry 37, 10420–10428. J. B. & Rees, D. C. (2002) Science 297, 1696–1700. 23. Schmid, B., Ribbe, M. W., Einsle, O., Yoshida, M., Thomas, L. M., Dean, D. R., 7. Noodleman, L., Lovell, T., Han, W.-G., Li, J. & Himo, F. (2004) Chem. Rev. Rees, D. C. & Burgess, B. K. (2002) Science 296, 352–356. 104, 459–508. 24. Arber, J. M., Flood, A. C., Garner, C. D., Gormal, C., Hasnain, S. S. & Smith, 8. Yang, T.-C., Maeser, N. K., Laryukhin, M., Lee, H.-I., Dean, D. R., Seefeldt, B. E. (1988) Biochem. J. 252, 421–425. L. C. & Hoffman, B. M. (2005) J. Am. Chem. Soc. 127, 12804–12805. 25. Malinak, S. M. & Coucouvanis, D. (2001) Prog. Inorg. Chem. 49, 599–662. 9. Eady, R. R. (1996) Chem. Rev. 96, 3013–3030. 26. Chen, J., Christiansen, J., Tittsworth, R. C., Hales, B. J., George, S. J., 10. Eady, R. R. (2003) Coord. Chem. Rev. 237, 23–30. Coucouvanis, D. & Cramer, S. P. (1993) J. Am. Chem. Soc. 115, 11. Ludden, P. W., Shah, V. K., Roberts, G. P., Ru¨ttimann-Johnson, C., Rangaraj, 5509–5515. P., Foulger, T., Allen, R. M., Homer, M., Roll, J., Zhang, X., et al. (1998) in 27. Krahn, E., Weiss, B. J. R., Kro¨ckel, M., Groppe, J., Henkel, G., Cramer, S. P., Biological Nitrogen Fixation for the 21st Century, eds. Elmerich, C., Kondorosi, Trautwein, A. X., Schneider, K. & Mu¨ller, A. (2002) J. Biol. Inorg. Chem. 7, 37–45. A. & Newton, W. E. (Kluwer, Dordrecht, The Netherlands), pp. 33–37. 28. Christiansen, J., Tittsworth, R. C., Hales, B. J. & Cramer, S. P. (1995) J. Am. 12. Frazzon, J. & Dean, D. R. (2002) in Metal Ions in Biological Systems, eds. Sigel, Chem. Soc. 117, 10017–10024. A. & Sigel, H. (Dekker, New York), pp. 163–186. 29. Muchmore, S. W., Jack, R. F. & Dean, D. R. (1996) in Mechanisms of 13. Rubio, L. M. & Ludden, P. W. (2002) in Nitrogen Fixation at the Millennium, Metallocluster Assembly, eds. Hausinger, R. P., Eichhorn, G. L. & Marzilli, L. G. ed. Leigh, G. J. (Elsevier Science, Amsterdam), pp. 101–136. (VCH, New York), pp. 111–133. 14. Dos Santos, P. C., Dean, D. R., Hu, Y. & Ribbe, M. W. (2004) Chem. Rev. 104, 30. Lovell, T., Torres, R. A., Han, W.-G., Liu, T., Case, D. A. & Noodleman, L. 1159–1173. (2002) Inorg. Chem. 41, 5744–5753. 15. Rubio, L. M. & Ludden, P. W. (2005) J. Bacteriol. 187, 405–414. 31. Cao, Z., Zhou, Z., Wan, H., Zhang, Q. & Thiel, W. (2003) Inorg. Chem. 42, 16. Shah, V. K., Allen, J. R., Spangler, N. J. & Ludden, P. W. (1994) J. Biol. Chem. 6986–6988. 269, 1154–1158. 32. Hinnemann, B. & Nørskov, J. K. (2004) Phys. Chem. Chem. Phys. 6, 843–853. 17. Allen, R. M., Chatterjee, R., Ludden, P. W. & Shah, V. K. (1995) J. Biol. Chem. 33. Plass, W. (1994) J. Mol. Struct. 315, 53–62. 270, 26890–26896. 34. Westre, T. E., Kennepohl, P., DeWitt, J. G., Hedman, B., Hodgson, K. O. & 18. Lee, S. C. & Holm, R. H. (2004) Chem. Rev. 104, 1135–1158. Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 6297–6314.

1242 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507853103 Corbett et al. Downloaded by guest on September 26, 2021 35. Musgrave, K. B., Angove, H. C., Burgess, B. K., Hedman, B. & Hodgson, K. O. 41. George, G. N. (1990) EXAFSPAK (Stanford Synchrotron Radiation Laboratory, (1998) J. Am. Chem. Soc. 120, 5325–5326. Stanford, CA). 36. Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. 42. Mustre de Leon, J., Rehr, J. J. & Zabinsky, S. I. (1991) Phys. Rev. B 44, (2001) Nucleic Acids Res. 29, 1097–1106. 4146–4156. 37. Johnson, M. K., Duderstadt, R. E. & Duin, E. C. (1999) Adv. Inorg. Chem. 47, 1–82. 43. Rehr, J. J. & Albers, R. C. (2000) Rev. Mod. Phys. 72, 621–654. 38. Burgess, B. K., Jacobs, D. B. & Stiefel, E. I. (1980) Biochim. Biophys. Acta 614, 44. Zhou, H.-C. & Holm, R. H. (2003) Inorg. Chem. 42, 11–21. 196–209. 45. Huang, J., Mukerjee, S., Segal, B. M., Akashi, H., Zhou, J. & Holm, R. H. 39. Ribbe, M. W., Hu, Y., Guo, M., Schmid, B. & Burgess, B. K. (2002) J. Biol. (1997) J. Am. Chem. Soc. 119, 8662–8674. Chem. 277, 23469–23476. 46. Vrajmasu, V., Mu¨nck, E. & Bominaar, E. L. (2003) Inorg. Chem. 42, 40. Ellis, P. J. & Freeman, H. C. (1995) J. Synchrotron Rad. 2, 190–195. 5974–5988. BIOCHEMISTRY

Corbett et al. PNAS ͉ January 31, 2006 ͉ vol. 103 ͉ no. 5 ͉ 1243 Downloaded by guest on September 26, 2021