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Structural Insights Into a Protein-Bound Iron-Molybdenum Cofactor Precursor

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Structural Insights Into a Protein-Bound Iron-Molybdenum Cofactor Precursor Structural insights into a protein-bound iron-molybdenum cofactor 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 nitrogenase 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 Azotobacter vinelandii strains express- FeMoco biosynthesis 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 ⌬ bioinorganic chemistry 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 nitrogen fixation, 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 proteins 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), carbon (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.
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