Biochemistry 2005, 44, 12955-12969
12955
NifS-Mediated Assembly of [4Fe-4S] Clusters in the N- and C-Terminal Domains of the NifU Scaffold Protein† Archer D. Smith,‡ Guy N. L. Jameson,§ Patricia C. Dos Santos,| Jeffrey N. Agar,‡ Sunil Naik,§ Carsten Krebs,§ Jeverson Frazzon,| Dennis R. Dean,| Boi Hanh Huynh,*,§ and Michael K. Johnson*,‡ Department of Chemistry and Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602, Department of Physics, Emory UniVersity, Atlanta, Georgia 30322, and Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061 ReceiVed June 30, 2005; ReVised Manuscript ReceiVed August 2, 2005
ABSTRACT: NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S]2+,+ cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in Vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and Mo¨ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S]2+ and [4Fe-4S]2+ clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S]2+ clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in Vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S]2+ clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.
Clusters of iron and inorganic sulfide are one of the most common and functionally diverse types of prosthetic groups in all of biology, with roles ranging from electron transfer and catalysis to sensing and regulation (1, 2). Moreover, the importance of this class of protein prosthetic group has generated considerable interest in understanding how they are fabricated within the cell. Major progress has been made in the past decade in understanding the complex process of Fe-S cluster biosynthesis using both in ViVo and in Vitro approaches (for recent reviews see refs 3-6). Three distinct types of biosynthetic machinery have emerged, termed NIF (nitrogen fixation) (7), ISC (iron-sulfur cluster) (8), and SUF (sulfur utilization factor) (9). However, following the model originally established for the NIF system (10, 11), each is believed to use a similar overall mechanism involving pyridoxal phosphate (PLP)1-dependent cysteine desulfurases (NifS/IscS/SufS) that supplies the inorganic sulfur for the biosynthesis of [2Fe-2S] or [4Fe-4S] clusters on a variety of scaffold proteins (NifU/IscU/SufU, NifIscA/IscA/SufA, and † This work was supported by grants from the National Institutes of Health (GM62542 to M.K.J. and GM47295 to B.H.H.) and the National Science Foundation (MCB-021138 to D.R.D.). * To whom correspondence should be addressed. Telephone: 404727-4295. Fax: 404-727-0873. E-mail:
[email protected] (B.H.H.); Telephone: 706-542-9378. Fax: 706-542-2353. E-mail: johnson@ chem.uga.edu (M.K.J.). ‡ University of Georgia. § Emory University. | Virginia Tech. 1 Abbreviations: DTT, dithiothreitol; PMSF, phenyl methyl sulfonyl fluoride; PLP, pyridoxal phosphate.
Nfu type). The preassembled clusters are generally believed to be transferred intact into apo Fe-S proteins, but thus far, this transfer has only been well-documented for [2Fe-2S] clusters (12-16). The NIF system comprises NifS, NifU, and NifIscA (10, 11, 17, 18) and functions primarily for the assembly of Fe-S clusters to be used in the maturation of the nitrogenase component proteins and hence is present in nitrogen-fixing bacteria. However, other NifS- and NifU-like proteins can also function in general Fe-S cluster biosynthesis in prokaryotes and eukaryotes, as evidenced by characterization of NifS and NifU homologues in nondiazotrophic bacteria, such as Helicobacter pylori (19), and in the anaerobic, amitochondrial protozoan parasite, Entamoeba histolytica (20). The ISC system is composed of IscS, IscU, IscA, Fdx (a [2Fe-2S] Fd), and two heat-shock cognate proteins, HscA and HscB (8, 21). This type of assembly machinery is very widespread, and partial or complete sets of the ISC components are conserved from bacteria to higher eukaryotes (36). Moreover, the involvement of HscA and HscB in the ISC system is particularly intriguing, because they exhibit ATPase activity and high-sequence homology to the family of molecular chaperones represented by the DnaK and DnaJ partner proteins. HscA and HscB have been shown to act in concert to bind IscU (22-25) and are proposed to facilitate cluster transfer from IscU to acceptor proteins in an ATPdependent process (26). The SUF system comprises SufA, SufU, SufB, SufC, SufD, SufS, and SufE and is found, in whole or in part, in a wide range of bacteria, archaea and
10.1021/bi051257i CCC: $30.25 © 2005 American Chemical Society Published on Web 09/10/2005
12956 Biochemistry, Vol. 44, No. 39, 2005
Smith et al.
Scheme 1: Primary Sequence and Modular Organization of A. Vinelandii NifUa
a Conserved cysteine and aspartate residues that are potential cluster ligands are highlighted in yellow and purple, respectively. Residues corresponding to NifU-1 and NifU-2 are overlined and underlined, respectively. The N-terminal, central, and C-terminal domains of NifU are indicted in green, red, and lilac, respectively.
plastids (4, 9, 27). SufBCD constitutes an unorthodox ATPase related to the ABC superfamily that stimulates the cysteine desulfurase activity of SufS, along with SufE (28, 29). SufE has recently been shown to have structural homology with IscU (30, 31) and to function as a sulfurtransferase that facilitates transfer of sulfane sulfur (S0) from SufS to acceptor proteins (29, 32). On the basis of the composition of operons (4, 9) or in Vitro studies (33, 34), SufA or SufU are likely to provide scaffold proteins for the SUF machinery in many organisms, but both are absent in many archaea that contain SufBCD (9). Thus far, three distinct types of Fe-S cluster scaffold proteins with distinct arrangements of cluster-ligating cysteine residues have been identified on the basis of sequence homology and in Vitro cluster assembly and/or transfer studies, and each has been shown to be able to accommodate [2Fe-2S]2+ or [4Fe-4S]2+ clusters. The A-type motif found in NifIscA, IscA, and SufA has three conserved cysteine residues in a C-X61-65-C-X-C arrangement that is common to all three cluster assembly machineries. In Vitro cluster assembly studies have provided evidence for assembly of [2Fe-2S]2+ and [4Fe-4S]2+ clusters on Azotobacter Vinelandii NifIscA (18, 35) and Erwinia chrysantemi SufA (33) and [2Fe-2S]2+ clusters on IscA (Isa in eukarya) proteins from Escherichia coli (13), Schizosaccharomyces pombe (15, 36), and Synechocystis (16, 37). The U-type motif is found at the N terminus of NifU, as well as in IscU (Isu in eukarya), and comprises three conserved cysteine residues in a C-X24-26-C-X42-43-C arrangement. SufUs are homologous to IscUs with the same three conserved cysteine residues, but they have some distinguishing characteristics (4): an 18-21 amino acid insert between the second and third conserved cysteine residues; a conserved lysine in place of the conserved histidine that coordinates Zn in the NMR structure of Haemophilis influenzae IscU, along with the three conserved cysteine residues (30); absence of the LPPVK motif that is required to interact with HscA cochaperone (25). Hence, in contrast to IscU, it seems likely that SufU does not require HscA/HscB cochaperones for Fe-S cluster assembly or
transfer. In addition, the differences in primary structure for SufU compared to IscU are likely responsible for differences in the reported NMR structures. Thermatoga maritima SufU is reported to have a molten globule-type structure that lacks a well-defined ternary structure (38, 39), whereas H. influenzae IscU has a well-defined central core that includes the Fe-S cluster-binding region (30). In Vitro cluster assembly studies have shown sequential assembly of [2Fe-2S]2+ and [4Fe-4S]2+ clusters on AV IscU (40) and assembly of [2Fe2S]2+ clusters on the N-terminal fragment of AV NifU (NifU1) (11), IscU, or Isu proteins from E. coli (41), S. pombe (42), and Homo sapiens (14, 43) and SufU from T. maritima (34). The third motif is found in the C-terminal domain of NifU, as well as in the “NifU-like” proteins termed Nfu or CnfU (for chloroplast NifU-like), and comprises a conserved C-X2-C motif. Absorption studies have provided evidence for a [2Fe-2S]2+ cluster in the as-purified Nfu protein from Synechocystis (12, 44), and the combination of absorption, analytical, and Mo¨ssbauer studies has demonstrated the assembly of a [4Fe-4S]2+ cluster on the human Nfu protein (45). The homodimeric NifU protein is unique among scaffold proteins in having a modular structure (46) comprising three distinct domains (see Scheme 1), two of which are potential sites for cluster assembly; an IscU-type domain at the N terminus that utilizes conserved residues C37, C62, and C106 in AV NifU and an Nfu-type site at the C terminus that utilizes conserved residues C272 and C275 in AV NifU. Indeed, recent genetic and biochemical studies indicate that both the N- and C-terminal domains are functional in mediating assembly of the [4Fe-4S] cluster on apo nitrogenase Fe protein (47). In addition, the central domain of NifU contains a permanent, redox-active [2Fe-2S]2+,+ cluster ligated by residues C137, C139, C172, and C175 in AV NifU (46, 48) with sequence homology to the bacterioferritin-associated E. coli [2Fe-2S] ferredoxin (49, 50). The role of the permanent [2Fe-2S]2+,+ cluster has yet to be determined but is likely to play a redox role in recruiting Fe for cluster assembly or in mediating the cluster assembly or release
NifU as a Scaffold for [4Fe-4S] Cluster Biosynthesis processes. However, in Vitro studies of NifS-mediated cluster assembly involving NifU have thus far been confined to the isolated N-terminal (IscU-like) domain of AV NifU (termed NifU-1, see Scheme 1), variants of NifU-1 in which a [2Fe2S]2+ cluster is stabilized via a D37A mutation, and fulllength AV NifU that is deficient in the permanent clusters as a result of C137A mutation. Hence, there is a pressing need to investigate the mechanism and type of clusters that can be assembled on full-length wild-type NifU and to establish how NifU facilitates cluster incorporation and maturation of nitrogenase proteins. We report here a spectroscopic investigation of the time course and products of NifS-mediated cluster assembly on wild-type NifU, wild-type and D37A NifU-1 (N-terminal domain fragment of NifU), and wild-type NifU-2 (central plus C-terminal domain fragments of NifU) (see Scheme 1). The approach involves analytical, UV-vis absorption, and Mo¨ssbauer studies, with particular emphasis on the use Mo¨ssbauer to address the nature of assembled clusters via NifS-mediated reconstitution of natural abundance or 57Feenriched NifU samples in the presence of added 57Fe. The results indicate that AV NifU can sequentially assemble labile [2Fe-2S]2+ and [4Fe-4S]2+ clusters in the N-terminal (IscUtype) domain and labile [4Fe-4S]2+ clusters in the Cterminal (Nfu-type) domain. Moreover, we demonstrate that [4Fe-4S]2+ clusters preassembled in either the N- or C-terminal domains can be rapidly transferred to apo nitrogenase Fe protein, indicating that both scaffolding domains of NifU are competent for maturation of nitrogenase component proteins. MATERIALS AND METHODS DNA Biochemistry, Plasmids, and Strain Constructions. The construction of plasmids used for the heterologous expression of NifU, NifU-1, D37A NifU-1, and NifU-2 has been previously described (11, 46, 48). The poly-histidine tagged nifS gene was engineered by PCR to contain NdeI and BamHI sites and ligated into pT7-7 NdeI-BamHI sites. The poly-histidine tag (seven histidines) is located near the C-terminal domain inserted into a SmaI-StuI deletion and has been shown to retain full physiological function by genetic rescue experiments. The nifShis7 pDB845 plasmid is ampicillin-resistant. Protein Biochemistry. AV NifU (pDB525) was heterologously overexpressed in E. coli strain BL21(DE3) as previously described (48). Typical purifications utilized 80120 g of E. coli cells that were resuspendend (1:3, w/v) in 50 mM Tris/HCl buffer at pH 7.4, with the buffer containing 2 mM β-mercaptoethanol and both RNAse and DNAse (Promega, 3.75 mg/L) in addition to 0.5% (w/v) phenylmethanesulfonyl fluoride (PMSF) (Sigma). After sonication and centrifugation, the soluble cell-free extract was decanted and NifU was subsequently precipitated at 33% ammonium sulfate saturation. The precipitated NifU pellet was resuspended in zero salt buffer, loaded onto a Q Sepharose (Pharmacia) column (50 mm inner diameter, ∼80 mL), and washed with 5 column volumes at 0.1 M NaCl before eluting with 0.1-0.6 M NaCl linear gradient over 600 mL. Fractions containing NifU were collected between 0.27 and 0.34 M NaCl and pooled as a single fraction. AV NifShis7 was heterologously expressed in E. coli strain BL21(DE3) as
Biochemistry, Vol. 44, No. 39, 2005 12957 previously described (10). NifShis7-containing cells were resuspended in 50 mM Tris/HCl buffer at pH 8.0, with the buffer containing 5 mM imidazole, 500 mM NaCl, DNAse (Promega, 3.75 mg/L), RNAse (Promega, 3.75 mg/L), and 0.5% PMSF, sonicated, centrifuged, and retaining the soluble cell-free extract. The cell-free extract was loaded onto a Ni2+charged 5-mL HiTrap (Pharmacia) metal-chelating column and washed with 10 column volumes of 50 mM Tris/HCl buffer at pH 8.0 containing 60 mM imidazole and 500 mM NaCl. NifShis6 was eluted with 50 mM Tris/HCl buffer at pH 8.0, with 1 M imidazole and 500 mM NaCl, and immediately loaded onto a 53-mL HiPrep (Pharmacia) desalting column equilibrated with 100 mM Tris/HCl buffer at pH 7.8, with 100 mM NaCl, to remove the imidazole. AV NifU-1, D37A NifU-1, and NifU-2 were purified as previously described (11, 46). All samples of NifS, NifU, and NifU variants were at least >95% homogeneous based on denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparation of NifU Samples for NifS-Mediated Cluster Assembly Experiments. Iron and protein content of wild-type AV NifU determined after purification typically ranged from 1.1 to 1.5 Fe/NifU monomer, significantly less than the 2.0 Fe/NifU monomer expected for 1 permanent [2Fe-2S]2+,+ cluster/NifU monomer. The substoichiometric cluster occupancy is most likely an artifact of either overexpression or cluster degradation during purification. Because substoichiometric occupancy of permanent clusters would complicate the interpretation of NifS-mediated cluster assembly studies, the following procedures were used to obtain NifU samples with fully replete natural abundance or 57Fe-enriched permanent clusters for NifS-mediated cluster assembly studies. Both procedures were carried out in a glovebox under argon (
