HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 24, 21397-21404, June 14, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21397    most recent M201439200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mansy, S. S. Articles by Cowan, J. A. Search for Related Content PubMed PubMed Citation Articles by Mansy, S. S. Articles by Cowan, J. A. Social Bookmarking                      What's this?

Iron-Sulfur Cluster Biosynthesis

THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN^*

Sheref S. Mansy^§, Gong Wu, Kristene K. Surerus^¶, and J. A. Cowan

From the  Evans Laboratory of Chemistry, Ohio State University, Columbus, Ohio 43210 and the ^¶ Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201

Received for publication, February 12, 2002, and in revised form, March 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU from Thermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. Mössbauer ( = 0.29 ± 0.03 mm/s, E_Q = 0.58 ± 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]²⁺ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% -helix, 13.1% antiparallel -sheet, 11.3% parallel -sheet, 20.2% -turn, and 19.1% other at 20 °C, with negligible spectral change observed at 70 °C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in ¹H-¹⁵N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritima IscU-mediated reconstitution of human apoferredoxin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Iron-sulfur (Fe-S) cluster proteins participate in a wide variety of physiologically essential processes including gene regulation, electron transfer, and catalytic reactions (1). In vitro it is possible to reconstitute Fe-S apoproteins by anaerobic incubation with iron, sulfide, and a suitable reducing agent. However, because of the cellular toxicity of free iron and sulfide, it is believed that Fe-S cluster biogenesis is mediated by specific protein-protein interactions rather than by spontaneous formation. Initial identification of essential Fe-S cluster maturation components was made within the nif¹ operon of Azotobacter vinelandii. Disruption of either nifS or nifU resulted in the loss of Fe-S coordination to nitrogenase (2). NifS has been shown to provide sulfur equivalents by catalytic cysteine desulfurization (3); however, the role of NifU has yet to be firmly established. NifU is a modular protein with three domains. The amino-terminal domain has three conserved Cys and binds a labile less stable [2Fe-2S] cluster, the central region contains four conserved Cys and coordinates a stable [2Fe-2S] cluster, and the carboxyl-terminal domain has an unknown function with two conserved Cys (4-6). Of these domains the central region has been the most thoroughly characterized. Through DNA sequencing it has been observed that nif homologues are encoded within the genomes of most organisms. These homologues have been named isc (for iron sulfur cluster) and are usually clustered together, encoding IscS, IscU, IscA, heat shock proteins HscB and HscA, and a ferredoxin (7). The NifU homologue, IscU, is only homologous to the amino terminus of NifU and analogously coordinates a reductively labile [2Fe-2S] cluster (6-9). In eukaryotes, Isc proteins have been identified in mitochondria (10). Results from genetic screens of A. vinelandii, in vitro characterization of NifU/IscU chemistry, and the high degree of conservation of NifU-like proteins from divergent species have led to the hypothesis that NifU/IscU proteins, in conjunction with NifS/IscS, deliver Fe-S cluster equivalents to target Fe-S apoproteins (4).

Thermatoga maritima is a hyperthermophilic bacterium. It represents one of the deepest and most slowly evolving eubacterial lineages (11), with an optimal growth temperature of 80 °C (12). The genomic sequence shows genes encoding both IscU (Tm IscU) and IscS located next to each other, although no other isc genes are found clustered within this region. Located elsewhere in the T. maritima genome are heat shock proteins that are homologous to those identified in other organisms (13) and several ferredoxins. Additionally, T. maritima expresses a second NifS-like protein that has been crystallographically characterized (14). Possibly, the functions of the missing Isc proteins are provided by other unidentified proteins involved in iron homeostasis, as has been identified in Escherichia coli (15, 16).

Herein we report the characterization of an evolutionarily ancient IscU. Similar to other bacterial and eukaryotic organisms (8, 17, 18), Tm IscU binds a [2Fe-2S] cluster. The holo form of the native protein proved to be relatively labile, but following prior precedent (8, 9) was stabilized by substitution of a conserved aspartate by alanine (D40A). Factors that promote stabilization through this substitution will be described elsewhere. Analysis of CD spectra over a wide temperature range demonstrate Tm IscU to possess significant secondary structure that is not dependent upon the coordination of an Fe-S cluster, whereas the dispersion of resonances in ¹H-¹⁵N HSQC spectra indicate significant tertiary structure. Evidence for secondary and tertiary structure was absent for the human and yeast homologues that we have examined in our laboratory, and reasons for this variation are discussed. Furthermore, Tm IscU proves to be competent for Fe-S cluster transfer to human ferredoxin (Hs Fd), consistent with a conserved role in Fe-S cluster biogenesis. This result provides strong support for a common conserved recognition mechanism for both prokaryotic and eukaryotic IscU-type proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

General Chemicals-- ⁵⁷Fe was from Pennwood Chemicals. All restriction enzymes and the copper staining kit were from Invitrogen. Pfu DNA polymerase and BL21CodonPlus(DE3)-RIL were from Stratagene (La Jolla, CA); PCR purification kit and Ni-NTA resin were purchased from Qiagen (Valencia, CA). Protein expression vectors pET21, pET28, and BL21(DE3) pLysS were from Novagen (Madison, WI). Oligonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA). CM-32 and DE-52 were from Whatman (Aston, PA). Homogenous-20 precast polyacrylamide gels and G-75 and Superose-12 resins were obtained from Amersham Biosciences.

Cloning, Mutagenesis, and Expression of T. maritima IscU-- T. maritima genomic DNA was obtained from the American Type Culture Collection (ATCC no. 43589D). DNA (50 ng), 0.2 µM amounts of each primer, 2.5 units of cloned Pfu DNA polymerase, 1× cloned Pfu buffer, and 0.2 mM each dNTP were used to amplify iscU (TIGR locus TM1372, www.tigr.org) via PCR. Primers were: 5'-GGGCCCGGCATATGGTTTTCAAGATGATG-3' and 5'-CCGGCCGGATCCTTAAGGCCGTGAAATCTTTTTG-3', where underlined regions denote NdeI and BamHI sites, respectively, and the bold position indicates a G to A substitution resulting in an amino-terminal Met rather than a Val for improved recombinant expression in E. coli. The thermocycle used was identical to that described in the Pfu DNA polymerase manual (Stratagene). PCR products were digested with NdeI and BamHI and ligated to similarly treated pET21 and pET28. Cloning into pET21 yielded a construct without a tag or additional residues (pTmIscU). Cloning into pET28 resulted in the addition of an amino-terminal His tag (pTmIscUHis).

The QuikChange technique (Stratagene) was employed for the D40A point mutation. Reactions contained 50 ng of template (either pTmIscU or pTmIscUHis), 2.5 units of cloned Pfu DNA polymerase, 1× cloned Pfu buffer, 0.5 mM DTT, and 125 ng of each primer. Primers were 5'-GGGAAAGAACATCTCTTGTGGCGCCGAAATCACACTCTAC-3' and 5'-GTAGAGTGTGATTTCGGCGCCACAAGAGATGTTCTTTCCC-3', where the bold positions indicate the mutation. The thermocycle was identical to that described in the QuikChange manual (Stratagene). An aliquot (27%) of the post-thermocycle sample was incubated with 7.5 units of DpnI at 37 °C for 2 h. Subsequently, CaCl₂-competent DH5 was transformed via heat shock with the mutant constructs (19). Cloning and mutagenesis results were confirmed by nucleotide sequencing at the Ohio State University Plant-Microbe Genomics Facility.

BL21CodonPlus(DE3)-RIL was used for protein expression. A 100-ml Luria-Bertani culture (supplemented with either 100 µg/ml ampicillin and 35 µg/ml chloramphenicol for pTmIscU constructs or 50 µg/ml kanamycin and 35 µg/ml chloramphenicol for pTmIscUHis constructs) was grown overnight as a starter culture. The entire starter culture was used as an inoculum for a 10-liter fermentation at the Ohio State University fermentation facility and grown to an A₆₀₀ ~ 0.6 prior to induction with 1 mM isopropyl-1-thio--D-galactopyranoside. Cells were pelleted 5 h after induction and stored at -80 °C for future use.

Protein Purification-- Cell pellets were resuspended in five volumes of 50 mM Tris-HCl, pH 7.4, 2 mM -mercaptoethanol, 20 µg/ml DNase, and 5 µg/ml RNase and lysed by sonication. Insoluble material was removed by centrifugation at 15,000 rpm, 4 °C for 1 h. For His-tagged constructs, the cleared lysate was applied to a Ni-NTA column equilibrated with binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 500 mM NaCl). The column was then washed with five column volumes of binding buffer and five volumes of binding buffer + 15 mM imidazole, and the protein was subsequently eluted with binding buffer + 100 mM imidazole. His-tagged protein was exchanged with 50 mM sodium phosphate, pH 7.4, via repeated ultrafiltration (Amicon).

The cleared lysate containing non-His-tagged protein was loaded onto a cation exchange column (CM32) equilibrated with 50 mM sodium phosphate, pH 7.4, and washed with one cleared lysate volume of phosphate buffer. The flow-through and wash fractions were combined. NaCl was added to 50 mM, -mercaptoethanol was added to 5 mM, and the solution was incubated at 85 °C for 0.5 h. The sample was then centrifuged at 15,000 rpm, 4 °C for 10 min and the supernatant loaded onto an anion exchange column (DE-52). The column was washed with three column volumes of 50 mM Tris-HCl, pH 7.4. The flow-through and wash fractions were combined and concentrated via ultrafiltration. Subsequently, protein solution was loaded onto a G-75 gel filtration column equilibrated with 50 mM sodium phosphate, pH 7.4. The fractions with a _max at 278 nm were pooled and confirmed to be pure IscU via SDS-PAGE. All apoprotein samples were stored at either 4 or -80 °C.

HuFd/pET3a (encoding human ferredoxin) was a gift from J. L. Markley. BL21(DE3) pLysS HuFd/pET3a was essentially expressed and purified as previously described (20). Hs apoFd was prepared as described by Nishio and Nakai (21).

Mass Spectrometry-- All mass spectra were acquired at the campus chemical instrument center at Ohio State University, and all solutions were made in Barnstead purified water. Molecular mass determination for extinction coefficient calculation was determined by electrospray ionization (ESI) using a Micromass Q-TOF^TM II (Micromass, Wythenshawe, UK) mass spectrometer equipped with an orthogonal electrospray source (Z-spray) operated in positive ion mode. Sodium iodide was used for mass calibration for a calibration range of m/z 100-2500. Salt buffers from the protein samples were cleaned using manual syringe protein traps from Michrom BioResources (Auburn, CA). Proteins were prepared in a solution containing 50% acetonitrile, 50% water, 0.1% formic acid at a concentration of 50 pmol/µl and infused into the electrospray source at a rate of 5-10 µl min¹. Optimal ESI conditions were: capillary voltage of 3000 V, source temperature of 110 °C, and a cone voltage of 60 V. The ESI gas was nitrogen. Q1 was set to optimally pass ions from m/z 100-2000 and all ions transmitted into the pusher region of the time-of-flight analyzer were scanned over m/z 100-3000 with a 1-s integration time. Data were acquired in continuum mode until acceptable averaged data were obtained (10-15 min). ESI data were deconvoluted using MaxEnt I provided by Micromass.

In-gel tryptic digests were performed on copper-stained protein bands (5, 10, and 20 µg of WT Tm IscU) separated on a 15% SDS-PAGE gel. Incised bands were washed with 50% methanol (HPLC-grade), 5% acetic acid (JT Baker UltrexII Ultrapure) and dried in HPLC-grade acetonitrile. Samples were then reduced with DTT (5 mg/ml in 100 mM ammonium bicarbonate) and alkylated with iodoacetamide (15 mg/ml in 100 mM ammonium bicarbonate). The protein band was dehydrated with acetonitrile, rehydrated with 100 mM ammonium bicarbonate, and dehydrated again with acetonitrile. Promega modified trypsin (20 ng/µl in 100 mM ammonium bicarbonate, total addition = 50 µl) was added to each gel piece and rehydrated on ice for 10 min. Samples were centrifuged and excess trypsin solution removed. Ammonium bicarbonate (50 mM) was added to 20 µl, vortexed and centrifuged briefly, and incubated at room temperature overnight. A solution of 50% acetonitrile, 5% formic acid (EM Science ACS 88%) was added to 30 µl, vortexed for 10 min, and centrifuged. The supernatant was isolated, and an additional 30 µl of 50% acetonitrile, 5% formic acid was added, vortexed for 10 min, and centrifuged. The supernatant was analyzed by matrix-assisted laser desorption/ionization time-of-flight performed on a Reflex III (Bruker, Bremen, Germany) mass spectrometer operated in linear, positive ion mode with a N₂ laser. Laser power was used at the threshold level required to generate signal. Accelerating voltage was set to 28 kV. The instrument was calibrated with protein standards bracketing the molecular weights of the protein samples (typically mixtures of bradykinin fragment 1-5 and ACTH fragment 18-39 as appropriate). Salt buffers from the protein samples were cleaned using ZipTips (Millipore, Bedford, MA) according to manufacturer directions. -Cyano-4-hydroxycinnamic acid was used as the matrix and prepared as a saturated solution in 50% acetonitrile, 0.1% trifluoroacetic acid (in water). Allotments of 1 µl of matrix and 1 µl of sample were thoroughly mixed together; 0.5 µl of this was spotted on the target plate and allowed to dry. Protein identification based on peptide masses was performed via ProFound (proteometrics.com).

Tm IscU Cluster Reconstitution-- Protein (0.5 mM) in 50 mM sodium phosphate, pH 7.4, 50 mM NaCl, 50 mM DTT was repeatedly degassed and argon-purged for 1 h. Fresh FeCl₃ and Na₂S were then slowly added to 1 mM. The mixture was allowed to incubate anaerobically at room temperature for 0.5 h. Insoluble material was removed by centrifugation at 15,000 rpm, 4 °C for 5 min. The supernatant was desalted by a G-25 column equilibrated with 50 mM Tris-HCl, pH 7.4, or 50 mM sodium phosphate, pH 7.4, and the colored protein fraction was collected. His-tagged holoprotein was concentrated and stored at -80 °C until further use. Non-His-tagged holoprotein was then loaded onto a DE-52 column equilibrated with 50 mM Tris-HCl, pH 7.4, and washed with five column volumes of the same buffer. The holoprotein was eluted with 50 mM Tris-HCl, pH 7.4, 100 mM NaCl.

Aggregation State Analysis-- FPLC gel filtration using a Superose-12 column (HR 16/50) at a flow rate of 0.5 ml/min was used for aggregation state determination. The running buffer was 50 mM HEPES, pH 7.4, 50 mM NaCl. A Gel Filtration Calibration Kit (Amersham Biosciences) was used to calibrate the column. The standards were RNase A (13,700 Da), chymotrypsinogen A (25,000 Da), ovalbumin (43,000 Da), and albumin (67,000 Da). Blue dextrin was used to determine the dead volume. Molecular sizes were determined by plotting log M_r of standards versus K_av where K_av = (V_e  V₀)/(V_t  V₀), V_e = elution volume, V₀ = dead volume, V_t = total column volume.

UV-visible Spectroscopy and Evaluation of Extinction Coefficients-- UV-visible spectra were recorded on a Hewlett-Packard 8425A diode array spectrophotometer using the On-Line Instrument Systems (OLIS) 4300S operating system software. A 1.0-cm path-length cuvette was used for all measurements. All solutions used for extinction coefficient determination were prepared in Barnstead purified deionized water. Apo D40A Tm IscU was initially dialyzed extensively against a volatile buffer (100 mM ammonium bicarbonate, pH 7.0) and then against unbuffered water. Finally, the protein was passed through a G-25 column equilibrated with water. After the absorption spectrum was collected, the sample was lyophilized and the mass of the protein sample determined. Using Beer's law and the molecular mass determined by ESI, the extinction coefficient of apo D40A Tm IscU was calculated. The concentration of protein used for holo D40A Tm IscU extinction coefficient calculation was determined by incubating holo D40A Tm IscU in 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA at 60 °C for 0.5 h followed by desalting via a G-25 column. Total protein concentration was then determined from the 278-nm absorption of apo D40A Tm IscU using the previously determined extinction coefficient.

Temperature Dependence of [2Fe-2S] Cluster Stability-- Holoprotein (30 µM) in 100 mM Tris-HCl, pH 7.4, 50 mM NaCl was repeatedly degassed and argon-purged in a stoppered cuvette while kept on ice. Absorption data at 412 nm was acquired on a Hewlett-Packard diode array spectrometer (HP 8453) with HP8453 Win system software. Temperature control was achieved with a Peltier temperature controller (HP 8909A). Absorption data at temperatures ranging from 20 °C to 86 °C was collected at 2 °C increments with a 0.5-min equilibration period at each temperature prior to measurement.

Mössbauer, EPR, and NMR Spectroscopy-- Mössbauer spectra of ⁵⁷Fe D40A Tm IscUHis was recorded on a constant acceleration spectrometer, model MS-1200D from Ranger Scientific, using a Janis SuperVaritemp cryostat (model 8DT), a Lakeshore temperature controller (model 340), and a ⁵⁷Co source from Isotope Products Laboratory. The ⁵⁷Fe-reconstituted protein was prepared as described above (see Tm IscU cluster reconstitution) except that ⁵⁷Fe³⁺ was used instead of regular FeCl₃. EPR signals were recorded with an X-band Bruker ESP 300 spectrometer equipped with an Oxford liquid helium cryostat at 15 K. Holo D40A Tm IscUHis concentrations were 0.9 mM for untreated samples, and 0.4 mM for ascorbate reduced samples. Holo D40A Tm IscUHis was reduced with 0.2 mM and 2 mM ascorbate and immediately frozen. ¹⁵N-Labeled apo D40A Tm IscU was expressed in minimal medium supplemented with ¹⁵NH₄Cl (22). ¹⁵N-HSQC spectra were recorded on a Bruker 600 Avance DMX spectrometer operating at 600.13 MHz. The pulse sequence was as described previously (23). A total of four scans were collected.

Iron Quantitation-- Iron content of holo D40A Tm IscU was determined by atomic absorption using a PerkinElmer Zeeman 5000 graphite furnace atomic absorption spectrometer. An iron standard solution (GFS Chemicals, Inc.) was used to construct a standard curve with a r² > 0.999. Sample loading was automated (AS40), and all data were run in duplicate and averaged. Absorption was measured at 305.9 nm and integrated for 7 s. All solutions were in Barnstead purified deionized water and 2% nitric acid. Background iron concentrations of solutions were measured and found to be negligible.

Circular Dichroism-- Circular dichroism spectra were measured on an Aviv model 202 circular dichroism spectrometer. Far-UV CD spectra were acquired with a 0.1-mm path-length cuvette. Experimental conditions were essentially as recommended by Johnson (24). Protein concentrations were 0.08 mM WT and D40A Tm IscU (based on monomeric protein) and 0.15 mM Hs Fd in 10 mM sodium phosphate, pH 7.0, 10 mM NaCl, except for holo D40A Tm IscU (10 mM Tris-HCl, pH 7.0, 50 mM NaCl). Spectra acquired at 20 °C were determined per 0.2 nm in triplicate and averaged. At elevated temperatures spectra were only recorded once. Secondary structure quantitation was determined via the self-consistent method (25) with the Dicroprot V2.5 version 5.0 package (26) obtained from www.ibcp.fr. Near-UV-visible CD spectra were recorded in a 3-mm path-length cuvette/1 nm. Protein concentrations were 40 µM in 50 mM Tris-HCl, pH 7.0. Buffer spectra were always subtracted.

D40A Tm IscU-directed [2Fe-2S] Cluster Transfer to Human Apoferredoxin-- Reactions were initiated by the addition of 0.1 mM holo D40A Tm IscUHis in 50 mM sodium phosphate, pH 7.4, to a solution containing 0.1 mM human apoferredoxin and 5 mM DTT in the same buffer. Reactions were incubated on ice, stopped by the addition of loading buffer, and immediately frozen at -80 °C. Reaction products were separated on a 7% native-PAGE and visualized by Coomassie Blue staining.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression and Aggregation State-- Tm IscU expression was high for all constructs in BL21CodonPlus(DE3)-RIL, greater than that obtained from BL21(DE3), with typical yields of greater than 700 mg of pure protein from a 10-liter fermentation. BL21CodonPlus(DE3)-RIL provides tRNAs for the rarely used E. coli codons of Arg, Ile, and Leu. These codons are more common in T. maritima and are found within the iscU gene. Surprisingly, expressed WT Tm IscU and His-tagged WT Tm IscU (Tm IscUHis) were observed to run as two closely spaced bands during SDS-PAGE. These bands were found in a 1:1 ratio and persisted from the time of lysis through all purification steps (Fig. 1) and give rise to similar mass spectrometric patterns (see below). Phenylmethylsulfonyl fluoride had no effect on the appearance or ratio of the two bands, and so they do not seem to be a result of endoproteinase activity. D40A Tm IscU and D40A Tm IscUHis only expressed as one band migrating at the same position as the lighter molecular mass band of the corresponding WT protein.

View larger version (77K): [in this window] [in a new window]   Fig. 1.   WT and D40A Tm IscU/His expression. Small 10-ml cultures were grown and induced as described under "Experimental Procedures" and used for lanes 2-6. Lanes 3-6 are cleared lysates after incubation at 85 °C for 0.5 h followed by centrifugation to remove insoluble material. Lane 1, Invitrogen low molecular mass markers; lane 2, cleared lysate containing WT Tm IscU, i.e. not subjected to a heat step; lane 3, WT Tm IscU; lane 4, WT Tm IscUHis; lane 5, D40A Tm IscU; lane 6, D40A Tm IscUHis; lane 7, WT Tm IscU after all the purification steps described under "Experimental Procedures." The mass markers are labeled in kDa. Lane 7 is from a different lane of the same gel.

Apo WT and D40A Tm IscU migrated as a monomer on an analytical gel filtration column, whereas holo WT and D40A Tm IscU eluted as a dimer with apparent masses of 18 and 35 kDa, respectively (Fig. 2). No high molecular mass aggregates were observed to elute.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Molecular weight determination by gel filtration. Diamonds are molecular size standards (see "Experimental Procedures"). Squares (filled is holo, and empty is apo) are D40A Tm IscU, and the triangle is apo WT Tm IscU.

Mass Spectroscopic Analysis-- ESI spectra of each of the two bands identified by SDS-PAGE for WT Tm IscU showed the same two major peaks corresponding to full-length protein and to protein lacking the amino-terminal Met in approximately a 1:1 ratio (16,070 and 15,940 Da, respectively), whereas D40A Tm IscU showed complete loss of the amino-terminal Met with a mass of 15,893 Da. These values are in close agreement with their predicted molecular masses of 16,071, 15,940, and 15,894 Da, respectively. In-gel tryptic digests of the two bands of WT Tm IscU yielded essentially identical matrix-assisted laser desorption/ionization-time of flight spectra for all three protein concentrations tested, i.e. peptide fragments with identical masses were observed for both SDS-PAGE bands without the appearance or disappearance of any signal. The search engine ProFound identified both bands as consisting of Tm IscU, further confirming that neither of the bands was caused by an impurity.

Cluster Coordination-- The bacterial pellets of induced cells were dark brown in color for all constructs. Following centrifugation to remove solids, His-tagged WT and D40A Tm IscU lysates yielded a red band that was bound by a Ni-NTA column. The non-His-tagged constructs had reddish-brown lysates, but no color survived the 85 °C purification step. These results suggest proper Fe-S cluster assembly in vivo in E. coli, and that the His tag does not interfere with Fe-S cluster coordination. Although a fraction of Tm IscU expressed as holoprotein, the overall holo concentration appeared to be quite low as judged by UV-visible spectroscopy. Therefore, attempts were made to reconstitute Tm IscU. Simple anaerobic incubation of either apo D40A Tm IscU or D40A Tm IscUHis with DTT and a 2-fold molar excess of iron and sulfide without the presence of denaturant resulted in a deep red protein with UV-visible spectra similar to [2Fe-2S]-containing proteins (Fig. 3). Increasing the iron and sulfide concentration only resulted in the appearance of adventitiously bound iron, as judged by Mössbauer analysis (data not shown). Holo D40A Tm IscU was further purified by anion exchange chromatography. Apo D40A Tm IscU did not bind to DE-52, whereas holo D40A Tm IscU was observed to bind. However, holo D40A Tm IscUHis did not stick to DE-52, presumably because of the increased pI of the protein resulting from the His tag. Iron content was measured by atomic absorption spectroscopy (monitoring the absorption at 305.9 nm) following control experiments to establish a standard calibration curve. Freshly purified holo D40A Tm IscU was found to contain 1.2 ± 0.1 Fe/monomeric subunit. Repeated attempts to reconstitute WT Tm IscU and WT Tm IscUHis were unsuccessful. This is in agreement with previous results, indicating increased cluster stability upon an Asp to Ala substitution at amino acid position 40 (Tm IscU numbering) (8, 27). Although it is not currently understood why a mutation at this position increases cluster stability, such a situation is not without precedent. A Leu to His substitution near one of the cluster ligands of E. coli FNR greatly increases the stability of its [4Fe-4S] cluster (28). As a result of the low yield of purified holo WT Tm IscU, and the relative instability of its cluster, all holoprotein studies were carried out with reconstituted D40A Tm IscU/His. In vivo cluster formation in WT Tm IscU is most likely facilitated by chaperones and IscS (17, 29, 30).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   UV-visible spectra of 60.6 µM apo (dashed line) and 24.5 µM holo D40A Tm IscU (solid line) in 50 mM Tris-HCl, pH 7.4.

Mössbauer and EPR Spectroscopy-- Mössbauer spectra of ⁵⁷Fe reconstituted D40A Tm IscUHis show a single iron-containing species with an isomer shift,  = 0.29 ± 0.03 mm/s, and a quadrupole splitting, E_Q = 0.58 ± 0.03 mm/s (Fig. 4). A similar spectrum was obtained at 4.2 K, with no hyperfine splitting observed, consistent with a diamagnetic diferric [2Fe-2S]²⁺ cluster, but cannot exclude the possibility of one or two non-cysteinyl cluster ligands (31). The data do, however, exclude formation of a [4Fe-4S] cluster. Holo D40A Tm IscUHis was EPR-silent, thus precluding the possibility of a rubredoxin-type center or a [3Fe-4S]⁺ cluster. Attempts to reduce and trap an EPR active species of holo D40A Tm IscUHis were unsuccessful as a result of the reductive lability of the cluster (4, 8, 9, 17). Although reduction is likely to be rapid, we were unable to trap the subsequent reduced species prior to degradation by manual freeze-quenching.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mössbauer spectrum of holo D40A Tm IscUHis at 100 K in a 450-G applied field. 57Fe-reconstituted IscU (~1.5 mM holo concentration) was in 50 mM sodium phosphate, pH 7.4.

UV-visible Spectroscopy-- Apo D40A Tm IscU had a _max at 278 nm with an extinction coefficient of 15,105 M¹ cm¹, which was determined by weighing a completely desalted and lyophilized protein where the absorbance spectrum had previously been measured. Holo D40A Tm IscU had _max at 278 and 412 nm with shoulders at approximately 320, 450, and 580 nm (Fig. 3). The known protein concentration, determined by converting holo- to apoprotein, yielded extinction coefficients of 41,097, 22,188, and 13,854 M¹ cm¹ at 278, 320, and 412 nm, respectively, for dimeric holo D40A Tm IscU. The absorption pattern and relative intensities are similar to those of previously studied IscU proteins and human ferredoxin (a [2Fe-2S]-containing protein); however, the absolute extinction coefficients for the latter two cluster-centered transitions are lower than expected for a one cluster per monomer ratio (5) and are consistent with one cluster per dimeric D40A Tm IscU.

Thermal Stability of the [2Fe-2S] Cluster-- Cluster loss from D40A Tm IscU and Hs Fd was monitored by visible absorption (Fig. 5). Hs Fd exhibited a typical profile with retention of cluster up to a critical temperature followed by rapid cluster loss, presumably because of loss of necessary structural elements. Hs Fd began to lose cluster at 54 °C and was completely apo by 64 °C. At higher temperatures Hs Fd began to precipitate even under anaerobic conditions, as evidenced by an increase in absorption caused by light scattering. D40A Tm IscU showed more unusual thermal behavior. Almost immediately upon increasing the temperature, the cluster of D40A Tm IscU began to degrade and was essentially absent by 55 °C. It is important to note that the cluster is thermodynamically unstable even at ambient temperatures, but takes longer to degrade. Fig. 5 highlights the instability of the IscU-bound cluster relative to the protein (compare below), and how the thermal stability of a ferredoxin cluster compares with that from IscU. At higher temperatures degradation is accelerated, and so a T_m calculation for such a thermal profile is meaningless because significant degradation occurs over a wide temperature range. Therefore, this experiment serves to illustrate the difference in thermal stability of the [2Fe-2S] cluster between these two proteins, rather than to assign a specific value.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Cluster thermal stability of D40A Tm IscU (solid line) and human ferredoxin (dashed line). Conditions are as described under "Experimental Procedures."

Circular Dichroism and NMR-- The secondary structural content of apo D40A and apo WT Tm IscU was determined by CD. Both were found to be have identical CD spectra indicating 36.7% -helix, 13.1% antiparallel -sheet, 11.3% parallel -sheet, 20.2% -turn, and 19.1% other at 20 °C with negligible spectral changes observed at 70 °C (Fig. 6). The experimental error of 8.6% in these measurements is defined as the difference between the calculated and the experimentally determined CD spectrum. Interestingly, holo D40A Tm IscU also had an identical CD spectrum consistent with no major structural rearrangement upon [2Fe-2S] cluster coordination. The far-UV CD spectra of holo D40A Tm IscU were only acquired to 195 nm, as opposed to 180 nm for the other samples, because of the presence of Tris-HCl rather than phosphate buffer. Because iron coordinates to Tris-HCl to a significantly lesser extent than to phosphate, Tris-HCl was used as a buffer for the holo D40A Tm IscU sample, even though it is less transparent than phosphate. In contrast to D40A Tm IscU, Hs holoFd showed obvious structural changes upon increased temperature from 20 to 60 °C, with the negative peak shifting from 204 to 200 nm and the positive peak moving from 195 to 187 nm. A temperature of 60 °C was chosen to avoid protein precipitation, as had been observed previously at higher temperatures (see "Thermal Stability"). After allowing the Hs Fd sample to cool slowly to 20 °C, the spectrum looked intermediate to the 20 and 60 °C spectra with negative and positive peaks at 203 and 188 nm, respectively, although the error in the positive peak is large.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Circular dichroism spectra of D40A Tm IscU and human ferredoxin. Panels A-C are far-UV CD spectra. Panel D shows near-UV-visible CD spectra. Spectra were acquired at 20 °C (solid lines) and 70 °C (for Tm IscU) or 60 °C (for human ferredoxin) (dashed lines). Panel A, apo WT Tm IscU; panel B, D40A Tm IscU (holoprotein is shown displaced for ease of comparison with apo); panel C, human ferredoxin (the middle spectral line is of protein cooled to 20 °C after incubation at 60 °C); panel D, the upper spectrum is of holo D40A Tm IscU and the lower is of human holoferredoxin.

The near-UV-visible portion of the CD spectrum for holo D40A Tm IscUHis was similar to that reported for E. coli IscU (18). The spectrum also has features in common with human and Anabaena ferredoxin consistent with [2Fe-2S] coordination (30, 32). In addition to the peak near 440 nm, there is an additional peak near 480 nm for D40A Tm IscUHis that is not present in WT ferredoxin. However, this peak is observed in C46S Anabaena ferredoxin (32).

High resolution ¹H-¹⁵N HSQC spectra for apo WT and D40A Tm IscU were essentially the same and the significant dispersion of signals indicated the presence of substantial tertiary structure. Previous measurements in our laboratory on the apo form of human and yeast homologues have shown ¹H-¹⁵N HSQC spectra that lack dispersion of cross-peaks, indicating the absence of significant tertiary structural elements.

Cluster Transfer to Human Ferredoxin-- The reconstitution of human apoferredoxin was easily achieved by incubation on ice with an equimolar concentration of holo D40A Tm IscUHis. The cluster transfer reaction was monitored by native PAGE (Fig. 7), exploiting the difference in migration of human apo- and holoferredoxin. Human holoferredoxin formation was quite rapid with completion of the reaction in less than 10 min. At this temperature and time scale, no significant D40A Tm IscU cluster degradation occurs. The yield of D40A Tm IscU-mediated reconstitution of Hs holoFd was determined to be greater than 70%, with cluster insertion into the remaining apoFd most likely precluded by disulfide bond formation. Furthermore, formation of Hs holoFd is not observed under conditions where standard solution reconstitution methods are used where free iron, sulfide, and DTT are incubated with apoprotein.

View larger version (88K): [in this window] [in a new window]   Fig. 7.   Cluster transfer from holo D40A Tm IscUHis to human apoferredoxin. Lanes 1 and 2, cluster transfer reactions (50 µM holo D40A Tm IscUHis and 50 µM Hs apoFd) terminated after 10 min and 1 h, respectively; lane 3, 0.1 mM D40A Tm IscUHis; lane 4, 0.1 mM Hs holoFd; lane 5, 40 µM Hs apoFd.

Sequence Comparison-- Fig. 8 shows that Tm IscU is homologous to other IscU proteins from higher organisms (26% identity and 47% similarity to human ISU) and contains the three conserved Cys and the conserved Asp at position 40 (T. maritima numbering). However, Tm IscU contains a 19-amino acid insertion in the middle of the protein not found in most other prokaryotic or eukaryotic IscU proteins. This insertion is found within the Bacillus subtilis IscU homologue named YurV (35% identity, 62% similarity to Tm IscU). These insertions are clearly homologous with 21% identity and theoretical pI values of 4.4 and 4.2 for Tm IscU and B. subtilis YurV, respectively.

View larger version (71K): [in this window] [in a new window]   Fig. 8.   IscU/NifU sequence alignment. Inverted text are amino acid identities, and bold positions are highly conserved residues. Only the IscU homologous region of NifU is shown. Organisms are as follows: Tm, T. maritima; Bs, B. subtilis; Ec, E. coli; Av, A. vinelandii; Hs, human; Sp, Schizosaccharomyces pombe.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression-- The expression and purification of D40A Tm IscU/His yielded large amounts of high purity protein with no complications. However, the expression of WT Tm IscU/His was more problematic. Although large amounts of highly purified WT Tm IscU/His were easily obtained, they were consistently present as two forms as judged by SDS-PAGE. The observance of two bands suggests some sort of post-translational modification, but we were only able to identify differences in amino-terminal Met processing. Small highly charged proteins have often been observed to behave oddly on SDS-PAGE (33, 34); however, WT Tm IscU and WT Tm IscUHis have predicted pI values of 5.7 and 7.1, respectively. The -subunit of T. maritima hydrogenase also recombinantly expresses as two forms with a molecular mass difference of greater than 7 kDa (35). The difference was shown to be the result of protein cleavage, as judged by mass spectroscopy and amino-terminal sequencing, and was not dependent upon a heating step during purification (35). This cleavage site is not found within Tm IscU. Mass spectroscopic analysis of an A. vinelandii ferredoxin cloned from its isc operon revealed that recombinant expression in E. coli yielded protein 52 Da larger than native protein, although the recombinant protein ran faster on a SDS-PAGE (36). The difference in molecular mass was not the result of amino-terminal cleavage, as judged by amino acid sequencing, and was not definitively characterized (36). The most reasonable explanation for the double band observed for WT Tm IscU, given the fact that each band is essentially the same species by mass spectrometric analysis, is that the protein binds SDS in two stoichiometries and therefore runs as a doublet, as previously observed and documented for the E. coli outer cell membrane protein OmpA (37, 38). It is interesting to note that a single point mutation known to stabilize cluster coordination yielded Tm IscU in a single form, perhaps by stabilizing a specific "conformer."

Cluster Coordination-- Reconstitution of D40A Tm IscU/His with iron and sulfide yielded holoprotein with Mössbauer, UV-visible absorption, and CD spectra characteristic of [2Fe-2S] coordination. The UV-visible spectra are similar to other reported IscU proteins in that their _max are roughly in the same positions. However, holo D40A Tm IscU had sharper, better defined peaks between 400 and 600 nm. Although the near-UV-visible CD spectrum had features indicative of a [2Fe-2S] cluster and was similar to WT ferredoxin (both Anabaena and human), the wavelengths and relative intensities of CD bands of peaks and troughs in the spectrum of holo D40A Tm IscUHis looked much like those observed for C46S Anabaena ferredoxin in which one of the Cys cluster ligands is substituted with a Ser (32). No naturally occurring [2Fe-2S] proteins to date have been shown to have an oxygen ligand, although primary amino acid sequence analysis of some [2Fe-2S] proteins suggests that it may exist (39). IscU proteins have three conserved Cys that upon mutation result in loss of Fe-S cluster coordination (8, 27). The nature of the remaining ligand has yet to be identified. Possibilities include solvent or an oxygen/nitrogen amino acid side chain; however, the absence of His in Tm IscU makes nitrogen ligation unlikely. Resonance Raman results from A. vinelandii IscU are consistent with either complete Cys ligation or single serinate ligation (17). Other than Asp-40, the conserved oxygen side chains derive from Asp-13, Asp-55, Ser-69, Ser-71, and Glu-83. Asp-55 is unlikely to be a ligand because D55A Tm IscUHis expresses as holoprotein (data not shown). Future structural and mutagenesis studies may help to better define the coordination environment of IscU-type clusters.

Cluster Stoichiometry-- The experimentally determined extinction coefficients suggest one cluster per dimer, consistent with direct estimation of the iron content of D40A Tm IscU, which gave 1.2 ± 0.1 Fe/monomer. Iron concentrations were accurately determined by atomic absorption and protein concentrations determined from the experimental extinction values. The occurrence of low concentrations of adventitiously bound iron, evidenced by Mössbauer data, and small losses in protein during desalting steps account for the slightly higher Fe:protein ratio identified. Correction for this results in close agreement with a single [2Fe-2S] cluster per dimer of protein. Certainly the dimerization of protein upon cluster acquisition and conversion from apo to holo states is attractively consistent with a single bridging cluster per dimer. Agar et al. (4) have isolated forms of A. vinelandii IscU with both one and two clusters per dimer and have shown that the one cluster form is more stable to iron chelators. Thus far, the only monomeric IscU identified is the human ISU, which binds a single cluster per monomer (8); however, other dimeric IscU-type proteins all appear to coordinate one cluster per protein subunit. Accordingly, the cluster stoichiometry for the holo form of Tm IscU may need to await further structural characterization for confirmation of a bridging cluster.

Thermal Stability-- The thermal stability of the [2Fe-2S] cluster in D40A Tm IscU is distinct from that of other ferredoxin-type clusters in Fe-S proteins from both thermophilic as well as mesophilic organisms. The [4Fe-4S] cluster of T. maritima ferredoxin is stable past the boiling point of water, with a calculated transition temperature of 125 °C (40). Even the [2Fe-2S] cluster of human ferredoxin shows greater thermal stability than D40A Tm IscU. The thermal profile of human ferredoxin is typical of Fe-S cluster proteins in which no cluster is lost up until a specific temperature is reached, after which the cluster is rapidly released following loss of necessary structural elements. Indeed, the thermal cluster profile of human ferredoxin corresponds to changes in structure observed by CD and is consistent with what is currently known regarding the influences of metal cofactor coordination on proper protein folding (41, 42). Interestingly, D40A Tm IscU did not exhibit a thermal profile similar to any Fe-S protein characterized thus far. Cluster loss was evident over a wide temperature range proceeding without an obvious thermal threshold. Such behavior is particularly surprising, considering that T. maritima can survive temperatures up to 90 °C (12), and suggests a stabilizing role by other proteins (such as chaperones) in the intracellular environment. Furthermore, no structural change was observed by CD over the tested temperature range or following cluster coordination. Accordingly, unlike many other Fe-S cluster-binding proteins, cluster loss does not coincide with loss of secondary and tertiary structural elements. The data are also consistent with the ability to reconstitute D40A Tm IscU in the absence of low levels of denaturant that are occasionally required to allow the protein to properly fold around the cluster (20).

Tm IscU Reconstitution of Human Apoferredoxin-- Cluster transfer between two proteins from extremely distant organisms such as T. maritima and Homo sapiens demonstrates the high degree of conservation of the Fe-S cluster assembly apparatus and is consistent with the notion of Fe-S proteins being evolutionarily ancient (43, 44). The cluster transfer reaction from D40A Tm IscUHis to human apoferredoxin was rapid even at low temperatures where background IscU cluster loss is negligible. Human apoferredoxin retains much of its secondary structure (Fig. 6C) (30) that presumably is recognized by IscU. This recognition motif must be conserved for such divergent sources of IscU to be competent in assisted Fe-S cluster maturation of ferredoxin. This is the first reported example of cluster transfer from an IscU-type domain to a target ferredoxin. It has been previously shown, in an example from a distinct protein family, that E. coli IscA can transfer an Fe-S cluster to E. coli apoferredoxin (45), and that a protein homologous to the carboxyl-terminal domain of NifU can similarly transfer a cluster to ferredoxin (21). The differences in the physiological roles between these different protein families are not currently known.

Structural Considerations-- The circular dichroism (Fig. 6) and NMR data reveal for the first time structural information regarding this family of proteins. Other IscU family members that have been studied in our laboratory (namely human and yeast homologues) lack CD and NMR features that support secondary and tertiary structural elements. Most likely, this reflects conformational flexibility that is of inherent functional relevance for these proteins. We had hoped that such a protein from a thermophilic organism might possess a degree of structural stability that is often associated with proteins from such organisms. We here demonstrate this to be, in fact, the case. The ability of the holo Tm IscU to functionally transfer cluster to a human apoFd speaks to the functional equivalence of this family of proteins. Undoubtedly other family members adopt key structural and functional states, but typically these are not detected by standard structural methods. The thermophile apparently diminishes the conformational flexibility sufficiently to allow these states to be identified.

	ACKNOWLEDGEMENT

We thank Jon-David Sears for help with bacterial fermentations.

	FOOTNOTES

^* This work was supported in part by a grant from the Petroleum Research Fund, administered by the American Chemical Society (to J. A. C.), and by National Science Foundation Grant CHE-0111161 (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by the National Institutes of Health Chemistry and Biology Interface Training Program at Ohio State University (Grant GM08512-03).

To whom correspondence should be addressed: Evans Laboratory of Chemistry, Ohio State University, 100 W. 18th Ave., Columbus, OH 43210. Fax: 614-292-1685; E-mail: cowan@chemistry.ohio-state.edu.

Published, JBC Papers in Press, April 3, 2002, DOI 10.1074/jbc.M201439200

	ABBREVIATIONS

The abbreviations used are: Nif, nitrogen fixation; HSQC, heteronuclear single quantum correlation; DTT, dithiothreitol; EPR, electron paramagnetic resonance; ESI, electrospray ionization; Fd, ferredoxin; Hs Fd, human ferredoxin; Isc, iron-sulfur cluster; WT, wild type; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit