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W2100489286 abstract "[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandiiferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions. [4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandiiferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions. One of the most interesting and important features of protein-bound [Fe-S] clusters is their ability to convert from one form to another (for reviews see Refs. 1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1470) Google Scholar, 2Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2313) Google Scholar, 3Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. Wiley Interscience, New York1994: 1896-1915Google Scholar, 4Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (293) Google Scholar, 5Howard J.B. Rees D.C. Adv. Protein Chem. 1991; 42: 199-280Crossref PubMed Google Scholar, 6Beinert H. FASEB J. 1990; 4: 2483Crossref PubMed Scopus (189) Google Scholar, 7Lindahl P.A. Kovacs J.A. J. Cluster Sci. 1990; 1: 29-73Crossref Scopus (36) Google Scholar). The simplest of these reactions involves the interconversion of [3Fe-4S]+/0 and [4Fe-4S]2+/+ clusters. These two cluster types are structurally very closely related and differ only by the presence or absence of a single iron atom at one corner of a cube. The physiological relevance of an iron loss or uptake mechanism is best illustrated by the example of aconitase and related dehydratases. In the best studied system, aconitase, the [3Fe-4S] cluster is inactive, and the spontaneous conversion to the [4Fe-4S] form of the enzyme represents a self-activation of the enzyme (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1470) Google Scholar, 8Kent T.A. Emptage M.H. Merkle H. Kennedy M.C. Beinert H. Münck E. J. Biol. Chem. 1985; 260: 6871-6881Abstract Full Text PDF PubMed Google Scholar). For the related iron-responsive element mRNA-binding protein, which is involved in iron homeostasis, the reverse reaction may be the first step on the route to apoprotein production (9Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1045) Google Scholar). In a quite different protein,Desulfovibrio gigas ferredoxin II (DgFdII), 1The abbreviations used are: DgFdII, D. gigas ferredoxin II; AvFdI, A. vinelandii ferredoxin I; TEMED, N,N,N′,N′-tetramethylethylenediamine; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; FdI, ferredoxin I; PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. 1The abbreviations used are: DgFdII, D. gigas ferredoxin II; AvFdI, A. vinelandii ferredoxin I; TEMED, N,N,N′,N′-tetramethylethylenediamine; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; FdI, ferredoxin I; PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. the 3Fe to 4Fe cluster conversion reaction, which is proposed to be controlled by the physiological effector pyruvate, is accompanied by a change in subunit composition of the protein, and the two forms participate in completely different electron transfer pathways (10Moura J.J.G. Moura I. Kent T.A. Lipscomb J.D. Huynh B.H. LeGall J. Xavier A.V. Münck E. J. Biol. Chem. 1982; 257: 6259-6267Abstract Full Text PDF PubMed Google Scholar, 11Moura J.J.G. LeGall J. Xavier A.V. Eur. J. Biochem. 1984; 141: 319-322Crossref PubMed Scopus (19) Google Scholar). ForBacillus subtilis amidotransferase the stability of the enzyme depends on the presence of a [4Fe-4S]2+/+ cluster, and exposure to dioxygen inactivates the enzyme by destroying its cluster (12Grandoni J.A. Switzer R.L. Makaroff C.A. Zalkin H. J. Biol. Chem. 1989; 264: 6058-6064Abstract Full Text PDF PubMed Google Scholar). Indeed numerous [4Fe-4S]2+/+-containing proteins are extremely sensitive to dioxygen, and the destruction of the cluster often initiates with the formation of a [3Fe-4S] cluster. Most proteins that contain [3Fe-4S]+/0 clusters do not undergo this type of cluster interconversion, and those that do vary in the ease and reversibility of the reaction (1Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1470) Google Scholar, 2Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2313) Google Scholar, 3Johnson M.K. King R.B. Encyclopedia of Inorganic Chemistry. Wiley Interscience, New York1994: 1896-1915Google Scholar, 4Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (293) Google Scholar, 5Howard J.B. Rees D.C. Adv. Protein Chem. 1991; 42: 199-280Crossref PubMed Google Scholar, 6Beinert H. FASEB J. 1990; 4: 2483Crossref PubMed Scopus (189) Google Scholar, 7Lindahl P.A. Kovacs J.A. J. Cluster Sci. 1990; 1: 29-73Crossref Scopus (36) Google Scholar). At present the structural features that are responsible for the variable reactivity of 3Fe and 4Fe clusters are not understood. In the majority of proteins that are known to undergo facile 3Fe to 4Fe cluster interconversion, including aconitase, the [4Fe-4S]2+/+ cluster has one noncysteine ligand, and it is the iron atom that is coordinated by that ligand that is labile (13George S.J. Armstrong F.A. Hatchikian E.C. Thomson A.J. Biochem. J. 1989; 264: 275-284Crossref PubMed Scopus (115) Google Scholar, 14Conover R.C. Kowal A.T. Fu W. Park J.-B. Aono S. Adams M.W.W. Johnson M.K. J. Biol. Chem. 1990; 265: 8533-8541Abstract Full Text PDF PubMed Google Scholar, 15Thomson A.J. Breton J. Butt J.N. Hatchikian E.C. Armstrong F.A. J. Inorg. Biochem. 1992; 47: 197-207Crossref PubMed Scopus (16) Google Scholar, 16Gorst C.M. Yeh Y.H. Teng Q. Calzolai L. Zhou Z.H. Adams M.W. La Mar G.N. Biochemistry. 1995; 34: 600-610Crossref PubMed Scopus (47) Google Scholar, 17Calzolai L. Gorst C.M. Zhao Z. Teng Q. Adams M.W.W. La Mar G.N. Biochemistry. 1995; 34: 11373-11384Crossref PubMed Scopus (88) Google Scholar, 18Calzolai L. Zhou Z.H. Adams M.W.W. La Mar G.N. J. Am. Chem. Soc. 1996; 118: 2513-2514Crossref Scopus (32) Google Scholar, 19Calzolai L. Gorst C.M. Bren K.L. Zhou Z. Adams M.W.W. La Mar G.N. J. Am. Chem. Soc. 1997; 119: 9341-9350Crossref Scopus (40) Google Scholar, 20Robbins A.H. Stout C.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3639-3643Crossref PubMed Scopus (223) Google Scholar, 21Robbins A.H. Stout C.D. Proteins. 1989; 5: 289-312Crossref PubMed Scopus (161) Google Scholar). In Desulfovibrio africanus FdIII, Desulfovibrio vulgaris FdI, and Pyrococcus furiosus Fd, the [4Fe-4S]2+/+ forms of the proteins appear to have three cysteine and one aspartate as ligands, and again the iron atom associated with the aspartate is the labile iron (13George S.J. Armstrong F.A. Hatchikian E.C. Thomson A.J. Biochem. J. 1989; 264: 275-284Crossref PubMed Scopus (115) Google Scholar, 14Conover R.C. Kowal A.T. Fu W. Park J.-B. Aono S. Adams M.W.W. Johnson M.K. J. Biol. Chem. 1990; 265: 8533-8541Abstract Full Text PDF PubMed Google Scholar, 15Thomson A.J. Breton J. Butt J.N. Hatchikian E.C. Armstrong F.A. J. Inorg. Biochem. 1992; 47: 197-207Crossref PubMed Scopus (16) Google Scholar, 16Gorst C.M. Yeh Y.H. Teng Q. Calzolai L. Zhou Z.H. Adams M.W. La Mar G.N. Biochemistry. 1995; 34: 600-610Crossref PubMed Scopus (47) Google Scholar, 17Calzolai L. Gorst C.M. Zhao Z. Teng Q. Adams M.W.W. La Mar G.N. Biochemistry. 1995; 34: 11373-11384Crossref PubMed Scopus (88) Google Scholar, 18Calzolai L. Zhou Z.H. Adams M.W.W. La Mar G.N. J. Am. Chem. Soc. 1996; 118: 2513-2514Crossref Scopus (32) Google Scholar, 19Calzolai L. Gorst C.M. Bren K.L. Zhou Z. Adams M.W.W. La Mar G.N. J. Am. Chem. Soc. 1997; 119: 9341-9350Crossref Scopus (40) Google Scholar). It is interesting in those cases that the aspartate replaces the cysteine at the central position of a [4Fe-4S]2+/+Cys-X-X-Cys-X-X-Cys-binding motif (Fig. 1). If the aspartate is replaced by a cysteine using site-directed mutagenesis, a stable 4Fe cluster results, but it can no longer be easily converted to a 3Fe cluster (22Busch J.L.H. Breton J.L. Bartlett B.M. Armstrong F.A. James R. Thomson A.J. Biochem. J. 1997; 323: 95-102Crossref PubMed Scopus (43) Google Scholar, 23Manodori A. Cecchini G. Schröder I. Gunsalus R.P. Werth M.T. Johnson M.K. Biochemistry. 1992; 31: 2703-2712Crossref PubMed Scopus (73) Google Scholar, 24Aono S. Bentrop D. Bertini I. Luchinat C. Macinai R. FEBS Lett. 1997; 412: 501-505Crossref PubMed Scopus (9) Google Scholar). Like these mutants, most native protein-bound [4Fe-4S]2+/+ clusters that have four cysteine ligands can be converted to the [3Fe-4S]+/0state only during destructive reactions en route to apoprotein, and stable 3Fe cluster forms are not available to study the reverse reaction (25Thomson A.J. Robinson A.E. Johnson M.K. Cammack R. Rao K.K. Hall D.O. Biochim. Biophys. Acta. 1981; 637: 423-432Crossref Scopus (69) Google Scholar, 26Bertini I. Briganti F. Calzolai L. Messori L. Scozzafava A. FEBS Lett. 1993; 332: 268-272Crossref PubMed Scopus (16) Google Scholar, 27Bell S.H. Dickson P.E. Johnson C.E. Cammack R. Hall D.O. Rao K.K. FEBS Lett. 1982; 142: 143-146Crossref PubMed Scopus (25) Google Scholar). An exception is DgFdII where the central Cys of the Cys-X-X-Cys-X-X-Cys binding motif is covalently modified by a thiomethane group and rotated away from the cluster and is therefore not used as a ligand in the [3Fe-4S]+/0 form of the protein (28Kissinger C.R. Sieker L.C. Adams E.T. Jensen L.H. J. Mol. Biol. 1991; 219: 693-715Crossref PubMed Scopus (143) Google Scholar). Azotobacter vinelandii FdI is an extremely well characterized 7Fe protein, which contains a [3Fe-4S] cluster that cannot be easily converted to a [4Fe-4S] cluster (29Stout G.H. Turley S. Sieker L.C. Jensen L.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1020-1022Crossref PubMed Scopus (103) Google Scholar, 30Stout C.D. J. Biol. Chem. 1988; 263: 9256-9260Abstract Full Text PDF PubMed Google Scholar, 31Stout C.D. J. Mol. Biol. 1989; 205: 545-555Crossref PubMed Scopus (137) Google Scholar, 32Morgan T.V. Stephens P.J. Burgess B.K. Stout C.D. FEBS Lett. 1984; 167: 137-141Crossref Scopus (24) Google Scholar, 33.Jensen, G. M., Azotobacter vinelandii Ferrodoxin I. Spectroscopic and theoretical studies.Ph.D. dissertation, 1994, University of Southern California.Google Scholar). As shown in Fig. 1 the AvFdI [3Fe-4S] cluster is ligated by cysteine residues at positions 8, 16, and 49. The 3Fe region of the protein therefore does not have either a Cys-X-X-Cys-X-X-Cys or a Cys-X-X-Asp-X-X-Cys motif. There is an additional free Cys at position 11 but that Cys is far removed from the cluster and is not used as a ligand. Our interest in converting the 3Fe cluster of AvFdI to a 4Fe cluster by mutagenesis is motivated both by a desire to understand the structural features that control the interconversion reactions and by the suggestion that the 3Fe to 4Fe conversion reaction, which is so difficult to accomplish with nativeAvFdI in vitro, might be physiologically relevant (34Thomson A.J. FEBS Lett. 1991; 285: 230-236Crossref PubMed Scopus (33) Google Scholar). That suggestion is based in part on the surprising observation that when apo-FdI is reconstituted in vitro with iron and sulfide the product is an 8Fe protein containing two [4Fe-4S]2+/+ clusters rather than the extremely stable and well characterized 7Fe form of the protein that contains one [3Fe-4S]+/0 and one [4Fe-4S]2+/+ cluster (32Morgan T.V. Stephens P.J. Burgess B.K. Stout C.D. FEBS Lett. 1984; 167: 137-141Crossref Scopus (24) Google Scholar). In a previous study we constructed a Y13C FdI variant that could easily be modeled with a 4Fe cluster in the 3Fe cluster position. That design was unsuccessful, resulting in a 7Fe protein possibly because A. vinelandii covalently modified the cysteine to form a persulfide that could not be used as a ligand (35Kemper M.A. Stout C.D. Lloyd S.E.J. Prasad G.S. Fawcett S. Armstrong F.A. Shen B. Burgess B.K. J. Biol. Chem. 1997; 272: 15620-15627Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). That interpretation is consistent with a recent report that the same mutation in a related ferredoxin from Bacillus schlegelii, which was heterologously expressed in Escherichia coli, appears to contain a 4Fe cluster in the 3Fe position (24Aono S. Bentrop D. Bertini I. Luchinat C. Macinai R. FEBS Lett. 1997; 412: 501-505Crossref PubMed Scopus (9) Google Scholar). 2That report did not, however, include direct UV-visible, CD, or EPR evidence to demonstrate the presence of the former cluster type and the absence of the latter. 2That report did not, however, include direct UV-visible, CD, or EPR evidence to demonstrate the presence of the former cluster type and the absence of the latter. In another study we were successful in constructing an 8Fe version of FdI by deleting residues Thr-14 and Asp-15 to create a Cys-X-X-Cys-X-X-Cys motif in the region of the 3Fe cluster (36Kemper M.A. Gao-Sheridan S. Shen B. Duff J.L.C. Tilley G.J. Armstrong F.A. Burgess B.K. Biochemistry. 1998; 37: 12829-12837Crossref PubMed Scopus (5) Google Scholar). However, like the four-Cys coordinated clusters discussed above, the conversion of the new 4Fe cluster back to the 3Fe cluster was a destructive reaction, and a pure sample of the 7Fe form of ΔT14/ΔD15 FdI could not be isolated (36Kemper M.A. Gao-Sheridan S. Shen B. Duff J.L.C. Tilley G.J. Armstrong F.A. Burgess B.K. Biochemistry. 1998; 37: 12829-12837Crossref PubMed Scopus (5) Google Scholar). Here we report the construction, characterization, and interconversion of a T14C variant of FdI that can be isolated both in 7Fe and 8Fe forms and the observation that it is the 7Fe form that accumulates in vivo. The oligonucleotide used for the mutagenesis had the sequence 5′-AAGTGCAAGTACTGCGATTGTGTTGAA-3′ which differs from the wild-type sequence by the substitution of TGC (encoding Cys) for ACC (encoding Thr). The oligo-directed mutagenesis, FdI overexpression inA. vinelandii, and cell growth were performed as described previously (37Shen B. Martin L.L. Butt J.N. Armstrong F.A. Stout C.D. Jensen G.M. Stephens P.J. La Mar G.N. Gorst C. Burgess B.K. J. Biol. Chem. 1993; 268: 25928-25939Abstract Full Text PDF PubMed Google Scholar). The parent A. vinelandii strain used for the expression of T14C FdI, designated DJ138, is lacking the genes encoding AvFdI and flavodoxin. The constructed T14C FdI variant strain is designated DJ138/pBS3A1. The purification of native FdI and T14C FdI follows a modification of the purification method described previously (38Stephens P.J. Jensen G.M. Devlin F.J. Morgan T.V. Stout C.D. Martı́n A.E. Burgess B.K. Biochemistry. 1991; 30: 3200-3209Crossref PubMed Scopus (47) Google Scholar). All steps were carried out anaerobically with or without (as specified below) 2 mm Na2S2O4 present in all buffers. A. vinelandii T14C FdI variant was grown under the N2-fixing condition, and cell-free extracts were prepared from about 1 kg of cell paste. The cell-free extracts were loaded onto a 5 × 20-cm DE52 cellulose column equilibrated with 0.025 m Tris·HCl, pH 7.4. A 3-liter linear 0.1–0.5m NaCl gradient was applied, and T14C FdI was eluted completely by a NaCl concentration of 0.45 m. The ferredoxin fraction, while being diluted with 2 volumes of 0.1m potassium phosphate buffer, pH 7.4, was immediately loaded onto a 2.5 × 12 cm DE52 cellulose column equilibrated with the same buffer. The column was then washed with 2 liters of 0.1m potassium phosphate buffer, pH 7.4, 0.12 m in KCl, at 240 ml/min. T14C FdI was eluted with 0.3 m KCl. Saturated ammonium sulfate in 0.05 m Tris·HCl, pH 7.4, was added very slowly with stirring to the T14C FdI fraction to 75% saturation in ammonium sulfate. The precipitated protein was recovered by centrifugation and dissolved in the minimum volume of 0.025m Tris·HCl, pH 7.4, 0.1 m NaCl. The T14C FdI solution was then loaded onto a 2.5 × 100-cm Sephadex G-50 superfine (Amersham Pharmacia Biotech) gel filtration column equilibrated with 0.025 m Tris·HCl, pH 7.4, 0.1m NaCl. The eluted T14C FdI fraction was concentrated using an Amicon ultrafiltration unit with a YM-10 membrane. For spectroscopic studies all protein samples were prepared anaerobically under argon in a Vacuum Atmospheres glove box (O2 <1 ppm) using degassed buffers. Where concentrated protein was required, the samples were concentrated and buffer exchanged using Centricon-10 microconcentrators. The photoreduction reaction was carried out by mixing T14C FdI, 5′-deazariboflavin and EDTA at final concentrations of 100, 200, and 20 μm, respectively, in 0.1 m Tris·HCl, pH 7.4, and then illuminating for 1 min using white light from a slide projector. UV-visible spectra were obtained with a Hewlett-Packard 8452 Diode Array UV-visible spectrophotometer. CD spectra were recorded using a JASCO J720 spectropolarimeter. EPR spectra were obtained using a Bruker 300 Ez spectrometer, interfaced with an Oxford Instruments ESR-9002 liquid helium continuous flow cryostat. Samples for whole cell EPR were prepared as described elsewhere (36Kemper M.A. Gao-Sheridan S. Shen B. Duff J.L.C. Tilley G.J. Armstrong F.A. Burgess B.K. Biochemistry. 1998; 37: 12829-12837Crossref PubMed Scopus (5) Google Scholar). To determine iron content, samples were digested, and the analysis was carried out as described elsewhere (39Burgess B.K. Jacobs D.B. Stiefel E.I. Biochim. Biophys. Acta. 1980; 614: 196-209Crossref PubMed Scopus (233) Google Scholar) using FeCl3·6H2O to generate a standard curve with the native FdI as control. A parallel Lowry assay (40Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) was carried out to confirm the quantities of proteins used in the iron analysis. The native polyacrylamide gel electrophoresis is a modification of the Tricine gel electrophoresis method described elsewhere (41Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). All steps were carried out anaerobically. The acrylamide gel mix contains acrylamide and bisacrylamide at a 37.5:1 ratio. Each gel consisted of a 12% separating layer and a 4% stacking layer. To the separating gel solution, freshly prepared 10% ammonium persulfate and TEMED were added to give final concentrations of 165 μm and 1.32 mm, respectively. Polymerization occurred after 5 min of gel casting. To the stacking gel solution, 10% ammonium persulfate and TEMED were added to give final concentrations of 300 μmand 5 mm, respectively. Polymerization occurred within 30 min. The gels were pre-run for 30 min at 70 V with the running buffer containing 2 mmNa2S2O4. After the protein samples were loaded, the gels were run for 2.5 h at 70 V. The cathode buffer was refreshed with fresh running buffer at 30-min intervals to ensure the continuous presence of sodium dithionite in the gel. The completed gels were treated with Coomassie Blue staining. Purified water of resistivity ∼18 MΩ·cm (Millipore) was used in all experiments. The buffers MES, HEPES, and TAPS, and co-adsorbates polymyxin B sulfate and neomycin sulfate were purchased from Sigma. Other reagents were purchased from Aldrich and were of at least analytical grade. An AutoLab electrochemical analyzer (Eco Chemie, Utrecht, The Netherlands) was used to record DC cyclic voltammograms (in the digital mode) and square-wave voltammetry. The three-electrode configuration featuring all-glass cells (typically holding 600 μl and either a single pot or multipot for pH dependence and metal-uptake experiments) has been described previously (42Armstrong F.A. Butt J.N. Sucheta A. Methods Enzymol. 1993; 227: 479-500Crossref PubMed Scopus (66) Google Scholar). The sample compartment was maintained at 0 °C. All potential values are given with reference to the standard hydrogen electrode. The saturated calomel reference electrode was held at 22 °C, which we have adopted asE(saturated calomel reference electrode) = +243 mVversus standard hydrogen electrode. Reduction potentials from cyclic voltammograms were calculated as the average of the anodic and cathodic peak potentials, E 0′ = 1/2(E pa + E pc). The pyrolytic graphite “edge” (PGE) electrode (surface area typically 0.18 cm2) was polished prior to each experiment with an aqueous alumina slurry (Buehler Micropolish: 0.3 μm for solution electrochemistry or 1.0 μm for protein film voltammetry) and then sonicated extensively to remove traces of Al2O3. All experiments and handling were carried out under anaerobic conditions in a glove box (Belle Technology, Poole, UK) with an inert atmosphere of N2(O2 <1.0 ppm). Cell solutions used for protein film voltammetry comprised a 60 mm mixed buffer system (15 mm each in HEPES, acetate, TAPS, MES, 0.1 m NaCl) except for experiments at pH 8.5–10.0 in which the buffer also included 7.5 or 15 mmCHES, respectively. Solutions also contained 0.1 mm EGTA and 200 μg/ml polymyxin, with 0.1 m NaCl as principal supporting electrolyte. The pH was adjusted with either HCl or NaOH and was measured at 0 °C before and immediately after experiments. Voltammograms were scanned first in a pot containing a cell solution at pH 7.0 in order to establish a film, following which the coated electrode was transferred to other pots containing solutions at a different pH values or containing Fe(II), Zn(II), or Tl(I). Solutions of metal ions Fe(II) ((NH4)2Fe(SO4)2·6H2O), Zn(II) (ZnSO4·6H2O), and Tl(I) (Tl(O2 CCH3)·6H2O) were made up in 20 mm HEPES, 0.1 m NaCl, and 200 μg/ml polymyxin solution at pH 7.0. For film experiments, the solutions used to coat the electrodes were as follows. For the 7Fe form, a 300 μm protein solution was prepared using 45 μl of the mixed buffer cell solution at pH 7.0, mixed with 10 μl of a stock solution of 1.6 mm protein in 25 mm Tris·HCl at pH 7.4. For films of the 8Fe ferredoxin, the solution contained 120 μm protein in 0.1m Tris·HCl, 0.1 m NaCl, 200 μg/ml polymyxin, with 2.5 mm sodium dithionite, and 1 mm Fe(II) at pH 7.4. Films were produced by painting a freshly polished electrode surface with about 1 μl of chilled protein solution using a Pasteur pipette drawn to a fine capillary tip, following which the electrode was placed promptly into the cell solution. For bulk solution voltammetry of the 7Fe form, the solution contained 160 μm protein, 25 mm Tris·HCl, and 0.1m NaCl at pH 7.4. For the 8Fe form, the solution contained 120 μm protein, in 0.1 m Tris·HCl and 0.1m NaCl with 1 mm Fe(II), and 2.5 mmsodium dithionite, at pH 7.4. Small aliquots of neomycin stock solution were added (final concentration 4.0 mm) to promote a strong and persistent electrochemical response. This solution was used for both cyclic and square-wave voltammetry. Crystals of oxidized T14C FdI in the 7Fe form were grown by seeding using small native FdI crystals as described previously (48Shen B. Jollie D.R. Stout C.D. Diller T.C. Armstrong F.A. Gorst C.M. La Mar G.N. Stephens P.J. Burgess B.K. J. Biol. Chem. 1994; 269: 8564-8575Abstract Full Text PDF PubMed Google Scholar). A large, single crystal, 0.5 × 0.7 × 0.9 mm in size, was mounted in a thin-walled glass capillary using a synthetic mother liquor of 4.8 m(NH4)2SO4, 0.45 mTris·HCl, pH 8.1. Data were collected at room temperature using CuKα radiation from a RU200 x-ray generator operated at 40 kV, 80 mA, and equipped with a graphite monochromator and Xentronics X-1000 area detector mounted on a Siemens P4 goniostat. Data were collected with a single phi scan of 180° with 0.25° oscillations and 6-min exposures per frame. The crystal did not exhibit significant deterioration in the 3 days required for data collection. The data were indexed, integrated, reduced, merged, and scaled using the Xengen suite of programs (TableI) (49Howard A.J. Nielsen C. Xuong N.-H. Methods Enzymol. 1985; 114: 452-472Crossref PubMed Scopus (280) Google Scholar).Table IData collection and refinement statistics for oxidized 7Fe T14C FdISpace groupP4(1)2(1)2Unit cell parameters, Åa = 55.88, b = 55.88,c = 96.40No. of observations33,677No. of independent reflections8,686R symm(F)0.059I/ς(I), all data18.8I/ς(I), last shell2.8Resolution of last shell, Å2.24–2.10Completeness, all data92.5%Completeness, last shell59.9%R factor0.206Resolution, Å8.0–2.10Reflections >0 ς(F)8225Average B factor, Å2840 protein atoms21.0[4Fe-4S] cluster17.3[3Fe-4S] cluster13.348 H2O molecules34.9Root mean square deviation from idealityBonds, Å0.014Angles, degree3.09Planes, Å1.48 Open table in a new tab The structure was solved by molecular replacement using Xplor version 3.8 (50Brünger A.T. Karplus M. Petsko G.A. Acta Crystallogr. A. 1989; 45: 50-61Crossref Scopus (271) Google Scholar). Starting coordinates of native FdI (31Stout C.D. J. Mol. Biol. 1989; 205: 545-555Crossref PubMed Scopus (137) Google Scholar) were modified to contain a Gly at both residues 11 and 14, and all H2O molecules were omitted from the model. This model was refined as a rigid body using data in the resolution range 8.0 to 4.0 Å and then by positional refinement using data to 4.0-, 3.5-, 3.0-, 2.7-, and 2.5-Å resolution. The resulting phases, unbiased for the side chains of residues 11 and 14, were used to calculate ‖F o‖ − ‖F c‖ and 2‖F o‖ − ‖F c‖. Fourier maps with all data in the resolution range 20.0 to 2.5 Å. The difference Fourier map revealed large positive 5ς peaks at the positions of Sγ of Cys-11 and C-γ2 of Thr-14 of native FdI, providing an internal check on the correctness of the phases and revealing the position of the Sγ atom in the mutated Cys-14 side chain. The 2.5-Å resolution 2‖F o‖ − ‖F c‖ map was consistent with Cys-11 and Cys-14 side chains and was used to model the Cys-14 side chain and remodel the Cys-11 side chain. Density fitting and model building were carried out with the Xtalview suite of programs (51McRee D.E. J. Mol. Graphics. 1992; 10: 44-47Crossref Google Scholar). The electron density and difference electron density did not indicate any significant changes in the [3Fe-4S] cluster or other portions of the structure. The structure was refined by Powell minimization and isotropic B factor refinement at 2.3 Å and then 2.1 Å resolution using Xplor version 3.8 (50Brünger A.T. Karplus M. Petsko G.A. Acta Crystallogr. A. 1989; 45: 50-61Crossref Scopus (271) Google Scholar). Side chains of three residues in contact with Thr-14 in native FdI, Asp-15, Lys-84, and Lys-85, were adjusted slightly to fit the 2‖F o‖ − ‖F c‖ electron density. Difference Fourier maps were used to locate peaks >3ς for H2O molecules; 48 H2O molecules refined withB factors <50.0 A2 were retained in the model. Statistics for the final refined structure are given in Table I. There are no non-glycine outliers in the Ramachandran plot. Coordinates for oxidized 7Fe T14C FdI have been deposited with the Protein Data Band code 1A6L. In an attempt to convert the [3Fe-4S] cluster into a [4Fe-4S] cluster in the crystal lattice, as observed in solution, two additional data sets were collected. A large single crystal of 7Fe T14C FdI was wedged on a capillary and bathed in a synthetic mother li" @default.
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W2100489286 date "1998-12-01" @default.
W2100489286 modified "2023-09-26" @default.
W2100489286 title "A T14C Variant of Azotobacter vinelandii Ferredoxin I Undergoes Facile [3Fe-4S]0 to [4Fe-4S]2+Conversion in Vitro but Not in Vivo" @default.
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W2100489286 doi "https://doi.org/10.1074/jbc.273.50.33692" @default.
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