“…However, the spectra of the three variant molecules are almost indistinguishable from each other and are indeed identical in the visible region above 350 nm, with a broad maximum at 415 nm and a shoulder at around 455 nm. Similar spectral features have been reported previously for [3Fe-4S] proteins, such as DgFd (41). The peak at 260 nm observed for BtFd V-II is consistent with the presence of a nucleotide moiety (CoA) as described above.…”
Section: Resultssupporting
confidence: 89%
“…More intriguingly, DgFd was purified from its native cell type in both [3Fe-4S] and [4Fe-4S] forms. In contrast, in Vitro chemical reconstitution of the Fe-S cluster from apo-DgFd yields only the [4Fe-4S] type, whereas recombinant DgFd expressed in E. coli is purified predominantly as a [3Fe-4S] form(18,41). To date, modification of Cys11 in recombinant [3Fe-4S] DgFd has not been examined.Physiological Considerations.…”
During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.
“…However, the spectra of the three variant molecules are almost indistinguishable from each other and are indeed identical in the visible region above 350 nm, with a broad maximum at 415 nm and a shoulder at around 455 nm. Similar spectral features have been reported previously for [3Fe-4S] proteins, such as DgFd (41). The peak at 260 nm observed for BtFd V-II is consistent with the presence of a nucleotide moiety (CoA) as described above.…”
Section: Resultssupporting
confidence: 89%
“…More intriguingly, DgFd was purified from its native cell type in both [3Fe-4S] and [4Fe-4S] forms. In contrast, in Vitro chemical reconstitution of the Fe-S cluster from apo-DgFd yields only the [4Fe-4S] type, whereas recombinant DgFd expressed in E. coli is purified predominantly as a [3Fe-4S] form(18,41). To date, modification of Cys11 in recombinant [3Fe-4S] DgFd has not been examined.Physiological Considerations.…”
During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.
“…Moreover, the Fe-S cluster geometry in the anaerobic structure is different from that in the aerobic structure, with a maximum increase and decrease of 0.15 and 0.14 Å for the Fe-S bond length ranges, respectively. A similar difference in the Fe-S cluster geometries was previously observed when recombinant DgFdII was characterized spectroscopically [81]: differences in the paramagnetic envelope of the NMR spectra of both the recombinant and the native proteins already pointed to structural changes in the [3Fe-4S] cluster geometries.…”
Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe 3 S 4 cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII int ) with four 1 H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII ox and FdII int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same 1 H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe-4S] core in the oxidized state (DgFdII ox ) was used here for the assignment of the same three ligands in the intermediate state (DgFdII int ). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm À1 and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII int . This study, together with the spin delocalization mechanism proposed here for DgFdII int , allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII ox and DgFdII int .
“…After purification with column chromatography, over 95% pure holoTtFd was obtained. Such a recombinant holo ferredoxin from the apo-ferredoxin has been reported. ,,− Holo TtFd gives a spot separated from apo TtFd in native PAGE analysis as shown in Figure . 1 Electrophoresis analysis of reconstituted TtFd and native TtFd.…”
Section: Resultsmentioning
confidence: 74%
“…Such a recombinant holo ferredoxin from the apoferredoxin has been reported. 6,10,[15][16][17] Holo TtFd gives a spot separated from apo TtFd in native PAGE analysis as shown in Figure 1.…”
Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin. The catalytic electron-transfer reactivity of the [7Fe-8S] ferredoxin between ferredoxin-NADP+ reductase and cytochrome c was recognized.
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