A Simple, Rapid, and Highly Efficient Gene Expression System for Multiheme Cytochromes c

Ozawa, Kiyoshi; Yasukawa, Fumiko; Fujiwara, Y.; Akutsu, Hideo

doi:10.1271/bbb.65.185

Cited by 34 publications

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“…Mutated cyt c 3 species were expressed in Shewanella oneidensis TSP-C transformed with a mutated plasmid, as described previously (17 adenosine, 20 mg/mL guanosine, 20 mg/mL cytidine, 20 mg/ mL thymidine, 20 mg/mL uridine, 20 mg/mL thiamine hydrochloride, 20 mg/mL R-biotin, and an amino acid mixture. The amino acid mixture (all per liter of culture) consisted of 300 mg of Ala, 200 mg of Arg, 400 mg of Asn, 400 mg of Asp, 50 mg of Cys, 400 mg of Gln, 1000 mg of Glu, 300 mg of Gly, 200 mg of His, 400 mg of Ile, 400 mg of Lue, 450 mg of Lys, 200 mg of Phe, 400 mg of Pro, 500 mg of Ser, 200 mg of Thr, 100 mg of Trp, 300 mg of Tyr, 300 mg of Val, and 150 mg of [d 3 -methyl]Met (98% d 3 -methyl, Cambridge Isotope Laboratories, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

et al. 2008

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Akutsu, H. (2008). Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3. Biochemistry, 47 (36), 9405-9415. Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3 AbstractTetraheme cytochrome c3 (cyt c3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c3 is higher than that of the wild type by ∼50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of ∼50 mV. In conclusion, the bishistidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3. © 2008 American Chemical Society. Koto, Kamigori, Hyogo 678-1297, Japan, The RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Japan, and Faculty of Engineering, Yokohama National UniVersity, Hodogaya-ku, Yokohama 240-8501, Japan ReceiVed April 2, 2008; ReVised Manuscript ReceiVed July 18, 2008 ABSTRACT: Tetraheme cytochrome c 3 (cyt c 3 ) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of DesulfoVibrio Vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3 . The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH ...

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Section: Methodsmentioning

confidence: 99%

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

et al. 2008

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“…Likewise, co-expression of cytochromes c maturation proteins of E. coli permitted the expression of holo-c-type cytochromes with multi-heme in E. coli , albeit at low levels (5). Alternatively, the holo-c-type cytochromes were expressed in Shewanella oneidensis MR-1, taking advantage of the host cell's own mechanism for heme insertion (6,7). In this latter case, much higher levels of expression were obtained for multi-heme c-type cytochromes (6).…”

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confidence: 99%

Overexpression of Multi-Heme C-type Cytochromes

et al. 2005

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“…[18] In addition, physical methods, i.e., NMR, and UV-vis, could be used to determine if the obtained cyt c 3 possesses the identical structure with the native cyt c 3 , as reported by Ozawa, et al [1] As stated above, the fraction of peak 1 in Figure 2, whose retention time is the same as native cyt c 3 , was preliminarily confirmed to contain the correctly refolded rcyt c 3 . To further confirm this, NMR were, therefore, used to determine the structure of the obtained rcyt c 3 .…”

Section: Characterization Of Rcyt Cmentioning

confidence: 63%

“…[1] Natural cyt c 3 are found almost exclusively in anaerobic bacteria, including sulfate reducing bacteria in the genus Desulfovibrio. At present, they can be produced by several expression systems, such as E. coli, [2,3] Shewanella oneidensi, [1,4] etc. In the S. oneidensis system, the highest yield of recombinant Desulfovibrio vulgaris, Miyazaki F cyt c 3 (rcyt c 3 ) was 8.3 mg/L-culture so far.…”

Section: Introductionmentioning

confidence: 99%

Refolding of Mis-folded Recombinant Cytochrome c ₃ with Strong Cation Exchange Chromatography

Shen

Takayama

Wei

et al. 2007

Journal of Liquid Chromatography & Related Technologies

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Mis-folded recombinant cytochrome c 3 (rcyt c 3 ) formed in the purification process was refolded with strong cation exchange chromatography, with tri-gradient of salt, urea, and pH in the mobile phase. The optimal concentration of urea was found to be 6.0 mol/L in mobile phase to refold the mis-folded rcyt c 3 under the experimental conditions. The recovery of the correctly refolded rcyt c 3 was calculated to be 57.0%.

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A Simple, Rapid, and Highly Efficient Gene Expression System for Multiheme Cytochromes c

Cited by 34 publications

References 1 publication

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

Overexpression of Multi-Heme C-type Cytochromes

Refolding of Mis-folded Recombinant Cytochrome c ₃ with Strong Cation Exchange Chromatography

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A Simple, Rapid, and Highly Efficient Gene Expression System for Multiheme Cytochromes c

Cited by 34 publications

References 1 publication

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c3

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c3

Overexpression of Multi-Heme C-type Cytochromes

Refolding of Mis-folded Recombinant Cytochrome c 3 with Strong Cation Exchange Chromatography

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Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c₃

Refolding of Mis-folded Recombinant Cytochrome c ₃ with Strong Cation Exchange Chromatography