Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics.
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.
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 Japan.
Maki
M
Kaneko
Ryoji
R
Masui
Kojiro
K
Ake
Yukihide
Y
Kousumi
Seiki
S
Kuramitsu
Minoru
M
Yamaguchi
Hiroki
H
Kuyama
Eiji
E
Ando
Shigemi
S
Norioka
Takashi
T
Nakazawa
Taka-Aki
TA
Okamura
Hitoshi
H
Yamamoto
Norikazu
N
Ueyama
D045304:Cytochromes c / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry; D005288:Ferredoxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005296:Ferrous Compounds / Q000737:chemistry; D005822:Genetic Vectors / Q000235:genetics; D009942:Organometallic Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods*; D011725:Pyridines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012428:Ruthenium / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013440:Sulfides / Q000737:chemistry; D016963:Thermus thermophilus / Q000235:genetics*
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D005288:Ferredoxins; D005296:Ferrous Compounds; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011725:Pyridines; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D013440:Sulfides; C026518:apoferredoxin; D012428:Ruthenium; D045304:Cytochromes c; C038178:ammonium ferrous sulfate; D005287:Ferredoxin-NADP Reductase; C013278:2,2',2''-terpyridine; C033479:sodium sulfide