Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics.

Maki Kaneko, Ryoji Masui, Kojiro Ake, Yukihide Kousumi, Seiki Kuramitsu, Minoru Yamaguchi, Hiroki Kuyama, Eiji Ando, Shigemi Norioka, Takashi Nakazawa, Taka-Aki Okamura, Hitoshi Yamamoto, Norikazu Ueyama

Index: J. Proteome Res. 3(5) , 983-7, (2004)

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Abstract

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.