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Volume 271, Number 49, Issue of December 6, 1996 pp. 31399-31406
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Site-specific Mutagenesis of Rhodobacter capsulatus Ferredoxin I, FdxN, That Functions in Nitrogen Fixation
ROLE OF EXTRA RESIDUES

(Received for publication, August 1, 1996)

Kazuhiko Saeki , Ken-ichiro Tokuda , Keiichi Fukuyama , Hiroshi Matsubara , Kazuhiko Nadanami , Mitiko Go and Shigeru Itoh ^**

From the  Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560, the  Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-01, and the ^** National Institute for Basic Biology, Myodaijicho, Oakazaki 444, Japan

One of the two [4Fe-4S]-type clusters of the Rhodobacter capsulatus ferredoxin I, FdxN, was modified through site-specific mutagenesis of the distinctive features of the second cluster-binding motif, Cys³⁸-X₂-Cys⁴¹-X₈-Cys⁵⁰-X₃-Cys⁵⁴-X₄-Cys⁵⁹. First, various mutagenized products were tested to learn whether they could rescue the decreased capacity of an fdxN-null strain MSA1 to fix nitrogen: the phenotype of MSA1 was reassessed to Nif^s (low growth by trogen ixation) from our previous description of Nif (Saeki, K., Suetsugu, Y., Tokuda, K., Miyatake, Y., Young, D. A., Marrs, B. L. and Matsubara, H. (1991) J. Biol. Chem. 266, 12889-12895). Substitution of Cys⁵⁹ to Ser yielded an almost fully active product, while that of Cys⁵⁴ did not. Gradual deletions and deletion-substitution of the 8 residues between Cys⁴¹ and Cys⁵⁰ also yielded active products. Second, three of the modified FdxN proteins were subjected to purification. Only the GA protein, whose 8 residues between positions 42 and 49 were replaced by the Gly-Ala sequence, was purified. The GA protein and the authentic FdxN showed similar optical properties. The two clusters in the former had E_m values of 490 and 430 mV, while those in the latter had an identical value of 490 mV, when determined by EPR analysis. It was concluded that: 1) Cys⁵⁹ is not a ligand to [4Fe-4S] clusters but is important for structural integrity, 2) the residues between positions 42 and 49 may form a "loop-out" from a structure analogous to the Peptococcus aerogenes ferredoxin, and 3) the loop-out region does not have functional significance in nitrogen fixation but may be responsible for maintaining the highly negative redox potential of one of the two clusters.

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