Site-specific mutagenesis of Rhodobacter capsulatus ferredoxin I, FdxN, that functions in nitrogen fixation. Role of extra residues.
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 Nifs (slow growth by nitrogen fixation) 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 Em 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.
Saeki
K
K
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560.
Tokuda
K i
Ki
Fukuyama
K
K
Matsubara
H
H
Nadanami
K
K
Go
M
M
Itoh
S
S
D000595:Amino Acid Sequence; D003553:Cystine; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010641:Phenotype; D011401:Promoter Regions, Genetic; D016354:Rhodobacter capsulatus; D013056:Spectrophotometry, Ultraviolet
D005288:Ferredoxins; D003553:Cystine; D009591:Nitrogenase