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 fe rredoxin I, FdxN, was modified through site-specific mutagenesis of th e distinctive features of the second cluster-binding motif, 2)-Cys(41) -X(8)-Cys(50)-X(8)-Cys(50)-X(3)-Cys(59). First, various mutagenized pr oducts were tested to learn whether they could rescue the decreased ca pacity of an fdxN-null strain MSA1 to fix nitrogen: the phenotype of M SA1 was reassessed to Nif(s) (slow growth by nitrogen fixation) from o ur previous description of Nif(-) (Saeki, K., Suetsugu, Y., Tokuda, H. , Miyatake, P., Young, D. A., Marrs, B. L. and Matsubara, H. (1991) J. Biol. Chem. 266, 12889-12895). Substitution of Cys(59) to Ser yielded an almost fully active product, while that of Cys(54) did not. Gradua l deletions and deletion-substitution of the 8 residues between Cys(41 ) and Cys(50) also yielded active products. Second, three of the modif ied 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 sho wed 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 identic al value of -490 mV, when determined by EPR analysis. It was concluded that: 1) Cys(59) is not a ligand to [4Fe-4S] clusters but is importan t for structural integrity, 2) the residues between positions 42 and 4 9 may form a ''loop-out'' from a structure analogous to the Peptococcu s aerogenes ferredoxin, and 3) the loop-out region does not have funct ional significance in nitrogen fixation but may be responsible for mai ntaining the highly negative redox potential of one of the two cluster s.