Identification of amino acids responsible for the oxygen sensitivity of ferredoxins from Anabaena variabilis using site-directed mutagenesis

• Published in: Biochimica et biophysica acta 1999-08, Vol.1412 (3), p.288-294
• Main Authors: Singh, B.B., Curdt, I., Jakobs, C., Schomburg, D., Bisen, P.S., Böhme, H.
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Amino Acid Sequence; Anabaena - chemistry; Escherichia coli - genetics; ferredoxin; Ferredoxins - chemistry; Ferredoxins - genetics; Ferredoxins - isolation & purification; Isoleucine - chemistry; Leucine - chemistry; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrogenase - chemistry; Oxygen - chemistry; Sequence Alignment; site-directed mutagenesis; Valine - chemistry
• ISSN: 0005-2728; 0006-3002; 1879-2650; 1878-2434
• DOI: 10.1016/S0005-2728(99)00069-9

The filamentous cyanobacterium Anabaena variabilis (ATCC 29413) possesses two molybdenum dependent nitrogenase systems, nif1 and nif2. The nif1 system is regulated by a developmental program involving heterocyst differentiation; the nif2 system is expressed in all cells only under anaerobic conditions and the expression is controlled environmentally. The genes fdxH1 and fdxH2, encoding two [2Fe–2S] ferredoxins, are part of the these two distinct and differently regulated nif gene clusters. The sensitivity of both ferredoxins to oxygen was different; the half-life of FdxH2 in air was only ≈1.5 h, while FdxH1 retained 80% of its nitrogenase activity after 24 h. We used site-directed mutagenesis to identify the role of individual amino acid residues responsible for oxygen sensitivity and found out that the FdxH2 double mutant I76A/V77L was much more resistant to oxygen than the wild-type ferredoxin (FdxH2) and similar to FdxH1. By modelling it was shown that the accessibility of the cavity around the iron–sulfur cluster was responsible for that.