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

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 approximately 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.