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.
Singh
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Botanisches Institut der Universität Bonn, Kirschallee 1, D-53315, Bonn, Germany.
Curdt
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Jakobs
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C
Schomburg
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D
Bisen
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PS
Böhme
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D017033:Anabaena / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry*; D016415:Sequence Alignment; D014633:Valine / Q000737:chemistry
D005288:Ferredoxins; D007532:Isoleucine; D009591:Nitrogenase; D007930:Leucine; D014633:Valine; D000409:Alanine; D010100:Oxygen