Abstract
Ferredoxins are small iron sulfur proteins necessary for electron donation. FdxH1 and FdxH2 are associated with two different nif gene clusters where they transfer electrons for the reduction of nitrogenase complex. FdxH1 was observed to be stable towards oxygen, whereas, FdxH2 was relatively unstable. We had identified the amino acid involved in oxygen sensitivity of ferredoxin protein using protein modeling. The exchange of valine to leucine at position 77 was critical for ferredoxin proteins in relation to its oxygen sensitivity. This exchange leads to a longer side chain, which inhibits the accessibility of oxygen to the iron sulfur cluster. Site directed mutagenesis and in vitro experiments confirms that valine indeed is involved in the oxygen sensitivity. The exchange of leucine to valine in FdxH1 makes it oxygen unstable. Thus, from the above results we can conclude that the position of leucine at position 77 is critical for oxygen sensitivity of ferredoxin and protein modeling can be used to identify specific amino acids in other oxygen-sensitive proteins.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Böhme H, Haselkorn R: Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. Strain PCC 7120. Mol Gen Genet 214: 278–285, 1988
Ullmann GM, Hauswald M, Jensen A, Knapp E-W: Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: Implications for their interactions with photo system I and ferredoxin-NADP reductase, proteins. Struct Funct Genet 38: 301–309, 2000
Tsutsui T, Tsukihara T, Fukuyama K, Katsube Y, Hase T, Matsubara H, Nishikawa Y, Tanaka N: Main chain fold of a [2Fe-2S] ferredoxin I from Aphanothece sacrum at 2.5 Å resolution. J Biochem (Tokyo) 94: 299–302, 1983
Kissinger CR, Sieker LC, Adman ET, Jensen LH: Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1.7 Å. J Mol Biol 219: 693–715, 1991
Freer ST, Alden RA, Carter CW Jr, Kraut J: Crystallographic structure refinement of chromatium high potential iron protein at two Angstroms resolution. J Biol Chem 250: 46–54, 1975
Rypniewski WR, Breiter DR, Benning MM, Wesenberg G, Oh BH, Markley JL, Rayment I, Holden HM: Crystallization and structure determination to 2.5-Å resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120. Biochemistry 30: 4126–4131, 1991
Petering D, Fee JA, Palmer G: The oxygen sensitivity of spinach ferredoxin and other iron-sulfur proteins. The formation of protein-bound sulfur-zero. J Biol Chem 246: 643–653, 1971
Schrautemeier B, Neveling U, Schmitz S: Distinct and differently regulated Mo-dependent nitrogen-fixing systems evolved for heterocysts and vegetative cells of Anabaena variabilis ATCC 29413: Characterization of the FdxH1/2 gene regions as part of the nif1/2 gene clusters. Mol Microbiol 18: 357–369, 1995
Schrautemeier B, Cassing A, Böhme H: Characterization of the genome region encoding an FdxH-type ferredoxin and a new 2[4Fe-4S] ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110. J Bacteriol 176: 1037–1046, 1994
Nakamura M, Saeki K, Takahashi Y: Hyperproduction of recombinant ferredoxins in Escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-O gene cluster. J Biochem (Tokyo) 126: 10–18, 1999
Casadaban MJ, Chou J, Cohen SN: In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143: 971–80, 1980
Sambrook J, Fritsch EF, Maniatis T: In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989
Jacobson BL, Chae YK, Markley JL, Rayment I, Holden HM: Molecular structure of the oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120 determined to 1.7-Å resolution. Biochemistry 32: 6788–6793, 1993
Bower MJ, Cohen FE, Dunbrack RL Jr: Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: A new homology modeling tool. J Mol Biol 267: 1268–1282, 1997
Pearlman DA, Case DA, Caldwell JC, Seibel GL, Singh UC, Weiner P, Kollman PA: AMBER 4.0. University of California, San Francisco, 1991
Schomburg D, Reichelt J: BRAGI: A comprehensive protein modelling program system. J Mol Graph 6: 161–165, 1988
Schmitz S, Martinez-Julvez M, Gomez-Moreno C, Böhme H: Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin: NADP+ reductase with ferredoxin probed by site directed mutagenesis. Biochim Biophys Acta 1363: 85–93, 1998
Schmitz S, Schrautemeier B, Böhme H: Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S] heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120. Mol Gen Genet 240: 455–460, 1993
Holden HM, Jacobson BL, Hurley JK, Tollin G, Oh BH, Skjeldal L, Chae YK, Cheng H, Xia B, Markley JL: Structure-function studies of [2Fe-2S] ferredoxins. J Bioenerg Biomembr 26: 67–88
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Singh, B.B., Curdt, I., Shomburg, D. et al. Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis strain ATCC 29413 is critical for its oxygen sensitivity. Mol Cell Biochem 217, 137–142 (2001). https://doi.org/10.1023/A:1007228929662
Issue Date:
DOI: https://doi.org/10.1023/A:1007228929662