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Purification, cofactor analysis, and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

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Abstract

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination.

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  1. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas-Universidad de Sevilla, Avda. Américo Vespucio, E-41092, Seville, Spain

    Luis M. Rubio, Enrique Flores & Antonia Herrero

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  1. Luis M. Rubio
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  2. Enrique Flores
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  3. Antonia Herrero
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Correspondence to Antonia Herrero.

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Rubio, L.M., Flores, E. & Herrero, A. Purification, cofactor analysis, and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase. Photosynthesis Research 72, 13–26 (2002). https://doi.org/10.1023/A:1016078700839

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