Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production

Abstract

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.

Nitrate ammonification by Nautilia profundicola AmH: experimental evidence consistent with a free hydroxylamine intermediate

“…The proposed uptake of the highly mutagenic hydroxylamine necessitates protection against DNA lesions, which may partially explain the extensive complement of DNA repair systems identified in N. profundicola (Campbell et al, 2009). Hydroxylamine has also been proposed as an intermediate of ammonium production from nitrite in plants and algae as ferredoxin-dependent nitrite reductase displays high reactivity and specificity for this substrate (Kuznetsova et al, 2004; Hirasawa et al, 2010). …”

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism

“…Nitrate and nitrite are taken up via transporters encoded by members of the NRT1, NRT2, and NAR1 families (Quesada et al, 1993;Fernandez and Galvan, 2007). Nitrate assimilation requires energy for reduction to the level of ammonium by enzymes, nitrate reductase in the cytosol, and nitrite reductase in the chloroplast, both encoded by single genes in C. reinhardtii, NIA1/ NIT1 and NII1, respectively (Romero et al, 1987;Fernández et al, 1989;Quesada et al, 1998;Fernandez and Galvan, 2008;Hirasawa et al, 2010). Many of the genes involved in nitrate/nitrite uptake and assimilation are under the control of a positive regulator, the NIT2 transcription factor (Fernández and Matagne, 1986;Camargo et al, 2007).…”

Remembering David B. Knaff (1941–2016)

“…The Knaff laboratory prepared a His-tagged nitrite reductase as well as the native reductase in E. coli, showed that the histidine-modified protein was similar to the native protein, and then used these preparations for sitedirected mutagenesis of amino acids previously shown to be involved in either catalysis or ferredoxin binding . A His-tagged enzyme was also prepared from Chlamydomonas (Hirasawa et al 2010). The site-directed mutants prepared with the Chlamydomonas protein were then used to construct a molecular model of the ferredoxin-binding site (Tripathy et al 2007).…”