Purification and characterization of ferredoxin–nitrite reductase from the eukaryotic microalga Monoraphidium braunii Javier Vigara a, *, María I. García-Sánchez b , Inés Garbayo a , Carlos Vílchez a , José M. Vega b a Departamento de Química y CC.MM., Universidad de Huelva, 21819 Huelva, Spain b Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de Sevilla, 41080 Sevilla, Spain Received 7 November 2001; accepted 9 January 2002 Abstract Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg –1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate K m values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Immunochemistry; Kinetic parameters; Molecular parameters; Monoraphidium braunii; Nitrite reductase 1. Introduction The assimilation of nitrate by eukaryotic microalgae proceeds through the following steps: (a) the uptake of nitrate, which is mediated by specific transporters; (b) the 2e reduction of nitrate to nitrite, catalyzed by the NADH–ni- trate reductase; (c) the transport of nitrite into the chloro- plast, mediated by a specific transporter [17]; (d) the 6e reduction of nitrite to ammonium, catalyzed by the ferre- doxin (Fd)–nitrite reductase (NiR); and (e) the incorporation of ammonium into carbon skeletons, as α-amino group of glutamate, which is catalyzed by the glutamine synthetase (GS)–glutamate synthase (GOGAT) cycle [20]. The NADH–nitrate reductase (EC 1.6.6.1) from some eukaryotic algae is, like the plant-type enzyme, a complex of 220 kDa containing two FAD, two b 557 -type hemes and two Mo-pterin, in two subunits, which are similar in size (110 kDa each) and functionality [3]. However, the enzyme from M. braunii shows 460 kDa, with four FAD, four b 557 -type heme and two Mo-pterin as prosthetic groups per enzyme molecule, which is composed of eight similar-sized subunits of 58 kDa [20]. Due to this singular nitrate reduc- tase, it appears very interesting to study the M. braunii NiR, the other enzyme involved in the nitrate-reducing system. Fd–NiR (EC 1.7.7.1) has been isolated from different plants, such as spinach [10], barley [12], rice [9] or pea [1], eukaryotic microalgae, like Chlamydomonas reinhardtii [18], Chlorella fusca [22] and Porphyra yezoensis [11], and from cyanobacteria, like Anabaena cylindrica [6] and Ana- baena sp. 7119 [16]. The enzyme is a monomeric protein with a molecular weight between 60 and 64 kDa, which contains one siroheme and one [4Fe–4S] cluster as pros- thetic groups [13]. The present work reports the purification to homogeneity of the Fd–NiR from M. braunii, and its characterization, which shows the enzyme is very similar to the plant-type Fd–NiR. Abbreviations: Fd, ferredoxin; GOGAT, glutamate synthase; MV, methyl viologen; NiR, nitrite reductase; pHMB, p-hydroxymercuribenzoate * Corresponding author. Fax: +34-959-017414. E-mail address: vigara@uhu.es (J. Vigara). Plant Physiol. Biochem. 40 (2002) 401–405 www.elsevier.com/locate/plaphy © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 3 8 5 - 2