NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway

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Abstract

Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV–visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe–2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Introduction

The nitrate assimilatory pathway represents a fundamental biological process in most bacteria [1], yeast [2], cyanobacteria [3], fungi [4], algae [5] and higher plants [6]. This pathway is mediated by nitrate reductase (Nas, EC 1.6.6.2) and nitrite reductase (NiR, EC 1.7.7.1), which catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia, respectively. Two classes of assimilatory nitrate and nitrite reductases are found in bacteria: NADH-dependent enzymes [7], [8], and the ferredoxin- or flavodoxin-dependent enzymes [9]. In non-photosynthetic organisms electron transfer is mediated by NAD(P)H [10]. On the contrary, a ferredoxin (Fd, hereafter) is typically found as the physiological electron donor in photosynthetic organisms [10]. Moreover, Fds are also present in anaerobic nitrogen-fixing bacteria [8], anaerobic parasitic and free-living protozoa, and even in vertebrates [11].

Ferredoxins are iron–sulfur electron-transfer proteins of low molecular weight (around 12 kDa), which are versatile from both structural and functional points of view [12]. These proteins are involved in a large number of physiological events, such as in the regulation of gene expression [13], oxygen and iron sensing, generation and stabilization of radical intermediates [14], or in peptide metabolism [15]. However, the most important function of ferredoxins is electron transfer. In fact, they play the important role of carrying one electron from the photosynthetic electron transport chain to several metabolic pathways in cyanobacteria and plant chloroplasts [16]. In the reduced state, ferredoxin transfer electrons to a number of different enzymes such as nitrate reductase, nitrite reductase, thioredoxin reductase, sulfate reductase, and glutamate synthase [17].

Fds have been typically classified in bacterial- and plant-type. Bacterial-type Fds contains clusters with four or three iron ions, while plant-type ferredoxins only possess two iron ions (bridged by two sulfide atoms, Fe2S2) per molecule [11]. This kind of Fds are highly acidic, being the acidic residues involved in the interaction of Fds with Fd-dependent enzymes [18]. Some ferredoxins isolated from non-plant sources, such as bacteria [19] and mitochondrial Fds also contain a 2Fe cluster. Halophilic archaea ferredoxins belong to the plant type [20], [21], although differences between these two groups have been emphasised [22]. Ferredoxins from halophilic Archaea such as Halobacterium halobium [20], Haloarcula marismortui [23], and Haloarcula japonica [24] have been purified and characterised. All these Fds contain a [Fe2S2] cluster.

We report here the purification and characterisation of a ferredoxin from Haloferax mediterranei (H. mediterranei). We had previously suggested that this Fd participates in the assimilatory reduction of nitrate and nitrite by nitrate reductase (Nas) [25] and nitrite reductase (NiR) [26], respectively. Here, we present the kinetic analysis of the interaction between these proteins. This study has allowed us to determine the specific role of this Fd in the assimilatory nitrate reduction pathway.

Section snippets

Protein purification

H. mediterranei culture growth and the purification of its Fd were performed as previously reported [20], [25], [26]. Assimilatory nitrate and nitrite reductase purification was carried out as described by the Martı́nez-Espinosa et al. [25], [26].

Protein determination and enzymatic assays

The protein content was determined by the Bradford method. Nas and NiR activities were measured according to Martı́nez-Espinosa et al. [25], [26], using the diazo coupling method. The salt concentration was 1 M NaCl for the Nas assay [25]

Isolation

H. mediterranei Fd was purified under aerobic conditions as described by Kerscher et al. [20]. The protein was easily recognised along the purification process by its dark brown colour. The ratio A420/A275 of the purified final sample was 0.21. A similar ratio has been found in other halophilic ferredoxins [20], [22], [24]. The apparent molecular weight (Mr) of the ferredoxin, determined by SDS-PAGE, was 21 kDa. This method is known to overestimate the molecular weight of halophilic proteins,

Acknowledgements

This work was supported by funds from CICYT PB98-0969.

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