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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 May 1; 64(Pt 5): 375–377.
Published online 2008 April 5. doi:  10.1107/S1744309108008038
PMCID: PMC2376390

Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

Abstract

Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P41212, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution.

Keywords: flavodoxins, nitrogen fixation, Rhodobacter capsulatus

1. Introduction

Rhodobacter capsulatus is a purple photosynthetic bacterium that is capable of reduction of atmospheric dinitrogen (N2) when ammonium ion levels in the medium are limiting (Weaver et al., 1975 [triangle]). Nitrogen fixation is carried out by the enzyme nitrogenase, which transfers electrons originating from low-potential electron carriers, such as flavodoxin or ferredoxin molecules, to molecular N2. In this work, we have centred our attention on the flavodoxin NifF, which has been identified as a candidate for nitrogenase reduction in R. capsulatus. NifF is the product of a single gene from the nif operon (where nif stands for ‘nitrogen fixation’) and is a 181-amino-acid polypeptide with a predicted molecular weight of 19.8 kDa (Gennaro et al., 1996 [triangle]). It carries a noncovalently bound flavin mononucleotide (FMN) molecule as the prosthetic group responsible for its redox properties. FMN is able to adopt three redox states: a fully oxidized state (quinone), a one-electron semi-reduced state (semiquinone) and a two-electron reduced state (hydroquinone). Flavodoxins behave as a mono-electron carrier and, in the case of NifF from R. capsulatus, the semiquinone and hydroquinone have been pro­posed to be the relevant redox states involved in electron exchange (Nogués et al., 2005 [triangle]).

The proposed electron donor to NifF is the enzyme ferredoxin/flavodoxin:NADP(H) oxidoreductase (FPR), as described by Bittel et al. (2003 [triangle]). Rhodobacter FPR belongs to the plant-type ferredoxin NADP(H) reductase family (EC 1.18.1.2) and contains a noncovalently bound FAD molecule as a prosthetic group. FPR is able to accept two electrons from the NADPH molecule and eventually transfers them to two NifF molecules in a reversible mechanism (Nogués et al., 2005 [triangle]; Bittel et al., 2003 [triangle]). The crystallization and determination of the three-dimensional structure of FPR has been reported recently by our group (Pérez-Dorado et al., 2004 [triangle]; Nogués et al., 2005 [triangle]). Here, we describe the initial results obtained for the crystallization of NifF and X-ray diffraction data to 2.17 Å resolution.

2. Experimental

2.1. Protein expression and purification

After amplification by the polymerase chain reaction, the nifF-coding sequence from R. capsulatus was cloned into pET-32a (Novagen), producing plasmid pET32-Fld, which was used to transform Escherichia coli BL21 (DE3) cells as described previously (Bittel et al., 2003 [triangle]). After transformant cultures reached exponential growth at 310 K, they were induced with 0.5 mM IPTG for 2 h. After centrifugation, the cleared cell lysate was applied onto an Ni–nitrilo­acetic acid (NTA) fast-flow affinity column (Qiagen). The fusion product carrying a His6-Trx tag (Novagen) was eluted with 300 mM imidazole in 50 mM Tris–HCl pH 7.0, digested with entero­kinase to remove the tag and purified as recommended by the supplier (see Bittel et al., 2003 [triangle]). Quantification of the isolated flavodoxin was performed spectrophotometrically using the absorption maximum of the FMN at 450 nm ([sm epsilon]450nm = 11.7 mM −1 cm−1) reported by Yakuning et al. (1993 [triangle]).

2.2. Crystallization

The protein was concentrated to 20 mg ml−1 in 50 mM Tris–HCl pH 8.0 using an Amicon ultrafiltration system with Amicon YM-10 membranes. Crystallization assays were carried out using the hanging-drop vapour-diffusion method at 291 K. The search for initial crystallization conditions was performed using Crystal Screens I and II and Index Screen from Hampton Research. Droplets formed by mixing 1 µl protein solution with 1 µl precipitant solution were equilibrated against 500 µl well solution. Microcrystals of NifF grew under several conditions containing polyethylene glycol (PEG) as the main precipitant agent. Consequently, we focused the search for crystallization conditions using the Synergy Screen (Majeed et al., 2003 [triangle]), which contains an extensive combination of different PEGs, salts and other organic compounds. The best crystals grew in one of these conditions, which consisted of 25%(w/v) PEG 3350, 5%(v/v) PEG 400 and 100 mM sodium acetate pH 5.5. After several optimization steps, we obtained large NifF crystals which grew in 5 µl droplets formed by mixing 2.5 µl of a solution containing 20 mg ml−1 protein buffered in 50 mM Tris–HCl pH 8.0 and 2.5 µl precipitant solution containing 20–25%(w/v) PEG 3350, 2–5%(v/v) PEG 400 and 100 mM sodium acetate pH 5.5. Crystals reached maximum dimensions of 1.0 × 0.7 × 0.7 mm in two weeks (Fig. 1 [triangle]).

Figure 1
Crystal of the flavodoxin NifF obtained with 20–25%(w/v) PEG 3350, 2–5%(v/v) PEG 400 and 100 mM sodium acetate pH 5.5. The approximate dimensions of the crystal are 1.0 × 0.7 × 0.7 mm.

2.3. X-ray data collection and processing

X-ray diffraction data were measured using Cu Kα X-rays (λ = 1.5418 Å) generated by an in-house Enraf–Nonius rotating-anode generator operated at 40 kV and 90 mA and equipped with a double-mirror focusing system. Images were collected on a MAR Research 345 image-plate detector. Crystals were soaked for 10 s in a cryoprotectant solution consisting of mother liquor with 15%(v/v) glycerol and then flash-cooled at 100 K in a nitrogen stream using a cryogenic system. Despite the fact that the NifF crystals reached large dimensions (around 1 mm in length), they were extremely fragile and cracked very easily when mounted in the cryo-loops. For this reason, high-quality X-ray data sets were difficult to obtain. The collected images were processed and scaled using the programs MOSFLM (Leslie, 1992 [triangle]) and SCALA from the CCP4 package (Collaborative Computational Project, Number 4, 1994 [triangle]).

3. Results

An X-ray data set was collected to 2.17 Å resolution from a single NifF crystal and displayed an intense and good-quality pattern (Fig. 2 [triangle]). Preliminary X-ray data processing showed that the crystal belonged to the tetragonal crystal system, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data-collection statistics are summarized in Table 1 [triangle]. Analysis of the intensities after scaling displayed reflections for Miller indices 00l when l = 4n and h00 when h = 2n, indicating space group P41212 or P43212 as the most probable. Specific volume calculations indicated the presence of one molecule in the asymmetric unit and a solvent content of 64% (V M = 3.4 Å3 Da−1; Matthews, 1968 [triangle]).

Figure 2
(a) Typical X-ray diffraction pattern of NifF crystals; (b) enlargement showing resolution rings.
Table 1
Data-collection statistics for NifF crystals

Structural resolution was carried out using the molecular-replacement method with the flavodoxin from Anabaena PCC7120 (PDB code 1flv), which shares 41% sequence identity and 60% sequence homology, as a model. The molecular-replacement method was performed using the program MOLREP (Vagin & Teplyakov, 1997 [triangle]) with reflections in the resolution range 25.6–3.0 Å. A single and unambiguous solution for the rotation and translation functions was obtained, which yielded a final correlation coefficient of 0.38 and an R factor of 0.53. The space group was confirmed to be P41212 and the asymmetric unit contained a single protein monomer. Structural refinement of the NifF model is currently in progress.

Acknowledgments

IP-D is a fellow of the Consejo Superior de Investigaciones Científicas (Spain). AB is a fellow of CONICET (Argentina) and NC is a staff investigator of the same institution. This work was supported by grants BFU2005-01645, BIO2003-01952 and BFU2006-10288 from Dirección General de Investigación, by 08.2/0030.1/2003 from the Comunidad de Madrid and by grant PIP 5124 from CONICET (Argentina). This is a product of the Project ‘Factoría Española de Cristalización’ Ingenio/Consolider 2010.

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