FEBS Letters 583 (2009) 325–329

journal homepage: www.FEBSLetters .org

A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii

Jessica Jacobs a, Susanne Pudollek b, Anja Hemschemeier a, Thomas Happe a,*

a Ruhr Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, ND2/169,Universitätsstrasse 150, 44780 Bochum, Germanyb Freie Universität Berlin, Fachbereich Physik, Arnimallee 14, 14195 Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 October 2008Revised 4 December 2008Accepted 6 December 2008Available online 26 December 2008

Edited by Stuart Ferguson

Keywords:AnaerobiosisFerredoxinGreen algaHydrogen metabolism

0014-5793/$ - see front matter � 2008 Federation ofdoi:10.1016/j.febslet.2008.12.018

Abbreviations: aa(s), amino acid(s); Aox, alternatiforward; Fnr, ferredoxin-NADPH-oxidoreductase; Ndinucleotide phosphate; orf, open reading frame; Pfl,PSI, photosystem II, photosytem I; qPCR, quantitativlarge subunit of Rubisco; Rubisco, ribulosebisphospSAM, S-adenosylmethionine.

* Corresponding author. Fax: +49 234 3214322.E-mail address: thomas.happe@rub.de (T. Happe).

We have found the transcript of one of at least six ferredoxin encoding genes of the green algaChlamydomonas reinhardtii, FDX5, strongly accumulating in anaerobiosis, indicating a vital role ofthe encoded protein in the anaerobic metabolism of the cells. According to absorption and electronparamagnetic resonance spectroscopy, Fdx5 is a plant-type [2Fe2S]-ferredoxin with a redox poten-tial similar to that of the ferredoxin PetF. However, although Fdx5 seems to be located in the chlo-roplast, it is not able to photoreduce nicotinamide adenine dinucleotide phosphate (NADP+) viaferredoxin-NADP-reductase, nor to be an electron donor to the plastidic [FeFe]-hydrogenase HydA1.Thus, Fdx5 seems to have a special role in a yet to be identified anaerobic pathway.� 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction as the electron acceptor of PSI [7]. C. reinhardtii PetF is also sup-

Ferredoxins are small soluble [FeS]-cluster containing proteinswhich can be found in every phylogenetic group. Roughly, ferre-doxins can be divided into plant-type ferredoxins containing[2Fe2S]-clusters and bacterial-type ferredoxins with [4Fe4S]-clus-ters [1]. They are mainly low-potential electron carriers involvedin various metabolic pathways having diverse redox partners [2].Maybe the best known ferredoxin is the ferredoxin of higher plantsacting as the electron acceptor of photosystem I (PSI) and involvedin nicotinamide adenine dinucleotide phosphate (NADP+) photore-duction as well as in nitrate and sulfate assimilation and in the reg-ulatory ferredoxin thioredoxin system [3].

In plants and algae, ferredoxins can be found in multiple iso-forms [4]. In higher plants, these isoforms may be expressed tissuespecific and show functional differences [5]. The unicellular chloro-phyte alga Chlamydomonas reinhardtii has at least six ferredoxinencoding genes [6]. Only the ferredoxin PetF, which is involvedin photosynthesis and which is a typical plant-type [2Fe2S] ferre-doxin, has been extensively studied and characterized [7]. It is nu-clear encoded but transferred to the chloroplast, where it functions

European Biochemical Societies Pu

ve oxidase; bps, basepairs; F,ADP, nicotinamide adeninepyruvate formate lyase; PSII,e RealTime PCR; Rubisco LS,hate carboxylase/oxygenase;

posed to be the connecting element between PSI and the [FeFe]-hydrogenase HydA1 [8], which is a central element of the complexanaerobic metabolism of the alga. Under anaerobic conditions inthe dark, C. reinhardtii produces H2, formate, acetate, ethanol, car-bon dioxide (CO2) as well as traces of glycerol and D-lactate. One ofthe key enzymes of this mixed acid fermentation is the pyruvateformate lyase (Pfl1) [9,10]. An induction of the anaerobic metabo-lism can also be observed in illuminated, but sulfur (S) deprived C.reinhardtii cells [11,12]. S deprivation leads to the establishment ofanaerobic conditions due to a down-regulation of photosyntheticoxygen (O2) evolution [11] and to an up-regulation of the tran-scripts encoding the key enzymes (Pfl1 and HydA1) of the anoxicpathways in C. reinhardtii [10].

In this study we have found that the mRNA level of PETF de-creased in S depleted algae, though the amount of PetF proteindid not change. In parallel, the transcript of the ferredoxin like geneFDX5 accumulated very strongly, indicating the encoded protein toplay an important role in the anaerobic metabolism of C. rein-hardtii. This study shows the first biochemical characterization ofthis novel plant-type [2Fe2S]-ferredoxin.

2. Materials and methods

2.1. Organisms and growth conditions

C. reinhardtii wild-type CC-125 (mt+ 137C) was obtained fromthe Chlamydomonas Culture Collection at Duke University. Cells

blished by Elsevier B.V. All rights reserved.

326 J. Jacobs et al. / FEBS Letters 583 (2009) 325–329

were grown photoheterotrophically in tris–acetate–phosphate(TAP) medium [13]. For anaerobic adaptation and for S deprivationof the algae, cells were prepared as described before [8,14].

The growth conditions of Escherichia coli DH5 and E. coliBL21(DE3)pLysS (Novagen, EMD Biosciences Inc., Madison, WI,USA) were reported elsewhere [15].

2.2. RNA analysis and PCR

Total RNA was isolated according to Hemschemeier et al [10].Northern blot analyses were conducted according to standardtechniques [15] using digoxigenin-labelled RNA probes. quantita-tive real time PCR (qPCR) experiments were conducted as de-scribed previously [10]. Oligonucleotides for the analysis of PETFwere 50-CCGCTGACACCTACATCCTG-30 (F) and 50-AACCGCTCA-TAGTGCTAGGC-30 (R), those for FDX5 were 50-CGGCTTGAAGAG-TCAAGAGT-30 (F) and 50-CCACACCAGAGCCATACATA-30 (R). Resultswere normalized to the transcript level of RPL10a, encoding thecytosolic 60S large ribosomal subunit protein L10a (GenBankXP_001699807, protein ID JGI3.0 195585).

2.3. Isolation of C. reinhardtii chloroplasts and mitochondria

S-deprived C. reinhardtii SAG 83.81 cells were harvested at2000�g in a GSA rotor (Sorvall/Thermo Fisher Scientific, Waltham,USA) for 5 min and resuspended in isotonic solution (0.3 M sorbi-tol, 10 mM tricine–HCl, pH 7.8, 5 mM MgCl2). The cell suspensionwas passed through a cell disruptor (BioNeb, Glas-Col, Terre Haute,USA) with N2-gas pressure at 20 p.s.i.. Cell lysate was shortly cen-trifuged to 5000 rpm in a SS34 rotor (Sorvall). The pellet was usedto prepare intact chloroplasts [16]. The supernatant was used forthe isolation of mitochondria [17].

2.4. Heterologous synthesis and purification of recombinant Fdx5

The FDX5 cDNA was amplified by PCR with Pfu DNA polymerase(Stratagene, La Jolla, CA, USA) using the oligonucleotides 50: ATGG-TAGGTCTCAGCGCTTTCAGGTGACGCTGCGCATGC and 30: ATGG-TAGGTCTCATATCACTGGTGCTTGCCGTACTCGCA on cDNA from aC. reinhardtii culture that had encountered 24 h of S-depletion astemplate. PCR products were cloned into pASK-IBA7 (IBA GmbH,Göttingen, Germany) using EcoNI restriction sites resulting in plas-mid pJJ18. For heterologous synthesis of Fdx5, E. coli BL21(DE3)-pLys was transformed with pJJ18. Protein production wasperformed in 3 l Vogel-Bonner medium containing 100 lg ml�1

ampicilline. For most analyses, pelleted E. coli hosts were placedin a glove box (Coy Laboratories, Detroit, USA) and Fdx5 was puri-fied under anaerobic conditions by Strep-TagII-chromatography asdescribed for Pfl previously [10].

2.5. Production of polyclonal Fdx5 antibodies

Polyclonal antibodies were produced by immunization of rabbitwith heterologously produced Fdx5 from which the Strep-TagIIwas removed by Factor Xa protease according to the manufactur-ers’ instructions (Novagen/Merck, Darmstadt, Germany). Antibodyproduction was carried out by BioGenes – Gesellschaft für Biopoly-mere mbH (Berlin, Germany) according to standard immunizationprotocols.

2.6. Purification of native Fdx5

For the isolation of native Fdx5 from C. reinhardtii, 10 l of S-de-pleted cells were harvested in the early H2 producing phase by cen-trifugation. Anaerobic purification was carried out in a glove box.Cells were lysed by sonification (4 � 30 s, output 3). Cell debris

was removed by centrifugation and the protein supernatant wasprecipitated with (NH4)2SO4. After the initial step (40% saturation),the protein solution was centrifuged (1 h, 6000 rpm, 4 �C) and thesupernatant saturated to 80% (NH4)2SO4 followed by further centri-fugation. The pellet was suspended in 15 ml 50 mM Tris–HCl, pH 8and dialyzed against 100 volumes of the same buffer over night.The solution was centrifuged (10 min, 10000 rpm) and loaded ona Q-Sepharose fast-flow column (30 � 60 mm) (Amersham Biosci-ence, München, Germany), pre-equilibrated with 50 mM Tris–HCl,pH 8. After washing with the same buffer, Fdx5 was eluted with alinear NaCl gradient (0–750 mM NaCl). Fdx5 containing fractionswere pooled and diluted 5-fold with 50 mM Tris–HCl, pH 8. Thesolution was loaded on a second Q-Sepharose fast-flow column(20 � 35 mm) and Fdx5 was finally eluted with a linear salt gradi-ent (0–500 mM NaCl).

2.7. Recording of the electron paramagnetic resonance (EPR) signals ofFdx5

Protein samples had a concentration of 200 lM and were re-duced with 20 mM Na2S2O4. They were transferred into EPR tubesand frozen in isopropanol. All experiments were performed on aBruker Biospin Elexsys E580 spectrometer (Bruker Corporation,Billerica, MA) equipped with a Super HighQ resonator. The EPRspectra were recorded at a sample temperature T = 10 K and at amicrowave frequency m = 9.38 GHz. A microwave power ofP = 4 mW and a modulation amplitude of 0.5 mT at 100 kHz mod-ulation frequency was used.

2.8. Protein analysis

Preparation of crude protein extracts of whole algal cells, so-dium dodecyl sulfate-polyacrylamide–gelelectrophoresis (SDS–PAGE) and Western blotting were conducted as described previ-ously [10]. Mass spectrometric identification was conducted as re-ported before [18]. N-terminal Edman sequencing was carried outby Proteome Factory AG (Berlin, Germany).

2.9. Antibodies

Anti-Aox1 antibody was purchased by Agrisera, Vännäs, Swe-den (http://www.agrisera.com). Anti-TcdE antibody was a giftfrom Gary Sawers (University of Halle-Wittenberg, Halle (Saale),Germany). Anti-HydA1-, anti-Rubisco LS- and anti-PetF-antibodieswere described before [19–21].

3. Results and discussion

3.1. FDX5 transcript accumulates strongly under anaerobic conditions

In Northern blot analyses examining the mRNA abundance ofseveral key genes of hydrogen metabolism and fermentation inS-deprived C. reinhardtii cultures, it turned out that the transcriptlevel of the gene encoding PetF, which is the ferredoxin knownto be involved in photosynthesis, decreased strongly (Fig. 1a). Thisprompted us to examine whether one of the at least five additionalferredoxin encoding genes [6] of C. reinhardtii is up-regulated in H2

producing algae. Northern blot analyses showed that FDX5 tran-script accumulated significantly in S-starved algal cultures thathad become anaerobic and started to express the genes encodingHydA1 and Pfl1 (Fig. 1a). In C. reinhardtii cells held under artificialanaerobic conditions, in which the transcript level of PETF did notsignificantly decline (Fig. 1b), FDX5 transcript levels increased, too(Fig. 1b).

Fig. 1. Northern blot analyses were used to monitor the abundance of HYDA1, PFL1,PETF and FDX5 transcripts. Total RNA was isolated daily from S-deprivedChlamydomonas reinhardtii cultures (a) or from cells incubated in artificial anaero-biosis (b) at the depicted time points after the start of the treatment.

Fig. 2. Western blot analyses showing the accumulation of Fdx5 in comparison toPetF, HydA1 and Pfl1 [10] in S-deprived C. reinhardtii. An equivalent of 5 lgchlorophyll was loaded on each lane of the SDS–PAGE. The respective proteins weredetected by polyclonal Fdx5 antibody, anti-spinach Ferredoxin-antibody, anti-C.reinhardtii-HydA1 antibody and anti-E. coli-TdcE antibody.

Fig. 3. Western blot analyses showing the subcellular localization of Fdx5 incomparison to Rubisco LS (R LS), Aox1 and HydA1. The analysis of crude proteinextract (R), purified chloroplasts (C) and mitochondria (M) from S-deprived cells isshown. An equivalent of 80 lg protein was loaded on lanes R and C. Lane Mcontained 40 lg of protein.

J. Jacobs et al. / FEBS Letters 583 (2009) 325–329 327

qPCR experiments confirmed the accumulation of FDX5 tran-script under S-depletion. They showed that the transcript level ofFDX5 increased 970-fold in parallel to the rising transcript levelof HYDA1 (11.80-fold) and PFL1 (4.92-fold) [10], while the tran-script level of PETF decreased 2,5- to 10-fold. The strong increasein FDX5 transcript abundance indicated a vital role of the encodedprotein in the anaerobic metabolism of C. reinhardtii cells. To eluci-date this role, the Fdx5 protein was characterized on the biochem-ical level.

3.2. Antibodies raised against the heterologously synthesized Fdx5confirmed the accumulation of Fdx5 in S-depleted C. reinhardtii cells

The FDX5 gene is annotated as a 1078 bps transcript on scaffold13:1459677–1461655 at JGI3.0 (GenBank ABC88604) comprising a393 bps open reading frame (orf) encoding a protein consisting of130 aas (protein ID 156833 at JGI3.0). Using the information aboutthe not very well conserved consensus sequences of chloroplastictransit peptides [22], we detected a putative transit peptide cleav-age site between positions 27 and 28 (sequence VQA27;F28).

A truncated cDNA encoding a 103 aas long Fdx5 variant, assum-ing the putative leader peptide to consist of 27 aas, was used forheterologous expression in E. coli BL21(De3)pLys and subsequentisolation of heterologously produced Fdx5 by Strep-TagII chroma-tography. The protein showed the typical brownish colour of ferre-doxins and tended two form oligomers when isolated and storedunder aerobic conditions (data not shown). This oligomerizationdid not appear under anaerobic conditions, indicating a severe ef-fect of O2 on the protein. O2 sensitivity is described for several FeScluster containing proteins, such as the fumarate nitrate reductiontranscriptional regulator of E. coli [23], and also for some ferredox-ins [24]. Because of the effect of O2 on recombinant Fdx5, bothpurification and storage of Fdx5 proteins were performed underanoxic conditions in all subsequent experiments.

To confirm the presence of Fdx5 in anaerobic C. reinhardtii cells,an anti-Fdx5 antibody was generated to be able to perform immu-nodetection studies. The antibody reacted with heterologouslyproduced Fdx5 and showed no crossreaction with PetF (data notshown). Applying this antibody to crude extracts of S-depleted C.reinhardtii cells, the accumulation of Fdx5 protein in parallel tothe accumulation of FDX5 transcripts (Fig. 1a) could be demon-strated (Fig. 2). The accumulation of Fdx5 occurred simultaneouslywith the synthesis of HydA1 and Pfl1 (Fig. 2, [10]). Interestingly,the amount of PetF protein remained fairly constant despite thestrong decrease of PETF transcript levels. The utilized anti-spin-ach-PetF antibody did not cross-react with Fdx5 (data not shown).Given that the antibody does not bind to one of the other fourputative ferredoxins of C. reinhardtii, which might accumulate un-der these conditions, this result indicates PetF to be a very stableprotein. A similar discrepancy between transcript levels and pro-tein abundance has been observed analyzing the protein and tran-

script abundance of cytochrome c6 (Cyc6) and the CYC6 gene,respectively, in copper deficiency [25].

3.3. NH2-terminal sequencing and localization of Fdx5

To verify the NH2-terminal sequence of the mature Fdx5 pro-tein, Fdx5 was partially purified from S-deprived C. reinhardtii.The obtained fractions were analyzed by Western blot analysis uti-lizing the polyclonal Fdx5 antibody and Fdx5 was subsequentlyidentified by mass spectrometry. Edman degradation allowed theidentification of the first five amino acids to be Phe-Gln-Val-Thr-Leu. This confirmed the recombinant protein representing the ma-ture Fdx5 protein. Consequently, the native protein has a length of103 amino acids and a calculated size of 11.56 kDa.

The determination of the N-terminus affirmed that the putativetransit peptide is cleaved off after alanine in the sequence Val-Gln-Ala. Val-X-Ala in position �3 to �1 to the cleavage site is a well-conserved motif in transit peptides targeting proteins to thechloroplast stroma in C. reinhardtii [26]. Additionally, the lengthof the putative transit peptide of Fdx5 (27 aas) is close to the meanlength (29 aas) of other C. reinhardtii chloroplastidic transit pep-tides [26]. In higher plants, a similar motif for chloroplastidic pro-teases is found [(Val/Ile)-X-(Ala/Cys)Ala] [22]. The occurrence ofthis motif in the Fdx5 leader peptide thus further implies a chloro-plastidic localization of the mature protein.

The site of action of Fdx5 was then investigated by protein blotanalysis of mitochondrial and chloroplast protein fractions. Toascertain the purity of the cellular fractions, the presence of spe-cific marker proteins was tested. Rubisco LS and HydA1 functionedas marker proteins for the chloroplast fraction, Aox1 as marker forthe mitochondrial fraction.

Western blot analysis revealed that mitochondrial fractionswere free of chloroplastidic proteins, whereas chloroplast fractionswere contaminated with mitochondrial proteins (Fig. 3). The prob-ing of the same blot with Fdx5 antibody showed that Fdx5 bands

Fig. 4. (a) Absorption spectrum of oxidized recombinant Fdx5 (1 lg ll�1). (b) EPR spectrum of reduced recombinant Fdx5 (200 lM). *: unspecific glass signal

328 J. Jacobs et al. / FEBS Letters 583 (2009) 325–329

exhibit the same pattern as the Rubisco LS and HydA1 bands andthat the occurrence of Fdx5 is restricted to the chloroplast fraction(Fig. 3). This result clearly indicates a chloroplastidic localization ofthe Fdx5 protein.

3.4. Fdx5 shows typical properties for a [2Fe2S] plant-type ferredoxin

The Fe content of recombinant Fdx5 was measured according tothe method of Fish [27]. Based on a molecular weight of 11.56 kDa,a ratio of 2.05 mol Fe per mol protein could be determined. Thisindicates that Fdx5 contains a [2Fe2S] cluster. The visible spectrum(Fig. 4a) of the purified protein showed absorption maxima at 333,419 and 462 nm, which are typical for plant-type ferredoxins in theoxidized state [7]. The EPR spectrum revealed g-values of 2.048,1.956, 1.905 (Fig. 4b). Similar g-values have been reported for otherplant-type [2Fe2S]-ferredoxins [2].

3.5. Fdx5 could not reduce ferredoxin-NADPH-oxidoreductase (Fnr) orHydA1

The redox potential of PetF has been reported to be �410 mVversus standard hydrogen electrode as determined by redox titra-tion [28]. Analyzing both PetF and Fdx5 by cyclic voltammetry, theredox potential of Fdx5 turned out to be very similar to that of PetF(data not shown).

This observation and the fact that Fdx5 is a typical plant-type[2Fe2S] protein prompted us to examine whether Fdx5 is activein typical electron transfer reactions like NADP+ photoreductionby PSI and Fnr [3]. Reaction batches according to Finazzi et al.(2005) [29] containing 5 lM Fdx5 as electron carrier showed nophotoreduction of NADP+ by Fnr, while in batches containing5 lM PetF the formation of NADPH could be detected. However,this assay gives no information whether Fdx5 is able to accept elec-trons from PSI.

It was also examined whether Fdx5 functions as the physiolog-ical electron donor of HydA1. Therefore, dithionite reduced Fdx5and PetF were compared concerning their ability to transfer elec-trons to HydA1 in vitro. With Fdx5 as electron donor, no significantH2 production could be measured. Using PetF as the electron donorresulted in a maximal hydrogenase activity of 500 lmol H2 h�1 mgprotein�1 and a Km(PetF) value of 5 lM. The latter results are inaccordance to literature [8]. Summarizing these results, it is obvi-ous that Fdx5 does not function as a specialized electron donorto Fnr nor HydA1 in anaerobic C. reinhardtii cells.

At this time point, the real function of Fdx5 remains an openquestion. The fact that this ferredoxin accumulates both in S-de-pleted anoxic algal cultures and cells anaerobically induced bydark incubation and argon flushing, respectively, indicates that

Fdx5 plays a special role in anaerobiosis in general, rather thanbeing essential for survival of C. reinhardtii in the absence of S.

There are several pathways in which C. reinhardtii could need aspecialized ferredoxin under anaerobic conditions. Especiallymembers of the super-family of radical S-adenosylmethionine(SAM) proteins depend on one-electron donors to fulfil their func-tion [30]. C. reinhardtii possesses several genes encoding radicalSAM proteins, such as Pfl activase [9,10] and two enzymes calledHydEF and HydG, which have been shown to be essential for thematuration of HydA1 [31]. Since most of the radical SAM proteinsand their partners, respectively, are active only under anoxic con-ditions [30], a role of the anaerobically accumulating Fdx5 proteinin donating electrons to one of these seems plausible. However,there are manifold metabolic and biosynthetic pathways in whichferredoxins are engaged and it is also known that different ferre-doxins fulfil analogue functions in different environments (e.g.[32]). Thus, Fdx5 could also function as a ferredoxin better suitedfor a common pathway acting in the altered environment in anaer-obic C. reinhardtii cells. Concluding, the drastic regulation of theFDX5 gene in response to anoxia strongly suggests a specializedfunction of Fdx5 in anaerobic C. reinhardtii cells, except H2-produc-tion and Fnr mediated NADP+ reduction. Obviously, Fdx5 plays arole in supporting specialized pathways utilized by anaerobic C.reinhardtii cells.

Acknowledgments

This work was funded by the Deutsche Forschungsgemeinschaft(SFB 480) and the European Commission (FP7, NEST STRP SOLARH2, contract 212508).

Furthermore, we want to thank several people for their contri-bution to this work: G. Sawers for TdcE-antibody, M. Nowaczykand M. Rögner for mass spectrometric analyses, H. Krassen fordetermining the redox potential of ferredoxins, M. Hippler for thecomponents of the photoreduction assay and C. Kamp for HydA1enzyme.

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