In the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcanii DS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependent assimilatory NO3-/NO2- reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential role in the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmic NarB- and NirA-type reductases respectively. UV–visible absorbance and EPR spectroscopic characterization of purified Hv-Fd demonstrate that this protein binds a single [2Fe–2S] cluster, and potentiometric titration reveals that the cluster shares similar redox properties with those present in plant-type ferredoxins

Bonete, Mari­a José

Butt, Julea N.

Gates, Andrew J.

Martínez‑Espinosa, Rosa María

Richardson, David J.

Zafrilla, Basilio

English

In the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcanii DS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependent assimilatory NO3−/NO2− reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential role in the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmic NarB- and NirA-type reductases respectively. UV–visible absorbance and EPR spectroscopic characterization of purified Hv-Fd demonstrate that this protein binds a single [2Fe–2S] cluster, and potentiometric titration reveals that the cluster shares similar redox properties with those present in plant-type ferredoxins.</jats:p

Basilio Zafrilla

Rosa María Martínez-Espinosa

María José Bonete

Julea N. Butt

David J. Richardson

Andrew J. Gates

Crossref

Biochemical Society Transactions

A haloarchaeal ferredoxin electron donor that plays an essential role in nitrate assimilation

University of East Anglia digital repository

1844 Biochemical Society Transactions (2011) Volume 39, part 6A haloarchaeal ferredoxin electron donor thatplays an essential role in nitrate assimilationBasilio Zafrilla*, Rosa Marı´a Martı´nez-Espinosa*, Marı´a Jose´ Bonete*, Julea N. Butt†‡§, David J. Richardson†‡ andAndrew J. Gates†‡1*Divisio´n de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias, Universidad de Alicante, Campus de San Vicente del Raspeig, 03080 Alicante, Spain,†Centre for Molecular and Structural Biochemistry, Norwich Research Park, Norwich NR4 7TJ, U.K., ‡School of Biological Sciences, University of East Anglia,Norwich Research Park, Norwich NR4 7TJ, U.K., and §School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.AbstractIn the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcaniiDS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependentassimilatory NO3 − /NO2 − reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential rolein the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmicNarB- and NirA-type reductases respectively. UV–visible absorbance and EPR spectroscopic characterizationof puriﬁed Hv-Fd demonstrate that this protein binds a single [2Fe–2S] cluster, and potentiometric titrationreveals that the cluster shares similar redox properties with those present in plant-type ferredoxins.The role of ferredoxins in nitrate andnitrite assimilationNitrogen is essential to all living organisms. This elementexists in many reservoirs on earth that are connectedby a set of biogeochemical processes collectively termedthe nitrogen cycle. Biological access to inorganic nitrogenis predominantly via ammonium (NH4 + ) and nitrate(NO3 − ). Ammonium provides the substrate for biosyntheticcellular pathways that form complex nitrogen-containingcompounds, such as nucleic and amino acids required forDNA and proteins.When the availability of NH4 + is limited, manyorganisms, including plants, fungi and bacteria, may expressassimilatory NO3 − reductase systems and instead assimilateinorganic nitrogen from NO3 − or NO2 − . The biochemicalbasis for this is NAS, a cytoplasmic NO3 − /NO2 −reductase system that can show a high degree of plasticitybetween different organisms, but commonly involves amolybdopterin cofactor-dependent nitrate reductase and asirohaem:ferredoxin-dependent nitrite reductase. In recentyears, archaea have been increasingly recognized as importantcontributors to the global nitrogen cycle. For example, manyhave been found to contain genes for denitrification andammonia oxidation [1,2]. Furthermore, Haloferax speciesare halophiles of the Euryarchaeota that inhabit salineenvironments and assimilate NO3 − or NO2 − via anassimilatory NO3 − /NO2 − reductase pathway [3].Although NAS systems are dependent on cytoplasmicnitrate and nitrite holoreductases, a high degree of flexibilityis observed regarding the nature of the physiologicalKey words: ferredoxin, haloarchaeon, nitrate assimilation, nitrate and nitrite reduction.Abbreviations used: Hm-Fd, ferredoxin from Haloferax mediterranei; Hv-Fd, ferredoxin fromHaloferax volcanii.1To whom correspondence should be addressed (email a.gates@uea.ac.uk).electron donor to these enzymes. In cyanobacteria, a [2Fe–2S](SγCys)4 ferredoxin is reduced by Photosystem I andprovides the electron donor to both the nitrate and nitritereductases NarB and NirA respectively [4]. Similarly, arecent biochemical investigation of the bacterial NAS systemfrom Paracoccus denitrificans has shown that a putative[2Fe–2S](SγCys)2(NδHis)2 ferredoxin, NasG, is essential forcoupling of the physiological electron donor, i.e. the reducedNADH pool, to both the nitrate and nitrite reductasesNasC and NasB respectively [5]. In haloarchaea such asHaloferax volcanii, the physiological electron donor to boththe assimilatory nitrate and nitrite reductases is proposedto be a small acidic [2Fe–2S](SγCys)4-type ferredoxin,termed Hv-Fd. This arrangement is reminiscent of plantand cyanobacterial systems, in which the Hv-Fd supplieselectrons to both a NarB- and NirA-type nitrate and nitritereductase respectively.In the present review, we explore the use of a combinationof UV–visible electronic absorbance and continuous waveEPR spectroscopy methodologies to identify the nature ofthe iron–sulfur site present in the purified Hv-Fd and alsoperform potentiometric experiments to define the redoxproperties of this metalloprotein.Puriﬁcation and spectroscopic properties ofHv-FdCurrently, 87 haloarchaeal genome sequences are available;of these, 13 correspond to Haloferax species (http://www.ncbi.nlm.nih.gov/bioproject?term=Haloferax). In allspecies, including Hfx. volcanii, it is possible to find morethan one open reading frame predicted to encode [2Fe–2S] proteins. The native Hv-Fd protein from Hfx. volcaniiDS2 (DM3757) was purified to homogeneity by anion-exchange (Sepharose CL-4B followed by Q-Sepharose) andC©The Authors Journal compilation C©2011 Biochemical Society Biochem. Soc. Trans. (2011) 39, 1844–1848; doi:10.1042/BST20110709Biochemical Society Transactions     www.biochemsoctrans.orgICoN2 and the NCycle16 1845Figure 1 UV–visible electronic absorbance spectrum (A) and SDS/PAGE analysis (B) of puriﬁed Hv-FdMolecular masses are indicated in kDa. The apparent molecular mass of Hv-Fd (∼14 kDa, conﬁrmed by analytical gel-ﬁltrationchromatography) is overestimated in SDS/PAGE analysis, behaviour that is commonly associated with haloarchaeal proteins.size-exclusion (Sephacryl S-200) chromatography (Figure 1).In order to confirm that the ferredoxin isolated was thatinvolved in assimilatory nitrate and nitrite reductase pathway,the peptide was treated with trypsin and analysed by nano-ESI (electrospray ionization)–LC (liquid chromatography)–MS/MS (tandem MS). The MS results obtained revealedthat this protein (129 amino acids) is encoded by thelocus tag ‘HVO_2995’ and the predicted molecular mass(14.4 kDa) was consistent with that calculated from gel-filtration chromatography (results not shown). A similarapproach was used to identify the previously characterizedHm-Fd, i.e. the physiological electron donor to NarB andNirA fromHaloferax mediterranei [6,7].The electronic absorbance spectrum of the purified proteincontained broad absorbance features between 300 and600 nm, consistent with the presence of a [2Fe–2S] clusteras described previously for other halophilic ferredoxins[6,7] (Figure 1). Protein concentration was determined bythe Bradford assay or spectrophotometrically using anabsorption coefficient of 9600 M − 1·cm− 1 at 420 nm [8]. Bothmethods gave comparable concentrations for the purifiedprotein sample, consistent with near-complete cofactoroccupancy.The X-band EPR spectrum of the oxidized (90 μM, asprepared) protein at 18 K showed no signal, but on additionof 1 equivalent of europium(II) chloride (E0′ = − 0.4 V[9]), a rhombic lineshape from a dominant paramagneticspecies was observed (Figure 2A), consistent with thepresence of a [2Fe–2S]1+ (S= 1/2) site. Further additionof europium(II) chloride or the stronger reducing agenteuropium(II) EGTA (E0’ = − 0.9 V, prepared as describedby Vincent and et al. [9]) failed to alter the magnitude orform of this signal and thus confirmed complete proteinreduction.Microwave power saturation curves alsomeasuredat 18 K, gave a half-saturation power value of 5.5± 0.3 mW,confirming that all spectra presented were recorded undernon-saturating conditions (Figure 2B). Simulation gaveFigure 2 Continuous wave X-band EPR spectrum (A, continuousline) and power-dependence (B) of the europium(II)chloride-reduced Hv-Fd (90 μM) in 2M NaCl, 50 mM Hepes (pH 7.0)Spectral simulation performed using WINEPR SimFonia (version 1.25,Bruker Analytische Messtechnik) is included and offset for clarity (A,broken line). Experimental conditions for the spectrum presented in (A)were: 9.68 GHz microwave frequency, 0.2 mW power, 1 mT (10 Gs)modulation amplitude, at 18 K.resonances at g1,2,3 = 2.07, 1.98, 1.91 and quantificationby comparison with a 1 mM copper(II) EDTA standardgave occupancy of approximately 0.9 spin/mol of protein,consistent withHv-Fd binding a single [2Fe–2S]2+ /1+ clusterco-ordinated by four conserved cysteine residues present inthe amino acid sequence. On addition of excess potassiumferricyanide, a featureless spectrum was observed similar tothat observed for the oxidized protein (results not shown),indicating reversible redox transformation of the cluster backto the diamagnetic [2Fe–2S]2+ (S= 0) state. Thus the low-temperature EPR spectrum and relaxation character of this[2Fe–2S]1+ cluster are similar to those determined for otherhaloarchaeal proteins.C©The Authors Journal compilation C©2011 Biochemical Society1846 Biochemical Society Transactions (2011) Volume 39, part 6Table 1 EPR g-values and redox properties of selected [2Fe–2S] proteinsOrganism g-values (g1,2,3) Midpoint potential (mV) ReferenceHfx. mediterranei 2.07, 1.98, 1.91 − 280 [7]Halobacterium halobium 2.07, 1.97, 1.90 − 345 [12]Hfx. volcanii 2.07, 1.98, 1.91 − 384 Our studySpinach 2.04, 1.95, 1.88 − 401 [11]EPR-monitoredpotentiometric titration experimentswereperformed to determine the midpoint potential of the iron–sulfur centre present in Hv-Fd. Samples were poised in ananaerobic sample vessel and solution potential wasmonitoredusing a multimeter that was referenced against a silver/AgClstandard (potentials herein are quoted compared with thestandard hydrogen electrode, by addition of + 197 mV [10]).The sample contained the followingmediators at 30μMeach:Methylene Blue, duroquinone, menadione, Indigo Carmine,9,10-anthraquinone-2,6-disulfonic acid, anthraquinone-2-sulfonic acid, phenosafranine, safranine-O, Benzyl Viologenand Methyl Viologen. Solution potential was decreasedby incremental addition of europium(II) chloride and, fordesired stable potentials, samples were withdrawn for EPRanalysis (Figure 3). Spectra were recorded, and the variationof signal magnitude with respect to solution potential couldbe fitted to a single n= 1 Nernstian process to give amidpoint potential (Em) of − 384± 10 mV at pH 7. ThisEm value is the lowest determined so far for a haloarchaealferredoxin and thus the redox properties are similar to other[2Fe–2S](SγCys)4 cluster containing ferredoxins presentin plants (Table 1). Significantly, the ferredoxins fromHaloarculamarismortui and plants also share a high degree ofstructural similarity, notably in the [2Fe–2S] environment andprotein core, although the haloarchaeal proteins have a highpreponderance of solvent-exposed acidic residues in additionto other surface adaptations that confer halotolerance[13].Wider roles for ferredoxins in nitrateassimilationThe purification and spectroscopic characterization of thenative ferredoxin isolated from Hfx. volcanii DS2 revealedthat this is a small ferredoxin that binds a single [2Fe–2S] cluster that can undergo a reversible single-electronredox transformation. Taking into account the similaritiesfound between Hv-Fd and Hm-Fd, this protein is likelyto be the physiological electron donor to both the NarB-and NirA-type nitrate and nitrite reductase, as has beendescribed previously for other haloarchaeal species such asHfx. mediterranei, in addition to cyanobacteria and plants[6,7]. As such this protein plays an essential role in theelectron-transfer cascade required for assimilatory nitratereduction inHfx. volcanii.Figure 3 EPR-monitored potentiometric titration of Hv-FdSamples were poised in an anaerobic glove box at the potentialsindicated, and EPR spectra were recorded (A). Experimental conditionsare as for Figure 2(A). The asterisk denotes the increasing contributionof reduced mediator radicals at g = 2 observed below approximately− 280 mV. A plot of signal intensity against solution potential was ﬁttedwith a single Nernst expression (n = 1) to give a midpoint potentialvalue of − 384± 10 mV at pH 7 (B). SHE, standard hydrogen electrode.Multiple sequence alignment (Figure 4) shows that theferredoxins in haloarchaea can be divided into two groups,on the basis of sequence. Like that of most Halobacteriaceae,theHfx. volcanii genome encodes both kinds of ferredoxins.We have characterized a member of the first group, i.e. Hv-Fd, which is defined by the N- and C-terminal sequencesMPTVTYLN and LQNRVI respectively. These proteinsC©The Authors Journal compilation C©2011 Biochemical SocietyICoN2 and the NCycle16 1847Figure 4 Sequence alignment of selected haloarchaeal, cyanobacterial and plant [2Fe–2S] ferredoxinsMultiple alignments of family I and II sequences are shown in (A) and (B) respectively. The conserved cysteine residues thatserve as ligands to iron atoms are highlighted.contain approximately 129 amino acids, whereas the secondgroup of ferredoxins are slightly smaller (∼108 amino acids)and distinguished by the N- and C-terminal sequencesMTEYTVEF and AGTAADDD respectively. The reason forthe presence of two forms of [2Fe–2S](SγCys)4 ferredoxin inthese micro-organisms is unclear at present. Whereas the roleof family I proteins as electron donor to both assimilatorynitrate and nitrite reductases has been established [6,7], a clearrole for the family II ferredoxins is yet to be demonstrated.It also remains to be determined whether these proteins areinterchangeable orwhether one family showsbias to a specificacceptor; however, one or both may act as electron donorC©The Authors Journal compilation C©2011 Biochemical Society1848 Biochemical Society Transactions (2011) Volume 39, part 6to glutamate synthase, which performs the final key step ininorganic nitrogen assimilation.AcknowledgementsWe thank Dr Myles Cheesman (University of East Anglia) and Dr M.Dolores Rolda´n (Universidad de Co´rdoba, Co´rdoba, Spain) for usefuldiscussions.FundingThis work was funded by research grants from the Ministerio deEducacio´n y Ciencia Spain [grant number BIO2008-00082] and Jose´Castillejo Fellowship.References1 Philippot, L. (2002) Denitrifying genes in bacterial and Archaealgenomes. 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(2001) Electrochemical Methods:Fundamentals and Applications, John Wiley and Sons, New York11 Aliverti, A., Hagen, W.R. and Zanetti, G. (1995) Direct electrochemistryand EPR spectroscopy of spinach ferredoxin mutants with modiﬁedelectron transfer properties. FEBS Lett. 368, 220–22412 Kerscher, L., Oesterhelt, D., Cammack, R. and Hall, D.O. (1976) A newplant type ferredoxin from halobacteria. Eur. J. Biochem. 71, 101–10713 Frolow, F., Harel, M., Sussman, J.L., Mevarech, M. and Shoham, M.(1996) Insights into protein adaptation to a saturated salt environmentfrom the crystal structure of a halophilic 2Fe–2S ferredoxin. Nat. Struct.Biol. 3, 452–458Received 23 August 2011doi:10.1042/BST20110709C©The Authors Journal compilation C©2011 Biochemical Society

A haloarchaeal ferredoxin electron donor that plays an essential role in nitrate assimilation

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