Biochimica et Biophysica Acta 1844 (2014) 1238–1247

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Biochimica et Biophysica Acta

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Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAMfamily member

Susana A.L. Lobo a, Andrew D. Lawrence b, Célia V. Romão a, Martin J. Warren b,Miguel Teixeira a,⁎, Lígia M. Saraiva a,⁎a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugalb Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK

Abbreviations: SAM, S‐adenosylmethionine; HPLC, higtography; MS, mass spectrometry; Ahb, alternative haemIII, iron-coproporphyrin III⁎ Corresponding authors.

E-mail addresses: miguel@itqb.unl.pt (M. Teixeira), lst

http://dx.doi.org/10.1016/j.bbapap.2014.03.0161570-9639/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 February 2014Received in revised form 28 March 2014Accepted 31 March 2014Available online 5 April 2014

Keywords:DesulfovibrioAlternative haem biosynthesisIron-coproporphyrin IIIS‐adenosylmethionineiron-sulfur protein

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genusand in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogenIII and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted intohaem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we re-port the biochemical characterisation of theDesulfovibrio vulgaris AhbD enzyme that catalyses the last step of thepathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM)and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD containstwo [4Fe–4S]2+/1+ centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin IIIinduces conformational modifications in both centres. Amino acid sequence comparisons indicated thatD. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-likemotif and two cysteine-rich se-quences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model ofD. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

All modified tetrapyrroles, which include some of the major cofac-tors of life such as haem, sirohaem, haem d1, chlorophylls and cobala-mins (vitamin B12) are synthesised along a branched biosyntheticpathway. Recently, the discovery of a new branch of the tetrapyrrolepathway revealed that haem b can be made by one of two distinctroutes. In the classic haem b synthesis pathway, found in eukaryotesand many bacteria, four enzymatic steps are required to transformuroporphyrinogen III, the primogenitor of all modified tetrapyrroles,into haem b. The first step of the classic pathway is catalysed byuroporphyrinogen decarboxylase (HemE), whichmediates the sequen-tial decarboxylation of the four acetic acid side chains of the substrate togive coproporphyrinogen III. Next, the two propionic acid side chains at-tached to C3 and C8 undergo oxidative decarboxylations to generateprotoporphyrinogen IX in a reaction catalysed by coproporphyrinogen

h-performance liquid chroma-biosynthetic proteins; Fe-Cp

@itqb.unl.pt (L.M. Saraiva).

oxidase (HemF or HemN). Subsequently the macrocyle is oxidised bythe removal of six hydrogens to give protoporphyrin IX in a reactioncatalysed by protoporphyrinogen oxidase (HemG or HemY) and finallyiron is inserted into the core of the substrate template by ferrochelatase(HemH) to give haem b [1,2]. However, in sulfate reducing bacteria ofthe Desulfovibrio genus and in methanogenic Archaea, haem b is pro-duced along a distinct branch of the modified tetrapyrrole pathwaywhere sirohaem acts as an intermediate precursor (Fig. 1) [3–5].Along this route uroporphyrinogen III is transformed into sirohaemby methylation at C2 and C7, oxidation of the macrocycle andferrochelation. Sirohaem is then converted into haem b by the enzymesof the alternative haem biosynthetic pathway (Ahb). Initially, sirohaemis acted upon by the AhbA and AhbB proteins that promote the decar-boxylation of the acetic acid chains attached to C12 and C18 yielding12,18-didecarboxysirohaem (Fig. 1). This decarboxylated form ofsirohaem is modified by AhbC, which catalyses the loss of the aceticacid side chains at C2 and C7 in an S-adenosylmethionine (SAM)-dependent reaction, to form iron-coproporphyrin III (Fe-Cp III)(Fig. 1). The final step is also a SAM-dependent reaction promoted byAhbD that converts the two propionate side chains attached to C3 andC8 of Fe-Cp III into vinyl groups forming haem b (Fig. 1) [3]; therefore,the conversion of sirohaem into haem b via the alternative haem path-way involves several reactions that are linked to radical SAM chemistry.

Fig. 1. Alternative haem b biosynthesis pathway in which sirohaem is converted to haem b via the AhbA, AhbB, AhbC and AhbD enzymes.

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Radical SAMenzymes are a vast family of proteinswith awide diver-sity of functions participating, for example, in the biosynthesis of cofac-tors, vitamins, DNA precursors, and antibiotics. One of the features ofthese enzymes is the presence of, at least, one [4Fe–4S]2+/1+ cluster co-ordinated by only three cysteine residues arranged in a conservedCx3Cx2C motif located at the N-terminal region of the protein [6–10].The catalytically active species of the cluster is the reduced [4Fe–4S]1+

form that promotes the reductive cleavage of SAM to yield methionineand a highly reactive 5′-deoxyadenosyl (dAdo) radical. Due to the oxy-gen lability of this iron–sulfur cluster these reactions usually occurunder anaerobic conditions. This large family of enzymes also includesthe oxygen-independent coproporphyrinogen oxidase, HemN, from thecanonical haem b pathway [11,12]. Radical SAM enzymes may have ad-ditional iron–sulfur centres, dubbed “auxiliary clusters” [13]. For exam-ple, the biotin synthase, BioB, contains an extra [2Fe–2S]2+/1+ cluster[14], whereas the methylthiotransferases RimO and MiaB bind anauxiliary [4Fe–4S]2+/1+ cluster coordinated by three invariant cysteines,which is proposed to be involved in activation of sulphide ormethylsulfide [15,16]. In the molybdenum cofactor biosyntheticenzyme MoaA, the additional [4Fe–4S]2+/1+ centre is also coordinatedby three cysteines [17] while in the anaerobic sulfatase maturatingenzyme AnSME from Clostridium perfringens the two auxiliary[4Fe–4S]2+/1+ centres are coordinated by four cysteine residues [18].

In this work, the AhbD enzyme involved in the last step of thealternative haem b biosynthetic pathway in Desulfovibrio vulgarisHildenborough was biochemically and spectroscopically characterisedand shown to be a member of the radical SAM superfamily of proteinshaving two [4Fe–4S]2+/1+ centres. Based on modelling studies, we

predicted the tridimensional structure of AhbD and propose the puta-tive pockets for the binding of the iron–sulfur centres and the SAMand iron-coproporphyrin III substrates.

2. Materials and methods

2.1. Cloning and expression of recombinant D. vulgaris AhbD

The gene encoding the D. vulgaris Hildenborough AhbD (gene locusDVU0855) was amplified in a PCR reaction using genomic DNA and twodifferent pairs of oligonucleotides to produce a His-tagged and a non-labelled protein. The DNA fragment amplified with the pair 5′-CACATA TGG GAG CGC ATC CCA CTG CCC-3′ and 5′-CAC TCG AGG CGGGAG GGC GTC ATA C-3′ was cloned into NdeI/XhoI pET23b+ vector(Novagen), which allowed production of a C-terminal 6x-HisTag fusedprotein. The DNA fragment amplified with primers 5′-CGG GCG GCCATA TGG GAG CG-3′ and 5′-CAT GTC GGT GGA GAA TTC CCC TGC-3′was cloned into NdeI/EcoRI pET28a+ vector (Novagen), excised withthe same restriction enzymes and cloned into pET23b+ to generate aproteinwith no fusion tags, designated AhbD. All recombinant plasmidswere sequenced to confirm the absence of errors in the gene sequencesand then transformed in E. coli BL21 Star(DE3)pLysS cells (Invitrogen).For protein expression, E. coli cells carrying the recombinant plasmidswere grown in 1 l flasks fully filledwith LBmedium, towhich the appro-priate antibiotics were added. Cells growing at 37 °C that reached anOD600 of approximately 0.5 were supplemented with 400 μM isopropylβ-D-1-thiogalactopyranoside (IPTG) and 30 μM Fe (in the form of

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FeSO4.7H2O), and after an overnight growth, at 20 °C,were harvested bycentrifugation.

2.2. Cell free lysates preparation

D. vulgaris cells were grown anaerobically in lactate/sulfate medium[19], at 37 °C, in 1 l closedflasks until an OD600 correspondent to the sta-tionary phase (OD600 ~1.2), at which point cells were collected by cen-trifugation and kept at−80 °C.

In an anaerobic chamber (Belle Technology, b2 ppm O2), D. vulgarisand the E. coli BL21 Star(DE3)pLysS cells overexpressing D. vulgarisAhbD were resuspended in degassed 50 mM Tris–HCl pH 8 (buffer A),lysed by sonication and centrifuged for 20min at ~30,000×g for remov-al of unbroken cells. The resulting cell free lysates of D. vulgaris and ofE. coli overexpressingAhbDwere used in the activity assays as describedbelow.

2.3. Protein purification

For protein isolation, E. coli BL21 Star(DE3)pLysS cells overexpress-ing D. vulgaris AhbD were resuspended in buffer A and disrupted bypassing three times through a French Press, at 900 Psi under aerobicconditions, after which the suspensions were centrifuged for 30 minat ~30,000 ×g. The resulting supernatant was submitted to an argon at-mosphere and further manipulated in a Coy model A-2463 anaerobicchamber containing a gas mixture of 95% argon plus 5% hydrogen.The supernatant was applied to a 3 ml Ni2+ saturated ChelatingSepharose resin (GE, Healthcare), previously equilibrated in buffer Awith 500mMNaCl and 10mM imidazole (binding buffer). A step gradi-ent of imidazole in the same buffer was applied and the protein waseluted at 250mM imidazole, concentrated in an Amicon Stirred Ultrafil-tration Cell using a 10 kDa membrane (Millipore), and submitted tobuffer exchange by means of a PD10 column (GE Healthcare) thatused buffer A as running buffer. The purity of the protein was evaluatedby SDS-PAGE and the protein concentration was determined by thePierce Bicinchoninc acid Protein Assay kit (Thermo Scientific) usingSigma protein standards. UV–visible spectra were recorded inside theanaerobic chamber in a Shimadzu UV-1800 spectrophotometer.

The molecular mass and quaternary structure of the protein weredetermined by passage in a Superdex 200 10/300GL column (GE,Healthcare), equilibrated with 50 mM sodium-phosphate pH 7 buffercontaining 150 mM NaCl. The iron content of the pure protein was de-termined by the 2,4,6-tripyridyl-1,2,3-triazine (TPTZ) method [20].

2.4. Analysis of the reaction products by high-performance liquidchromatography–mass spectrometry (HPLC–MS)

Independent assays were performed using the purified protein aswell as the cell free lysates of D. vulgaris and of E. coli BL21Star(DE3)pLysS cells overexpressing D. vulgaris AhbD.

Anaerobically purified D. vulgaris AhbD (15 μM) was mixed with15 μMFe-Cp III, 1 mM SAM and 0.5mM sodium dithionite in a final vol-ume of 1ml and left to react for 3 h and overnight, at room temperature,under anaerobic conditions.

Reaction mixtures of 4 ml in buffer A containing D. vulgaris cell ly-sate, 20 μM Fe-Cp III (Frontier Scientific), 0.1 mM SAM, 10 mM sodiumdithionite and 0.5mMNADHwere prepared and anaerobically incubat-ed overnight, at room temperature. Reactionmixtures of 4ml in buffer Awith E. coli cell lysate, 20 μM Fe-Cp III, 0.5 mM SAM and 10mM sodiumdithionite were also prepared and incubated overnight.

In all cases, after the completion of the reaction, the tetrapyrrole in-termediates were purified by a haem extraction protocol [21] andanalysed by reverse phase chromatography and mass spectrometry.To this end, the final extraction solvent, ethyl acetate, was evaporatedand the intermediates were resuspended in a mixture of 90% acetoni-trile–10% methanol (HPLC grade) and resolved on an Ace 5 AQ column

(2.1 × 150mm; Advanced Chromatography Technologies Ltd) attachedto anAgilent 1100 seriesHPLC equippedwith a diode array detector andcoupled to a micrOTOF-Q II (Bruker) mass spectrometer. The tetrapyr-role derivatives were detected at 390 nm and separation was achievedby applying a binary gradient at a flow rate of 0.2 ml min−1 with 0.1%trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.The column was first equilibrated with 20% solvent B and after sampleinjection the concentration of solvent B was increased to 100%. Thetotal duration of each run was 50 min [3].

For the analysis of SAMderived products, reactionmixtures contain-ing ~40 μM D. vulgaris AhbD, 1 mM sodium dithionite and 15 μM Fe-CpIII in a final 0.5 ml volume of buffer A were anaerobically prepared, andthe reactionwas initiated by adding 1mMSAM. After overnight incuba-tion, 100 μl aliquots were taken and immediately quenched by additionof an equal volume of 100 mM H2SO4. The mixtures were then centri-fuged for 20 min at 16,000 ×g, and 50 μl of the supernatants containingthe SAM related products were analysed by HPLC–MS. As control, a mixof SAM, 5′-deoxyadenosine (dAdo), 5′-methylthioadenosine (MTA) andS-adenosyl-L-homocysteine (SAH) was also prepared and the productswere separated and analysed by mass spectrometry as previously de-scribed [3], at a flow rate of 0.2 ml/min and with a gradient of acetoni-trile in 0.1% TFA as follows: 0–1 min isocratic 0% B (acetonitrile), 1–15 min ramp to 10% B, 15–20 min ramp to 30% B, 20–25 min ramp to50% B, 25–30 min ramp to 100% B, 30–35 min isocratic 100% B, 35–40min ramp to 0% B and 40–50min to 0%. The elution profile wasmon-itored by UV–visible spectroscopy at 260 nm.

2.5. Binding assay

The binding of Fe-Cp III to AhbD was analysed by monitoring thechanges of the UV–visible spectrum of the reduced Fe-Cp III (15 μM)upon successive additions of reducedAhbDprotein (2 to 36 μM). The re-duced form of Fe-Cp III was obtained by previous incubation with2 molar equivalents of sodium dithionite in buffer A, while reductionof AhbD was achieved by addition of 8 molar equivalents of sodiumdithionite. The shift of the Soret band and the variation of the absor-bance at 550 nm were monitored. The association constant (KA) andthe maximal number of binding sites (Bmax) values were determinedby fitting the data to the equation Y=Bmax * X / (X+ KA) by nonlinearregression using GraphPad Prism software version 5.0 for Windows,GraphPad Software, San Diego, California, USA (www.graphpad.com).In this equation Y was calculated from the absorbance at 550 nm orthe shift of the Soret bandmaximumandX represents the concentrationof the protein. The titration data were normalised between 0 and 100 inrelation to the smallest and the largest values of each data set.

2.6. EPR studies

Four samples prepared under anaerobic conditions were analysedby EPR spectroscopy, all containing the same concentration of AhbD(160 μM): sample 1, constituted by the as-purified AhbD; sample 2,consisting of purified enzyme reduced with sodium dithionite(2 mM); sample 3, a mixture of AhbD with SAM (1 mM) and sodiumdithionite (2 mM); and sample 4, a mixture of AhbD, SAM (1 mM),Fe-Cp III (175 μM) and sodiumdithionite (2mM). All sampleswere pre-pared in buffer A with a final volume of 300 μl, loaded into EPR tubes,sealed with caps and immediately frozen in liquid nitrogen. EPR spectrawere acquired at several temperatures and microwave powers in aBruker EMX spectrometer equipped with an Oxford Instruments con-tinuous flow helium cryostat. Spectra simulation was done with theSPINCOUNT program (http://www.chem.cmu.edu/groups/hendrich/facilities/index.html). Several independent protein preparations fromdistinct cell cultureswere studied yielding consistently the same results.

The reduction potential of AhbDwas determined bymeans of an an-aerobic redox titration (under constant flux of argon) of the purifiedAhbD, monitored by EPR. To this end, 200 μM AhbD resuspended in

Fig. 3. Titration of Fe-Cp III with D. vulgaris AhbD. UV–visible spectra of reduced Fe-Cp III(15 μM) obtained upon successive additions of AhbD (spectral shifts are indicated byarrows) and respective titration curve (inset) considering the shift of the Soret bandmax-imum (black squares) and the absorbance increase at 550 nm (open diamonds); thevalues were normalised to their final stage. The solid line corresponds to the fitting ofthe curves with an equation for a 1:1 binding stoichiometry and a binding constant of8 μM (see Materials and methods).

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buffer Awas titrated by stepwise addition of buffered sodiumdithionite(10, 50 and 100 mM), in the presence of a standard redox mediatorsmixture (100 μM): 1,2 naphtoquinone (E′0 = 180 mV), 1,4-naphtoquinone (E′0 = 60 mV), methylene blue (E′0 = 11 mV), mena-dione (E′0= 0mV), indigo tetrasulfonate (E′0=−30mV), plumbagin(E′0 = −40 mV), resorufin (E′0 = −51 mV), indigo trisulfonate(E′0 = −70 mV), indigo disulfonate (E′0 = −110 mV), phenazine(E′0 = −125 mV), 2-hydroxy-1,4-naphtoquinone (E′0 = −152 mV),anthraquinone sulfonate (E′0 = −225 mV), phenosafrine (E′0 =−255 mV), safranine (E′0 = −280 mV), neutral red (E′0 =−325 mV), benzyl viologen (E′0 = −345 mV) and methyl viologen(E′0 = −440 mV). A mixture of catalase (130 U/ml), glucose oxidase(4 U/ml) and glucose (3 mM) was also added to the sample mixtureto further ensure that anaerobic conditionsweremaintained. Combinedsilver/silver chloride and platinum electrodes calibrated against a satu-rated quinhydrone solution at pH 7 were used. Potentials are reportedrelative to the standard hydrogen electrode. The experimental datawere fitted to a single one-electron Nernst curve (n = 1).

3. Results

3.1. Biochemical and UV–visible spectroscopic analysis of D. vulgaris AhbD

The recombinantD. vulgarisAhbDwaspurified under anaerobic con-ditions in order to protect any putative oxygen-sensitive clusters. Nev-ertheless, the stability of the AhbD clusters was tested by exposure ofthe protein to oxygen and no degradation was observed. The proteinwas eluted from an analytical gel filtration chromatography as a mono-merwith an apparentmolecular mass of ~40 kDa, in agreementwith itsgene-derived amino acid sequence. The as-isolated protein exhibits abrown colour with a visible spectrum displaying two absorption bandsat 325 and 415 nm (Fig. 2). These features are similar to those of theoxidised form of iron–sulfur cluster containing proteins. Indeed, metalanalysis also showed that AhbD contains 6 ± 2 mol of iron per mol ofprotein, suggesting the presence of more than one iron–sulfur cluster.

3.2. D. vulgaris AhbD binds Fe-Cp III

In the alternative biosynthetic pathway operative in D. vulgaris,AhbD converts iron-coproporphyrin III into haem b. In this work, wehave analysed the binding of the substrate by following changes in thevisible spectrum of Fe-Cp III upon addition of AhbD. The mixing ofAhbD with oxidised Fe-Cp III caused no alterations in the spectrum ofFe-Cp III. However, addition of reduced AhbD to the sodium dithionitereduced iron-tetrapyrrole induced shifts in all the porphyrin-associated absorption bands (Fig. 3). Note that these shifts were not

Fig. 2. UV–visible spectrum of the as-isolated D. vulgaris AhbD (5 μM) in 50 mM Tris–HClpH 8, recorded under anaerobic conditions.

seen in control samples that contained all components except AhbD.We observed that the Soret band of Fe-Cp III shifted from 405 nm to413 nm, concomitantly with the development of bands at 528and 550 nm. These alterations are consistent with the binding of theFe-Cp III to the protein and the formation of a low-spin hexa-coordinated Fe-Cp III. Furthermore, the trace resultant from the subtrac-tion of the final titration spectrum from that of the iron-coproporphyrinalone exhibits maxima at 424, 518 and 550 nm. These maxima are sim-ilar to those previously observed for D. desulfuricans ATCC2774bacterioferritin, which is naturally isolated with iron-coproporphyrinas the haem cofactor and that has two haem-iron axial ligands [22,23].

We also evaluated the affinity of the Fe-Cp III substrate to D. vulgarisAhbD by performing titration experiments of AhbDwith Fe-Cp III whichwere monitored by following the alteration of the Soret band and theabsorbance increase at 550 nm (see Materials and methods). The dataallowed the determination of an association constant (KA), consistentwith a single (1:1) binding stoichiometry, of 8 ± 1 μM (Fig. 3).

3.3. D. vulgaris AhbD promotes cleavage of SAM and converts Fe-Cp III intohaem b

Since the step promoted by AhbD is proposed to be a SAM-dependent reaction, the products of SAM cleavage were analysed byHPLC–MS after incubation of the purified protein with SAM and sodiumdithionite. Under these conditions, the formation of 5′-deoxyadenosine(dAdo) productwas detected (Fig. 4A). The reactionmixture containingthe same concentration of all components but done in the presence ofthe Fe-Cp III substrate was also analysed.We observed that the additionof the second substrate led to an approximately 10-fold increase of theamount of the dAdo formed, indicating that the metalloporphyrin po-tentiates the SAM cleavage (Fig. 4C). The data show that D. vulgarisAhbD performs a reaction that is typical of the radical SAM enzymes,namely the reductive cleavage of SAM into methionine and the dAdoradical. As with other members of this family of proteins [24] the SAMderived products were observed in the absence of the second substrate.

The products formed during the conversion of Fe-Cp III into haem bcatalysed by D. vulgaris AhbD were also determined by HPLC–MS. Thereaction was performed separately with D. vulgaris free cell lysates,E. coli cell extracts overproducing an untagged form of D. vulgarisAhbD and purified D. vulgaris AhbD. The reaction mixtures consisting

Fig. 4.HPLC–MS analysis of the cleavage derived products of SAM promoted byD. vulgarisAhbD. From top to bottom: HPLC trace of the standards 5′-deoxyadenosine (dAdo) andS-adenosylhomocysteine (SAH); (A) AhbD incubatedwith SAM in the presence of sodiumdithionite; (B) AhbDand SAMdone in the absence of the reducing agent (control sample);(C) AhbD incubated with SAM and Fe-Cp III under reducing conditions; (D) AhbD mixedwith SAM and Fe-Cp III without the reducing agent (control reaction). HPLC traces wereacquired at 390 nmafter an overnight incubation of the reactionmixture. In reactionmix-tures A-D SAM was eluted as two peaks with the same m/z value of 399.

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of Fe-Cp III, SAMand the purifiedAhbDprotein or theD. vulgaris free celllysates overproducing AhbDwere incubated overnight, and the extract-ed samples separated and analysed. Peaks typically corresponding tohaem b (m/z 616), a mono-vinyl derivate of Fe-Cp III (m/z 662) andthe unreacted form of Fe-Cp III (m/z 708) were detected indicatingthat the conversion was incomplete (Fig. 5A and D). However, whenE. coli cell free extracts overexpressing AhbD were used in the presenceof SAM, a complete conversion of Fe-Cp III into haem b was observedafter an overnight incubation (Fig. 5B), while control reactions per-formed in the absence of SAM contained essentially only Fe-Cp III(Fig. 5C).

Altogether, these results show that AhbD converts Fe-Cp III intohaem b and that the reaction occurs in two consecutive steps.

3.4. D. vulgaris AhbD contains two [4Fe–4S]2+/1+ centres

The nature of the iron–sulfur clusters in AhbD was studied in detailby EPR spectroscopy. The EPR spectrumof the as-isolated AhbD exhibits

a quasi-isotropic signal with g-values at around 2.02 (2.03, 2015, 2.005)as determined by simulating the spectrum using the programSPINCOUNT (Fig. 6Aa), characteristic of an oxidised [3Fe–4S]1+/0 clus-ter. This resonance accounts 10–15% of the spectral intensity of thedithionite reduced sample, under the same non saturating conditions,and most likely results from a minor oxidative degradation of one ortwo of the tetranuclear centres, a common feature in low-reduction po-tential [4Fe–4S]2+/1+ centres. Reduction of AhbD with sodiumdithionite led to development of a more complex signal with g-valuesranging from g ~2.06 to 1.92, which are typical of reduced bi- ortetranuclear iron–sulfur centres (Fig. 6Ab). These resonances were de-tected without significant line broadening only up to a temperature of~30 K. Hence, we concluded that are not originated from binuclear cen-tres but from tetranuclear iron–sulfur clusters which agrees with thecysteine motifs present in the AhbD amino acid sequence (see below).The resonances were deconvoluted by varying the temperature andmi-crowave power. When, at 10 K, the microwave power was increased aresonance with gmax = 2.083 developed. This is was clearly discernibleat 40mW(Fig. 6Ba)while at lower g-values no further resonances weredetected. Also, resonances at low magnetic fields, which can originatefrom cluster spin states higher than ½, were not observed even athigh microwave powers and low temperatures down to 4.6 K (datanot shown). The experimental data at 10 K could be rationalised asresulting from two reduced tetranuclear centres, with distinctly dis-cernible gmax values and quite different relaxation properties: a fastrelaxing species, only observable at low temperatures and high micro-wave power, with g-values at 2.083, 1.921 and 1.84, and a slowerrelaxing one with g-values at 2.057, and 1.925 (Fig. 6Ba). Within thelimitations resulting from the distinct relaxation properties, these spe-cies are present essentially in equimolar amounts.

To further characterise D. vulgaris AhbD, the redox potential ofthe iron–sulfur centres was determined by performing a redox titrationfollowed by EPR. The results were analysed with a one-electron Nernstcurve and allow determining a single reduction potential value of−390± 10mV (Fig. 7). The data also indicate that the reduction potentials ofthe two centres are similar as the individual reduction potentials of thetwo iron–sulfur centres could not be deconvoluted. Moreover, the lackof a plateau observed under the tested conditions shows that themetal centres of AhbD have very low reduction potentials, whoseupper limit is −390 mV.

We have also analysed the EPR spectra of dithionite-reduced AhbDsamples upon addition of the substrates. In the presence of SAM,some modifications of the spectra occurred and the data could againbe interpreted as resulting from two main components, with g-valuesonly slightly distinct from those of the reduced sample (Fig. 6Ac andBb). The minor alterations observed upon addition of SAM only arenot unexpected as EPR spectroscopy, although sensitive tominutemod-ifications of the paramagnetic centre electronic structure, does not nec-essarily reflect specific changes of, e.g., the coordination of the iron ionsin the clusters.

Major differences were observed when the substrates SAM andFe-Cp III were both added: two sets of resonances are clearly affected,but to differing extents. By varying the microwave power, two majorspecies were defined, one that was more similar to the previouslyidentified slow relaxing centre, with g-values at 2.064 and 1.92, anda second one from a faster relaxing centre with very broad reso-nances and g-values of 2.13, and ~1.92, underneath the gmed of thefirst species (Fig. 6Ad and Bc).

Above ~40 K, the spectra display solely a signal with g-values of2.035, 2.023, and 2.01, quite distinct from that of the [3Fe–4S]1+ centredetected in the oxidised protein; this species is also observed in the 10 Kspectra (Fig. 6A and B, marked by an asterisk). This species, which has aslow relaxation rate (it is not detected at high microwave powers) andof unknown origin, was observed in all spectra of the reduced protein(including in the presence of the substrates), and in different prepara-tions, albeit accounting always to less than 10% of the total spectral

Fig. 5. HPLC–MS analysis of tetrapyrrole derivatives derived from reaction mixtures of cell free extracts or purified D. vulgaris AhbD. The reaction mixtures contained: (A) D. vulgaris cellfree extracts, Fe-Cp III, SAMand sodiumdithionite; (B) E. coli cell free extracts overexpressingD. vulgarisAhbD, Fe-Cp III, SAM and sodiumdithionite; (C) purifiedD. vulgarisAhbD, Fe-Cp IIIand sodium dithionite; (D) purified D. vulgarisAhbD, Fe-Cp III, SAM and sodium dithionite. HPLC traces were acquired at 390 nm after an overnight incubation of the reactionmixture. Fe-Cp III eluted from the HPLC column as two peaks (marked with an asterisk) with the same m/z value of 708.

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intensity at 10 K. Since this species corresponds to a minor component,its origin was not further investigated.

Altogether, the data show that D. vulgaris AhbD contains two [4Fe–4S]2+/1+ centres that are perturbed upon addition of the substratesSAM and Fe-Cp III. It should be noted that all experiments were per-formed in the presence of excess sodium dithionite, and for this reasonreoxidation of the clusters was not observed upon addition of SAM; theiron of Fe-Cp III will be in the 2+ state and is therefore EPR silent in theconditions used, which is consistent with our previous observation thatthe ferrous Fe-Cp III adopts a low-spin (S = 0) configuration whenbound to the protein. The largest changes at one of the clusters probablyreflect the binding of SAM to the non-cysteine coordinated iron of thecluster in the protein N-terminal domain, as has been shown for severalother radical SAM enzymes (e.g. [15,25–28]. The major effects after ad-dition of the Fe-Cp III substrate suggest that it induces alterations in theconformation of the AhbD protein, and that the Fe-Cp III binding pocketis probably close to the iron–sulfur clusters.

3.5. Amino acid sequence analysis of D. vulgaris AhbD

A BLAST search against Desulfovibrio spp. and Archaea genomesshowed that homologs of D. vulgaris AhbD are widespread inDesulfovibrio spp., sharing 27%–89% identity and 42%–92% similaritywhen considering the first 100 retrieved amino acid sequences. For ex-ample, AhbD from D. vulgaris and D. desulfuricans ATCC 27774 have 70%identity and 77% similarity between them. Orthologs ofD. vulgarisAhbDalso occur inmethanogenic Archaea (e.g.,Methanosarcina barkeri AhbD,50% identity, 68% similarity). In agreement with our data, recent studiesshowed that theM. barkeri AhbD also catalyses the conversion of Fe-CpIII into haem and the authors proposed that it contains [4Fe–4S]2+/1+

centres [29]. Interestingly, D. vulgaris AhbD shares ~26% identity and44% similarity with the enzyme involved penultimate step of the

alternative haem b pathway, namely the D. vulgaris AhbC that converts12,18-didecarboxysirohaem into Fe-Cp III (Figs. 1 and 8).

Fig. 8 compares the amino acid sequence of D. vulgaris AhbD withthose of enzymes involved in the synthesis of tetrapyrroles and decar-boxylation reactions (Fig. 8). D. vulgaris AhbD shares 25% identity, 39%similarity with Paracoccus denitrificans NirJ, which participates inthe synthesis of haem d1. In relation to E. coli HemN, the oxygen-independent coproporphyrinogen III oxidase of the classical haem bpathway that performs the oxidative decarboxylation of the propionateside chains of rings A and B of coproporphyrinogen III to vinyl groupsproducing protoporphyrinogen IX, a low degree of similarity is seen(10% identity, 20% similarity) [3,26,30]. The low degree of similarityamong these proteins suggests that despite the small structural differ-ences of the several tetrapyrrole substrates their conversion requiresenzymes with highly specific structures.

A comparative analysis of the amino acid sequence of D. vulgarisAhbD against the NCBI protein database shows that the protein belongsto the radical SAM enzymes superfamily. The protein has a radicalSAM motif in the N-terminal region and a SPASM-like motif in theC-terminus (Fig. 8). The SAM motif contains a cysteine rich region(C31x3C35xHC38) typical of SAM proteins, namely the Cx3CxXC se-quence, where X is an aromatic residue; these three cysteines bindthe catalytic [4Fe–4S]2+/1+ centre, leaving the fourth iron free to in-teract with SAM (e.g., [6,18,31]). The N-terminal region of D. vulgarisAhbD has also a GGD triad in place of the GGEmotif, which is present inseveral radical SAM proteins and is involved in the binding of SAM [31].The C-terminal region of AhbD houses a second cysteine rich regionC326x2C329x5C335x2C338, characteristic of a SPASM motif, whosename is derived from the following biochemically characterised mem-bers of this subfamily: subtilosin A maturase AlbA, pyrroloquinolinequinone E PqqE, anaerobic Sulfatase maturating enzyme, andmycofactocin peptide maturase [32–34]. The SPASM domain is quite

Fig. 6. EPR spectra ofD. vulgarisAhbD: oxidised and reduced forms andupon incubationwith SAMand Fe-Cp III. (A) EPR spectra of AhbD: as prepared, oxidised form (a); sodium-dithionitereduced (b); sodium-dithionite reduced AhbD mixed with SAM (c); sodium-dithionite reduced AhbD incubated with SAM and Fe-Cp III (d). Spectra were recorded at 10 K and 2 mW.(B) Power dependence of the EPR spectra of AhbD: sodium-dithionite reduced form (a); sodium-dithionite reduced AhbDmixedwith SAM (b); sodium-dithionite reduced AhbD proteinmixed with SAM and Fe-Cp III (c). Spectra were recorded at 10 K and 2, 20, 40 and 63 mW, as indicated. Asterisk denotes the unknown species (b10%).

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conserved in members of the radical SAM family and is describedto bind auxiliary [4Fe–4S]2+/1+ centres. The biochemical andspectroscopic studies presented above indicate that in D. vulgarisAhbD the SPASM motif is also involved in the binding of one auxiliary[4Fe–4S]2+/1+ cluster, most possibly coordinated by four cysteine resi-dues. For most of the enzymes, the function of the additional cluster re-mains, so far, unclear [13].

Fig. 7. Redox titration of the [4Fe–4S] 2+/1+ clusters of D. vulgaris AhbD. Titration wasmonitored by EPR spectroscopy, measuring the peak-to peak intensity of the g=1.91 res-onance, at 10K. The solid line corresponds to a one-electronNernst curve,with a reductionpotential of−390 mV.

4. Discussion

In this work, D. vulgaris AhbD is shown to be a monomeric cytoplas-mic protein and a member of the radical SAM superfamily. Theprotein harbours two [4Fe–4S]2+/1+ centres, one located at the N-terminal domain and coordinated by three cysteines and the other atthe C-terminus putatively ligated by four cysteines. Under reductiveconditions and in the presence of SAM, AhbD promotes the sequentialdecarboxylation of the two propionate side chains attached to C3 andC8 of Fe-Cp III to form vinyl groups, i.e., generating haem b.

Using two of the more similar structural protein analogues ofD. vulgaris AhbD, namely the AnSME from C. perfringens [18] andMoaA of S. aureus [17], we predicted a three dimensional modelfor the structure of AhbD (Fig. 9). AnSME, which has two auxiliary[4Fe–4S]2+/1+ clusters each coordinated by four cysteines located atthe C-terminus, shares with D. vulgaris AhbD 15% identity, 31% similar-ity, while MoaA that has one additional [4Fe–4S]2+/1+ coordinated byonly three cysteines shares 14% identity and 34% similarity with theD. vulgaris enzyme (Fig. 8). The D. vulgaris AhbD structure model hasβ/α motifs arranged in a TIM barrel-like motif, the common feature ofmost radical SAM enzymes [6], although some of the beta strands ap-pear as loops probably due to the low amino acid sequence similarity.As predicted from the amino acid sequence, the N-terminal region cor-responds to the SAM radical protein domain, which includes the threecysteines that bind the catalytic [4Fe–4S]2+/1+ cluster, the two glycineresidues, Gly75 and Gly76, and the aspartate residue Asp77 that inter-acts with the SAM molecule. The C-terminal domain contains four cys-teines (Cys326, Cys329, Cys335 and Cys356) that are predicted to be

Fig. 8. Amino acid sequence alignment of D. vulgaris Hildenborough AhbD (gene locus DVU0855) with D. desulfuricans ATCC 27774 AhbD (Ddes0287), Methanosarcina barkeri AhbD(MbarA1458)D. vulgarisHildenboroughAhbC (DVU0857),Methanosarcina barkeriAhbC (MbarA1793),D. desulfuricansATCC27774AhbC (Ddes0289), P. denitrificans haemd1 biosyntheticenzyme NirJ (Pden2494), Clostridium perfringens AnSME (CPF_0616), Staphylococcus aureus MoaA (SA2063) and Escherichia coli HemN (b2955). The two domains of D. vulgaris AhbDnamely, the AdoMet radical domain and the auxiliary iron–sulfur binding SPASM domain are indicated. The amino acid residues of the AdoMet radical motif for the binding of the con-served iron–sulfur centre CX3CX2C are shaded in black and marked with stars. Residues of the GG(E/D)P motif involved in the binding of SAM are shaded in dark grey and indicatedwith open circles. Cysteine residues of the SPASMmotif are shaded in light grey. The alignment was constructed using the amino acid sequence of D. vulgaris AhbD to search for similarsequences against all microbial genomes using NCBI Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/), performed in ClustalX 2.1 [37] and edited in GeneDoc [38].

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Fig. 9. 3D model of D. vulgaris AhbD. (A) Cartoon representation of the predicted D. vulgaris AhbD structure. (B) Tridimensional structure of C. perfringens AnSME displayed in the sameorientation as in panel A. (C) Surface representation of D. vulgaris AhbDmodel. (D) Surface representation of D. vulgaris AhbDmodel rotated by 90° from the view in panel C. The figuredepicts a channel (markedwith an arrow) and the predicted binding site of the SAMmolecule which is located at the end of the channel. Themodel was generated by the I-TASSER server[39–41] using as template the structure of C. perfringens AnSME (PDB ID: 4K36). The root-mean-square deviation between the two models is 0.987 Å. In panels A and B, the monomer iscoloured in gradient, starting from blue (N-terminal) to red (C-terminal) and the region of the conserved cysteines involved on the iron–sulfur clusters is boxed and zoomed. The aminoacid residues, the SAM molecule and the iron–sulfur clusters are represented as sticks. Pictures were built with the PyMOL program [42].

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the ligands of the auxiliary [4Fe–4S]2+/1+ cluster equivalent to auxiliarycentre II in the C. perfringens AnSME (Fig. 9A). Both iron–sulfur clustersare expected to be close to the protein surface, which may allow

exchange of electrons with yet unknown physiological partners. Inter-estingly, in the place where C. perfringens AnSME binds the auxiliarycluster I, the D. vulgaris enzyme has still two cysteine residues

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(Cys288 and Cys338) (Fig. 9B), which are conserved in all AhbD homo-logues so far sequenced. In the protein surface representation of AhbD(Fig. 9C and D) it is striking the presence of a cavity that remarkably re-sembles the haem binding cavity of hemophores [35,36], and which isabsent in the model templates. We hypothesize that it will be in thiscavity that the substrate Fe-Cp III binds pointing the two propionateside chains to the interior of the protein and being close to the SAMbinding pocket which will enable their decarboxylation; the Fe-Cp IIIis also predicted to be located near theD. vulgarisAhbD auxiliary cluster,in a pocket resembling that containing the auxiliary cluster I binding sitein C. perfringens AnSME.

In conclusion, we show that D. vulgaris AhbD is a two [4Fe–4S]2+/1+

containing haem synthase that adds a new function to the widespreadsuperfamily of radical SAM enzymes.

Funding

The work was funded by PEst-OE/EQB/LA0004/2011, FCT projectPTDC/BBB-BQB/0937/2012 and the FCT student fellowship SFRH/BPD/63944/2009.

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