Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP+ reductase

Article abstract of DOI:10.1016/j.bbagen.2014.07.021

Background Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans. Methods A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV-vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners. Results A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis. Conclusions Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans. General significance Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.

Full text of DOI:10.1016/j.bbagen.2014.07.021

Biochimica et Biophysica Acta 1840 (2014) 3208–3217
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Heme-iron utilization by Leptospira interrogans requires a heme
oxygenase and a plastidic-type ferredoxin-NADP+ reductase
Anabel Soldano a, Huili Yao b, Mario Rivera b, Eduardo A. Ceccarelli a, Daniela L. Catalano-Dupuy a,
⁎
a
Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Ocampo y Esmeralda, 2000 Rosario, Argentina
Department of Chemistry and Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Multidisciplinary Research Building, 2030 Becker Dr, Room 220 E, Lawrence, KS 66047, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 May 2014
Received in revised form 4 July 2014
Accepted 28 July 2014
Available online 1 August 2014
Background: Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin
employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes
to virulence in Leptospira interrogans.
Methods: A phylogenetic analysis was performed using heme oxygenases sequences from different organisms in-
cluding saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned,
overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectro-
photometry and 1H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied
with different redox partners.
Keywords:
Leptospira interrogans
Heme oxygenase
Ferredoxin-NADP+ reductase
Iron
Results: A plastidic type, high efficiently ferredoxin-NADP+ reductase (LepFNR) provides the electrons for heme
turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover,
LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products.
The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the
functional role of this enzyme in L. interrogans pathogenesis.
Electron transfer
Conclusions: Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we
report a new substrate of ferredoxin-NADP+ reductases different to ferredoxin and flavodoxin, the only recog-
nized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of
heme degradation in L. interrogans.
General significance: Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since
iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic
applications.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
HO, which employs heme as both substrate and a prosthetic group
[3], catalyzes the conversion of heme to biliverdin IX, carbon monoxide
Iron is an essential nutrient for most organisms. Pathogenic microor-
ganisms require host-iron for the metabolic processes that allow them to
colonize their hosts and cause disease. Since free iron is not readily avail-
able and heme represents the most abundant source of iron in mammals,
many pathogens have developed different mechanisms to acquire
and utilize this molecule as iron source. A family of monooxygenases
known as heme oxygenases (HO; EC 1.14.99.3) carries out heme degra-
dation and enables microbes to use heme-iron [1,2].
(CO) and free iron (Fe2+) utilizing oxygen and reducing equivalents [4].
HOs are present in a wide range of organisms including mammals
[5], higher plants [6], algae [7,8], cyanobacteria [9], fungi [10], and vari-
ous pathogenic microorganisms [11–15]. HOs play important roles in
different physiological processes as iron homeostasis, defense against
oxidative and cellular stress, neurotransmission by the generation
of CO as a physiological messenger molecule [16] and biosynthesis of
photoreceptive pigments in photosynthetic organisms. In pathogenic
microorganisms the major function of HOs has been attributed to iron
acquisition from host heme during infection [17], although some of
them have been identified to protect against heme toxicity [1].
Several HOs structures are available [18–24]. HOs are classified as
“all alpha” proteins. They fold into single compact multi-helical do-
mains containing two structural repeats of 3-helical motif. The heme
is sandwiched between two helices called proximal and distal, and
is coordinated by a His side chain in the proximal side and by a water
molecule in the distal side [25]. Heme degradation is a complex set of
Abbreviations: HO, heme oxygenase; LepHO, Leptospira interrogans heme oxygenase;
apo-LepHO, apo form of Leptospira interrogans heme oxygenase; FNR, ferredoxin-NADP+
reductase; LepFNR, Leptospira interrogans ferredoxin-NADP+ reductase; Fd, ferredoxin;
LFd2, Leptospira interrogans 2[4Fe-4S] ferredoxin; BV, biliverdin
⁎
Corresponding author at: IBR, CONICET, Facultad de Ciencias Bioquímicas y
Farmacéuticas, UNR, Ocampo y Esmeralda, 2000 Rosario, Argentina. Tel.: +54 341
4237070.
E-mail address: catalano@ibr-conicet.gov.ar (D.L. Catalano-Dupuy).
http://dx.doi.org/10.1016/j.bbagen.2014.07.021
0304-4165/© 2014 Elsevier B.V. All rights reserved.

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reactions that requires the input of several electrons originating from
NADPH and three molecules of oxygen. While the mechanism of
heme cleavage is broadly conserved between HOs from most organisms
[2] (see above), the source of reducing equivalents is variable. In mam-
mals the electrons required to drive the heme oxygenase reaction are de-
rived from NADPH cytochrome P450 reductase [26]. Plant, cyanobacterial
and bacterial heme oxygenase activity may be ferredoxin-dependent, as
it has been suggested that the NADPH/ferredoxin-NADP(H) reductase/
ferredoxin system may function to deliver electrons to these enzymes
[27–29]. In some cases, the requirement of a second (auxiliary) reductant,
such as ascorbate or trolox, has been proposed [28]. However, it was
reported that the catalytic activity of Pseudomonas aeruginosa heme oxy-
genase requires only a ferredoxin-NADP+ reductase [30].
Since iron is required for pathogen survival and infectivity, iron
uptake and utilization is an attractive target for antimicrobial drug
development [31]. Consequently, HO may be a potential target for
new antimicrobials that hamper bacterial heme-iron utilization [32]. A
knockout of the heme oxygenase gene in P. aeruginosa (pigA) shows
severe growth defects [13]. Similar observations have been made with
the pathogen Leptospira interrogans [14]. A mutant lacking the heme
oxygenase gene (LB186) is growth impaired when hemoglobin is the
only iron source in the medium, suggesting that HO is essential for
heme-iron utilization by the spirochete. It has also been shown that
L. interrogans HO contributes significantly to virulence in the hamster
model of infection [33].
(v3.1.2) [38]. A fixed preset Whelan–Goldman model of amino acid sub-
stitution was used [39]. The Markov Chain Monte Carlo method was run
with the following settings: 4 chains; temperature 0.25; 4,000,000 gener-
ations, sampling every 200 generations; and "burn-in” to discard the first
5,000 trees. Finally, a consensus tree was obtained (50% majority rule)
and plotted using Dendroscope V 3.2.5 [40].
2.2. Construction of the LepHO expression vector
The gene encoding LepHO (LB186) was amplified by PCR using the
genomic DNA from L. interrogans serovar Lai 56601, kindly provided
by Dr Xiao-Kui Guo from the Dept. of Microbiology Shanghai Second
Medical University, Shanghai, China. Oligonucleotides sequences 5’-
cgcggatccatgagtttagcaactattttacg-3’ and 5’-cccaagcttttaaccttttccaagaac
ggaatc-3’ were designed to introduce BamHI and HindIII restriction
enzyme sites at the 5´ and 3´ ends, respectively. The expression plasmid
was constructed by inserting the amplification product, previously cut
with the indicated enzymes, into the similarly restricted pET-TEV
vector, a modified pET28a vector that contains a Tobacco Etch Virus
(TEV) protease cleavage site between the N-terminal hexahistidine
(His6) tag and the multiple cloning site.
2.3. Protein expression and purification
The pET-TEV vector harboring the LepHO gene was transformed into
E. coli BL21 (DE3) for protein expression. A single colony was cultured
overnight in 10 ml of LB medium supplemented with kanamycin
(50 μg/ml); the cell suspension was subsequently transferred to 1 L of
fresh LB-kanamycin medium and grown at 37 °C until the OD600 reached
0.7–0.8. Protein expression was induced by addition of isopropyl 1-thiol-
D-galactopyranose (IPTG) to a final concentration of 0.5 mM, and the cul-
ture was maintained during 16 h at 20 °C with mild agitation. E. coli cells
were harvested by centrifugation, and disrupted by sonication in 50 mM
Tris–HCl (pH 8), 100 mM NaCl, 1 mM benzamidine. The lysate was cen-
trifuged, and the resulting supernatant was loaded onto a TALON-cobalt
affinity chromatography column (Clonthech Laboratories) previously
equilibrated with 50 mM Tris–HCl (pH 8), 100 mM NaCl. Following a
washing step, the recombinant protein was eluted with the same buffer
containing 150 mM imidazole. The His6-tag was removed from apo-
LepHO by adding the recombinant TEV protease during the dialysis, and
the proteins were further separated by a subsequent TALON-cobalt affin-
ity chromatography procedure. TEV protease was obtained as described
[41]. Apo-LepHO concentration was determined using the bicinchoninic
acid method supplied in a Pierce BCA® protein assay kit (Thermo Scien-
tific). The purified protein was stored at −80 °C until use.
L. interrogans is a parasitic bacterium that infects humans and causes
leptospirosis, also known as Weil's disease [34]. In the past few years
this zoonotic disease has emerged as a major public health problem in
much of the developing world. Leptospira also infects other mammals
including rats, cattle, horses, pigs and dogs and some wild animals
which are reservoir hosts of this pathogen. Leptospirosis is acquired
via skin abrasions or the mucous membranes through contact with con-
taminated soil, water, or urine. L. interrogans colonizes the tissues of the
host resulting in disease syndromes ranging from a mild flu-like illness
to severe hemorrhagic disease.
The reaction catalyzed by heme oxygenase is dependent on reducing
equivalents, since it requires NADPH and a reductase. Previously,
we have proposed that this crucial function might be played by the
ferredoxin-NADP(H) reductase (FNR; EC 1.18.1.2) in L. interrogans
[35], as it has already been observed for the FPR enzyme of P. aeruginosa
[30]. In this work, we studied the heme oxygenase from L. interrogans
(LepHO). The location of LepHO gene and nearby open reading frames
(ORFs) in the L. interrogans serovar Lai 56601 genome indicates that
this locus participates in the acquisition and utilization of heme and
probably in virulence. We also performed a phylogenetic analysis to
investigate how this enzyme is related to other HOs. To infer the metabol-
ic role of LepHO we studied its functional and structural properties.
We found that the plastidic-type ferredoxin-NADP+ reductase is able to
efficiently support the catalytic activity of LepHO in vitro. Moreover, the
reaction proceeds to completion producing biliverdin and free iron, with-
out the need of a ferredoxin. These results suggest that the flavoenzyme is
the redox partner of L. interrogans heme oxygenase in vivo.
The ferredoxin-NADP+ reductase (LepFNR) and ferredoxin LB107
(LFd2) from L. interrogans were expressed and purified as described
previously [35]. Protein concentrations were determined by spectrosco-
py using the published extinction coefficient for LepFNR (ε459 nm
=
9.5 mM−1 cm−1) or employing the above mentioned bicinchoninic
acid method, in the case of LFd2.
2.4. Reconstitution of LepHO-heme complex and binding affinity of heme
2. Materials and methods
The LepHO-heme complex was prepared by adding small incre-
ments of hemin (Fluka, prepared in 5 mM NaOH) to the purified apo-
LepHO and following the optical absorbance ratio at 280/403 nm.
Once saturation was achieved, the unbound hemin was removed by
size exclusion chromatography using Sephadex G50 and SuperdexTM
75 10/300 GL columns pre-equilibrated with 50 mM Tris–HCl (pH 8),
100 mM NaCl.
The incorporation of heme to apo-LepHO was monitored spectro-
photometrically. Once the ferric heme complex is formed it gives a char-
acteristic spectrum with a Soret absorbance peak at 403 nm that is
highly distinguishable from the free heme spectrum (Soret band at
385 nm). Heme binding assay to apo-LepHO was performed by addition
2.1. Sequence alignment and phylogeny
The HO amino acid sequence from L. interrogans NP_714730.1
(GI:24217247) was used as query sequence to perform standard
tBLASTn through the complete protein and translated NCBI database
(Release 195, Apr 2013). The phylogenetic analysis was performed
using 107 sequences. After sequence alignment using ClustlX (2.0) [36]
poorly aligned positions and divergent regions of the alignment were re-
moved using the more conservative settings of the program Gblocks [37].
The extracted multiple alignment blocks were then used for obtaining
a tree by the Bayesian inference method using the MrBayes package

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A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
of increasing concentrations of hemin (0.01 to 0.15 μM) while maintain-
ing the enzyme concentration at 0.13 μM in 1 ml of 50 mM Tris–HCl
(pH 8), 100 mM NaCl. After each addition, the spectrum between 300
and 800 nm was recorded, and the complex absorbance at 403 nm
was calculated. A mathematical deconvolution was applied as follows:
the absorbances of free hemin and LepHO-heme complex at 403 nm
are proportional to their absorbances at 385 nm isosbestic point multi-
plied by a factor x and y respectively (Eqs. (1) and (2)). These values
were calculated from a set of measurements at different concentrations
of free hemin or LepHO-complex as x = 0.85 and y = 1.94.
water peak at 4.7 ppm. Proton spectra were acquired with pre-
saturation of the residual water peak over 10,000 data points, with a
spectral width of 24 kHz, a 125 ms acquisition time, a 50 ms relaxation
delay, and 20 k scans.
2.8. Spectrophotometric analysis of heme turnover by LepHO
Optical absorption spectra were obtained using a Shimadzu UV-
2450 spectrophotometer, and all the measurements were carried out
in 600 μl of 25 mM HEPES-KOH (pH 7.5) at 25 °C in a cell with 1 cm
path length. Electron transfer reactions from NADPH to LepHO were
evaluated using as redox protein partners LepFNR and LFd2. The stan-
dard assay contained 6 μM LepHO, 0.1 mg/ml catalase (Sigma), 3 mM
glucose 6-phosphate, 300 μM NADP+, 1 unit/ml glucose-6-phosphate
dehydrogenase and 0.5 μM; 1 μM or 1.5 μM LepFNR. When the system
FNR/Fd was evaluated, the final protein concentrations were 0.5 μM
LepFNR/0.5 μM LFd2; 0.5 μM LepFNR/1 μM LFd2 or 1 μM LepFNR/0.5
μM LFd2. Reactions were started by addition of the NADPH reductase
system. Spectral changes between 300 and 800 nm were monitored
over a 30 min time period. Biliverdin formation was followed using
the absorbance change at 680 nm.
A403 free hemin ¼ xA385
ð1Þ
A403 complex ¼ yA385
ð2Þ
During titrations the spectra of bound and unbound hemin are
superimposed over the entire range of analysis. Therefore, to determine
the dissociation constant of the LepHO-heme complex was necessary to
differentiate and quantify the A403 of the complex from the A403 of free
hemin (Eq. (3)).
Ferrous iron release during LepHO-catalyzed heme degradation was
analyzed by addition of 250 μM ferrozine to the reaction medium. Time
dependent spectral changes were monitored, and the absorbance
at 562 nm was followed for formation of the ferrozine-ferrous iron
complex.
When ascorbate was employed as reductant, 5 mM ascorbic acid
was added to 6 μM LepHO, and the spectral changes between 300 and
800 nm were recorded for 70 min.
A403 total ¼ A403 complex þ A403 free hemin
ð3Þ
We then considered that the recorded absorbances at 403 nm
(A403 total) were composed of two components: the A403 hemin multi-
plied by “a” and A403 LepHO-heme multiplied by “b” (Eq. (4)). The A403
hemin and A403 LepHO-heme are the estimated absorbances at that
wavelength if all added hemin is free or in complex with LepHO respec-
tively. The variables “a” and “b” are the fraction of free or bound hemin.
2.9. Kinetic parameters of the LepFNR-LepHO electron transfer reaction
A403total ¼ aA403hemin þ bA403 LepHO−heme
ð4Þ
For kinetic analysis 0.25 μM LepFNR was added to reactions contain-
ing 0.7 to 11.5 μM LepHO-heme, 0.1 mg/ml catalase and 300 μM
NADPH. The decrease of the Soret peak at 403 nm was monitored during
200 s, and slopes determined by linear regressions 30 s after addition of
the reductase were taken as the initial reaction rates. Enzyme activity
(μmol LepHO reduced min−1 mg LepFNR−1) was plotted against the
concentration of LepHO, and a non-linear regression of the data was
calculated to fit Michaelis–Menten kinetics.
Substituting Eqs. (1) and (2) in Eq. (4) and rearranging:
b ¼ ðA403 total −xA385Þ=ðy−xÞA385
ð5Þ
Consequently, the absorbance of the complex at 403 nm is obtained
by Eq. (6):
A403 complex ¼ byA385
ð6Þ
Steady-state kinetic and binding data were fitted to the theoretical
curves using SigmaPlot (Systat Software Inc., Point Richmond, CA).
The calculated absorbance of the complex was then plotted against
the hemin concentration to construct titration curves, from which the
dissociation constant was obtained using a one site binding model fit.
2.10. HPLC analysis of LepHO reaction products
2.5. Size exclusion chromatography
Upon completion of the LepFNR-mediated heme oxygenase reac-
tion, glacial acetic acid (50 μl) and 5 M HCl (100 μl) were added to
500 μl aliquots of the assay mixture. The products were extracted into
chloroform (500 μl), the organic layer was washed further with distilled
water (3 × 500 μl) and reduced to dryness. The resultant residue was
resuspended in 100 μL of 5% (v/v) HCl in methanol and esterified for
16 h at 4 °C. The products were diluted with distilled water (400 μl)
and extracted into chloroform. The organic layer was then reduced to
dryness and solubilized in 100 μl of mobile phase consisting of metha-
nol:water (85:15, v/v). The sample was analyzed by HPLC on ÄKTA ex-
plorer system (Amersham Biosciences) equipped with a Hypersil™ ODS
C18 column (3 μm, 150 × 4.6 mm, Thermo Scientific). The flow rate was
0.4 ml/min, and eluates were monitored at 380 nm [28]. The mixture of
all four biliverdin IX isomers was synthesized by oxidative cleavage of
hemin [43]. Hemin (5 mg) dissolved in 50% pyridine was treated with
0.1 M sodium phosphate buffer (pH 7.0) containing 50 mg of ascorbate
and incubated at 37 °C for 5 h. The reaction mixture was then acidified
and esterified as described above. The same treatment was carried out
with a biliverdin hydrochloride standard (Santa Cruz Biotechnology).
Additionally, a liquid chromatography-tandem mass spectrometry anal-
ysis was performed using an Agilent 1200 HPLC system, coupled to a
Gel filtration chromatography was performed on ÄKTA explorer sys-
tem (Amersham Biosciences) equipped with a Superdex™ 75 10/300
GL size-exclusion column (GE Healthcare). Buffer containing 50 mM
Tris–HCl (pH 8), 100 mM NaCl at a flow rate of 0.5 ml/min was used
for equilibration of the column and eluent. Standards of known molec-
ular weight were loaded to the column, and their elution volumes were
determined spectroscopically at 280 nm. Apo-LepHO and LepHO-heme
complex were loaded on the column and chromatographed using the
same conditions as the molecular weight markers.
2.6. Determination of the extinction coefficient
The extinction coefficient at 403 nm (ε403 nm) for the LepHO-heme
complex was determined by the pyridine hemochrome method [42].
2.7. NMR spectroscopy
NMR spectra were acquired on a Bruker Avance spectrometer oper-
ating at a frequency of 599.740 (1H) MHz and referenced to the residual

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3211
G1314C VWD UV detector and a micrOTOF mass spectrometer (Bruker
Daltonik, Bremen, Germany) operating in the positive-ion mode.
posterior probabilities of 60% or more. Results are shown in Fig. 2
in the form of an unrooted tree. Our analysis clearly displays well-
defined groups of eukaryotes and prokaryotes. Major bacterial groups
were properly clustered in monophyletic lineages. The two Firmicutes
sequences included in the analysis were clustered together distantly
from all other sequences. Other Firmicutes were highly divergent and
therefore not included in the analysis. Likewise, during tree inference,
we found that isoforms from land-plants were rather distant from all
other HOs analyzed, as was previously observed [53,54]. It was there-
fore not possible to obtain convergence in our phylogenetic tree infer-
ence when plant HO sequences were included. Consequently, these
HOs were omitted from our study.
As was previously found for other Leptospira proteins [35], Spiro-
chaetes HOs share a common ancestor with their homologues from
Cyanobacteria. However, it is evident that both groups diversified very
early. Interestingly, the Eukaryotes HOs are closer to Cyanobacteria
than other bacterial sequences, suggesting a monophyletic origin from
a unique ancestor. HOs from Cyanobacteria and Eukaryotes containing
two HO isoforms were segregated differentially.
Spirochaetes are all included in a well-defined group from a common
ancestor. The saprophytes Leptospira biflexa and Leptospira meyeri are
more closely related to each other than to pathogenic Leptospira. Inter-
estingly, all saprophyte HO sequences have three residue insertion
(Q86K87K88) not observed in other HOs. All pathogenic Leptospira HOs
clustered in a closely related group. Leptospira liceraseace, which has
been defined as opportunistic or intermediate, is placed apart between
the saprophytes and pathogenic Spirochaetes. This distinct separation of
the different Leptospira HO orthologes may suggest a possible functional
role in pathogenesis.
3. Results
3.1. Genome localization, primary sequence analysis and molecular phylog-
eny of LepHO
The locations of LepHO gene and nearby ORFs in the L. interrogans
serovar Lai 56601 genome [44] are shown in Fig. 1. LepHO (LB186) is
coded in the forward strand of a region between position ~176,600
and ~187,800 of chromosome II. Overlapping the 3´ end of the LepHO
gene is ORF LB187, which codes for a putative protein similar to those
of the major facilitator transporter superfamily. These proteins consti-
tute one of the largest groups of membrane transporters found in
many bacterial species [45]. The other six ORFs in the region are coded
in the reverse (minus) strand. ORF LB191 codes for an iron-regulated
heme binding protein known as HbpA. This protein functions as a
TonB-dependent receptor responsible for mediating acquisition of
heme-containing molecules and was found only in some pathogenic
Leptospira serovars [46]. The ORF LB183 codes for one of the four FUR
homologues, regulatory proteins responsible for modulating the ex-
pression of genes involved in iron acquisition [47]. In the same region,
we found ORF LB181 which codes for a putative membrane protein re-
lated to dolichyl-phosphate-mannose-protein mannosyltransferase. Al-
though O-mannosylation was thought to be restricted to fungi, in recent
years it has become clear that the activity is conserved in all kingdoms
and that in bacteria it may contribute to evade the defense mechanisms
of the infected host [48]. LB190 codes for a homolog to YbaB/EbfC DNA-
binding family proteins. This family may play a role in DNA replication-
recovery following DNA damage, but the function(s) of these proteins
remains to be determined [49]. Finally, ORF LB192 is related to the pro-
tein HmuY, a heme-binding protein that retrieves heme from the host
and then delivers it to outer-membrane transporters, the TonB-
dependent receptor [50]. Clearly, LepHO is located in a genome locus
that participates in the acquisition and utilization of heme, which is
likely related to infection and host persistence.
To search for HO family members, the protein and translated data-
bases available at NCBI were examined by BLAST [51]. Using the
amino acid sequence from L. interrogans HO as query, sequences were
retrieved and 107 manually selected (Table S1). The sequences were
aligned using ClustalX2 [36] as described in experimental procedures,
and relevant blocks were extracted from the multiple sequences align-
ment for phylogeny (Fig. S1). LepHO sequence contains a homologous
His relevant for heme-iron coordination at position 15, as observed in
many HOs [52]. Other amino acids reported to be involved in heme-
protein interactions are completely conserved; these include Tyr 124,
Gly 128 and 133, Lys 167 and Arg 171. The Gly rich region from residue
127 to 134, which is important for substrate (heme) entry and product
(biliverdin) release, is also totally conserved in the LepHO (numbers as
in LepHO, conserved residues are highlighted in red in Fig. S1).
3.2. Overexpression and purification of LepHO
LepHO overexpression in E. coli BL21(DE3) causes a bright green
color pigmentation of the cells due to biliverdin accumulation (data
not shown), as has been observed upon overexpression of other HOs
[29,55]. The apo form of LepHO was entirely recovered from soluble
fraction of cell lysate. The protein was purified using cobalt-affinity
chromatography and then excised from the His6-tag by digestion with
a recombinant TEV protease. The apo-LepHO was obtained in homoge-
neous form as judged by a single band in SDS PAGE, which corresponds
to ~26 kDa, a value close to the molecular mass predicted from the
amino acid sequence (Fig. 3A). Mass spectrometry analysis confirmed
the LepHO molecular weight to be 26.3 kDa. Typical purifications pro-
duced ~80 mg of protein per liter of culture.
3.3. Reconstitution of LepHO-heme complex
HOs use heme both as a substrate and a prosthetic group, and can
form a stable complex with heme [3]. To inquire whether LepHO
could form such a complex, the purified apo-protein was incubated
with hemin and the absorption spectrum recorded after removal of
the unbound hemin by filtration on Sephadex G50. The electronic
absorption spectrum of the LepHO-heme complex differs from that of
We performed a Bayesian inference of a tree for the 107 HO se-
quences using a Markov Chain Monte Carlo approach, with Bayesian
Fig. 1. Location of the gene encoding heme oxygenase (LB186) in the genome of L. interrogans. Genes that are close to HO coding sequence LB186 are shown as arrows with the correspond-
ing identifier. See the text for details.

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A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
Fig. 2. Phylogenetic relationship between different heme oxygenases. The tree was constructed as described in Materials and methods. The distance between two sequences is represented
as the lengths of the connecting branches. The database accession numbers and the full names of the HO source organisms are stated in the Table S1.

A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
3213
Fig. 3. Purification of apo-LepHO and its complex with heme. (A) SDS-PAGE analysis: lane MW, molecular weight markers; lane 1, soluble cell lysate of E. coli BL21 (DE3) expressing LepHO
with the His6-tag; lane 2, purified apo-LepHO by cobalt affinity chromatography and subsequently digested with TEV protease. Numbers on the left indicate the molecular mass of the
standards. (B) Optical absorption spectra of apo-LepHO (solid line) and its ferric heme complex (dashed line), which displays typical peaks at 403, 500 and 630 nm. The inset shows
an enlargement of the region between 450 and 800 nm. (C) Gel-filtration chromatography profiles of apo-LepHO (solid line) and LepHO-heme complex (dashed line). The elution volumes
of molecular mass standards are shown as triangles in the upper horizontal axe (in kDa: blue dextran, 2000; GST, 52; carbonic anhydrase, 29; GFP, 27 and aprotinin, 6.5).
the purified apo-LepHO (Fig. 3B). The ferric heme complex gives
the characteristic spectrum of heme oxygenases, with a Soret band at
403 nm and peaks in the visible at 500 and 630 nm, which are compa-
rable with those reported for bacterial HO-heme complexes [17]. The
LepHO millimolar extinction coefficient at 403 nm was calculated to
be 111.13 mM−1 cm−1, a value that is close to those reported for
other HOs [56].
The purified apo-LepHO and its ferric heme complex were subjected
to gel permeation chromatography on a Superdex™ 75 column. Moni-
toring the absorbance at 280 nm (protein) confirmed that both samples
elute mainly as single peaks corresponding to 28 kDa, thus indicating
that the apo-protein and its heme complex are monomeric (Fig. 3C).
The incorporation of heme to apo-LepHO was determined by
absorption spectrophotometry. Incremental addition of hemin to apo-
LepHO allows visualization of the stoichiometric complex as reported
for other HOs [57]. Since the spectra of heme and LepHO-heme complex
overlap it was necessary to deconvolute the data as detailed in Materials
and methods. Addition of hemin to a solution 0.13 μM apo-LepHO
revealed a 1:1 stoichiometric relationship between protein and heme
a value lower than those previously determined for all known HOs
[10,28,58].
3.4. NMR spectroscopic characterization of LepHO
It has been shown that the 1H NMR spectra of cyanide-inhibited
heme oxygenase (HO-CN) is a diagnostic tool of heme in-plane orienta-
tion, and hence regioselectivity of meso carbon oxidation [29,59].
The 1H NMR spectra of α-biliverdin producing HO-CN enzymes are
nearly identical and show only one heme methyl resonance (3 methyl,
or 3Me) ca. 20 ppm in the paramagnetically shifted, downfield portion
of the spectrum. Consequently, the fact that the 1H NMR spectrum of
LepHO-CN exhibits only the 3Me resonance ca. 20 ppm indicates that
LepHO oxidizes heme to α-biliverdin (Fig. 5A). The presence of two
methyl resonances originating from the 3-methyl group in LepHO-CN
(3Me and 3me) indicates that heme binds in two orientations that differ
by rotation about the α-γ-meso axis, as has been observed with human
[60] and Corynebacterium diphtheriae HO [61,62]. The production of α-
biliverdin by LepHO was further confirmed by HPLC analysis (Fig. 5B).
The product extracted upon completion of the LepFNR-mediated
heme oxygenase reaction showed a peak with a retention time of
15.1 min corresponding to the α-isomer of biliverdin standard.
(Fig. 4). From these data a Kd of 0.017
0.002 μM was estimated,
3.5. Heme-degrading activity of LepHO
Ascorbate can serve as the electron donor in the oxidative degrada-
tion of heme by HO [63]. Therefore, the heme-degrading activity of
LepHO was monitored by UV-visible spectroscopy after the addition of
5 mM ascorbic acid (data not shown). As the reaction proceeded, the
LepHO Soret peak at 403 nm decreased, and a broad absorption band
centered near 680 nm appeared, indicating that LepHO was able to
cleave heme to produce biliverdin. As control, the same experiment in
the absence of reductant did not shown any spectral change.
It is thought that cyanobacterial and eubacterial HOs are ferredoxin-
dependent. However, it was stated that HO activity in cell extracts con-
taining reduced ferredoxin also requires the presence of a second reduc-
tant, such as trolox or ascorbate [9,28,29,64]. In fact, in the absence of
such auxiliary reducing agents, HO activity is very slow [9,64] or is
arrested at the oxyferrous heme stage [29]. More recently, it has been
observed that in P. aeruginosa a NADP+-dependent ferredoxin reduc-
tase efficiently supports the catalytic activity of HO in vitro, without
the need for a mediating ferredoxin [30]. In our case, incubation of
LepHO with LepFNR and NADPH resulted in decreased Soret band inten-
sity, with a concomitant shift to 410 nm and the appearance of two
Fig. 4. LepHO-heme binding affinity. Optical absorption spectra of 0.13 μM apo-LepHO
after addition of increasing amounts of hemin (0.01 to 0.15 μM). The inset shows
LepHO-heme complex absorbance at 403 nm plotted as function of hemin concentration.
The dissociation constant (Kd = 0.017 0.002 μM) was obtained by fitting the data to a
one site binding model.

3214
A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
release of free iron and biliverdin in FPR/HO system from P. aeruginosa,
requires the addition of acid or a chelating agent such as desferroxiamine
[30]. To further characterize the LepFNR/LepHO reaction, activity mea-
surements were performed using 0.25 μM LepFNR and increasing con-
centrations of LepHO (0.7–11.5 μM), monitoring the decrease in
absorbance at 403 nm. Because the decay of Soret band intensity is the re-
sult of several reactions in the process of heme degradation, apparent
Michaelis constants were estimated. Under the aforementioned condi-
tions, a Vmax app value of 0.177 0.007 μmol LepHO reduced min−1 mg
LepFNR−1 with a Km app for LepHO of 1.77 0.21 μM were obtained.
To investigate the ferrous iron release by the NADPH/LepFNR/LepHO
system the formation of an iron-ferrozine complex was monitored [65].
Reactions were performed as described before except that 250 μM
ferrozine, a ferrous iron chelator, was added to the reaction media,
and the spectral changes were followed during 45 min. As control, the
same reaction was performed without LepFNR. Results from these ex-
periments showed that addition of 0.5 μM LepFNR to the reaction mix-
ture produced the typical disappearance of the Soret band at 403 nm
and the simultaneous formation of a peak at 562 nm (Fig. 6B), demon-
strating that under these conditions LepHO can release heme-iron.
The production of 0.75 mol of ferrous iron per mol of heme after
45 min of reaction was determined using the extinction coefficient of
the iron-ferrozine complex (27.9 mM−1 cm−1) (Fig. 7C).
Heme-degrading activity of LepHO was also assayed with the
L. interrogans FNR/Fd system. L. interrogans contains two different ferre-
doxins but only one of them, a 2[4Fe-4S] Fd (LFd2), can exchange elec-
trons with LepFNR [35]. Initial measurements using 0.5 μM LepFNR
showed a detectable increased rate of heme degradation by LepHO
when 1 μM LFd2 was added (Fig. 7A). Although these results seem to
suggest participation of LFd2 and LepFNR in relaying electrons from
NADPH to LepHO, the dependency of LepFNR and LFd2 concentration
on heme degradation was further analyzed at different LepFNR/LFd2 ra-
tios. Results from these experiments showed that when LepFNR molar
concentration is equivalent to that of the LepFNR/LFd2 system the rate
of heme degradation by LepHO is similar (Fig. 7A). The same was ob-
served when the reaction was monitored by following biliverdin forma-
tion (Fig. 7B). The increase of LepFNR concentration in the reaction
medium overcame the effect caused by LFd2 addition (compare fill
inverted triangles, which represent the 1.5 μM LepFNR condition, with
open squares corresponding to 1 μM LepFNR/0.5 μM LFd2, in Fig. 7). Fur-
thermore, when various LepFNR and LepFNR/LFd2 concentrations were
analyzed different heme/Fe2+ conversion rates were obtained. Howev-
er, final ferrous iron amounts produced were the same in all cases
(Fig. 7C). These findings suggest that LepFNR efficiently supports the
catalytic activity of LepHO in vitro, without the need of a ferredoxin, in-
dicating that this flavoenzyme is the redox partner of LepHO in vivo.
Fig. 5. Analysis of α-biliverdin production by LepHO. (A) Downfield portion of the 1H NMR
spectrum of cyanide inhibited LepHO obtained at 305 K from a 1 mM solution of LepHO-
CN in 50 mM sodium phosphate buffer (pH 7.0). The presence of only one heme methyl
resonance (3Me, or 3me) in this spectral region is diagnostic of α-meso carbon hydroxyl-
ating HOs. (B) LepHO reaction products were analyzed by HPLC after esterification as de-
scribed in Material and methods. The absorbance of the eluate was monitored at 380 nm.
BV IX isomers: mixture of the four biliverdin isomers obtained by chemical oxidative deg-
radation of hemin; BV IXα: commercial α-biliverdin standard; LepHO: products extracted
from LepFNR-LepHO reaction. Retention times for individual isomers were 15.1 min for BV
IXα, 17.2 min for BV IXδ, 19.3 min for BV IXβ and 24.1 min for BV IXγ.
4. Discussion
Iron acquisition by pathogenic bacteria is a critical step in the infec-
tion process and a central mechanism for host colonization. This creates
a confrontation between the vertebrate defense mechanisms aimed at
sequestering available iron and the pathogen strategies for scavenging
the metal. The latter include the secretion of specific molecules, the par-
ticipation of membrane transporters, sensors and regulators, and the
involvement of metabolic systems for the extraction of iron from bio-
molecules [2,66].
One of the most important sources of iron in vertebrates is heme
bound to proteins such as hemoglobin or myoglobin. HO in Leptospira
has been reported as essential for host heme-iron utilization and for
virulence [33]. The heme degradation reaction catalyzed by HO requires
an efficient redox system to provide the necessary electrons. Hence, the
main objectives of this work were to biochemically characterize LepHO,
to analyze in depth the catalytic activity of heme degradation, and to
elucidate the protein partner that delivers the necessary reducing
equivalents.
peaks with maxima at 538 and 575 nm (Fig. 6A). These spectral changes
are consistent with the formation of a ferrous dioxyheme complex
[Heme(Fe2+)-O2] as has been previously observed [28]. Conversion
of the latter to biliverdin is evident in the time-dependent decay of
the 538 and 575 nm peaks and growth of a broad band at 680 nm. To
establish that biliverdin was present in the reaction mixture a mass
spectrometry positive-ion mode analysis was performed. Pigments
extracted upon LepHO reaction showed a single high abundance peak
with a mass-to-charge ratio of (1+) 583.25. In addition, the chemical
formula of the molecule was predicted to be [C33H35N4O6]. These data
were identical with the positive ion of authentic biliverdin standard.
Taken together, obtained results indicate that LepFNR supports the
LepHO catalyzed heme oxidation to biliverdin and free iron. In contrast,

A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
3215
Fig. 6. Heme degradation carried out by LepHO. (A) Time dependent absorption spectra of LepHO before (dashed line) and after the addition of 0.5 μM LepFNR in the presence of 300 μM
NADPH and 0.1 mg/ml catalase recorded at 1 min intervals (solid lines). Directions of spectral changes are indicated by arrows. The inset shows an enlargement of the region between 500
and 800 nm. (B) Same reaction as (A) but in presence of 250 μM ferrozine. The inset shows a time-dependent plot of the formation of the ferrozine-ferrous iron complex (562 nm) upon
addition of LepFNR.
4.1. Phylogeny indicates that both LepHO and LepFNR arrived from a
common ancestor
4.2. LepHO shows distinctive features not previously observed in other HOs
With the aim of studying the structural and functional properties of
L. interrogans HO we first analyzed the interaction between the enzyme
and its prosthetic group/substrate heme. Previously, by titrating hemin
into 10 μM LepHO it has been estimated a Kd of 4 μM for the binding pro-
cess [14]. We observed that reducing LepHO and hemin final concentra-
tions during the titration experiments allows obtaining data that better
adjust to a hyperbolic curve. We performed simulations of equilibrium
conditions establishing that 0.13 μM apo-LepHO was the most adequate
protein concentration to achieve saturating and spectrophotometrically
measurable conditions. Using this setup, we estimated a Kd of 17 nM,
significantly lower than all other reported Kd values [10,28,58]. There-
fore, it is possible that if many of the dissociation constants previously
published for HOs were recalculated using more appropriate conditions,
lower values of them will be obtained. Recently, Koga et al. through de-
velopment of a heme sensor using fluorescently labeled heme oxygen-
ase estimated a Kd of ~1.5 nM for the rat HO-1-heme complex. In this
case, the authors employed a protein concentration of 50 nM [70]. In a
previous study using the absorbance titration of 3 μM wild-type HO-1,
they had determined a Kd of 0.35 μM [71]. The difference of the Kd values
between the fluorescence titration and the absorption titration was
attributed to the changes in protein and hemin concentrations used in
the two experiments. However, a possible effect introduced by fluores-
cent labeling cannot be disregarded.
From our phylogenetic analysis we concluded that important
divergences are observed among different HOs, in contrast to previous
reports [17]. Bacterial HOs are clearly grouped according to their phy-
lum, and cyanobacterial enzymes are closely related to the eukaryotic
HOs, as has been reported previously [17]. The HO from Leptospira
appears to have separated from their ancestors in an ancient event.
Previously, we studied the phylogenetic relationships of LepFNR,
also noting in this case that the reductase may have arrived from an
ancient common ancestor [35]. These observations are coherent with
the hypothesis that major evolutionary changes within prokaryotes
may have occurred in a directional manner, being Spirochetes older
species than Proteobacteria [35,67]. The fact that HO grouped differen-
tially among pathogenic and non pathogenic leptospiras could be
interpreted as evidence of some specialization of function. Probably,
both proteins (LepHO and LepFNR) have improved their functional rela-
tionship and their role in pathogenesis in parallel. An evident similarity
is observed when phylogenies of both LepHO and LepFNR were com-
pared (data not shown). We have previously suggested that LepFNR
may have arrived form a lateral gene transfer event [68]. However,
the evidence provided here indicates that both LepFNR and LepHO
have been present already in the proto-spirochete [69], from which all
leptospiras come.
Fig. 7. Heme cleavage by LepHO with LepFNR and LFd2 as redox partners. Time dependent absorbance changes at 403 nm (A) and 680 nm (B) during heme degradation for reaction mix-
tures containing 6 μM LepHO, 300 μM NADPH, 0.1 mg/ml catalase and 0.5 μM LepFNR (●); 1 μM LepFNR (○); 1.5 μM LepFNR (▼); 0.5 μM LepFNR/0.5 μM LFd2 ( ); 0.5 μM LepFNR/1 μM LFd2
(■); or 1 μM LepFNR/0.5 μM LFd2 (□). The decrease of absorbance at 403 nm and the increase at 680 nm are diagnostic of the heme degradation and formation of biliverdin, respectively.
Time-dependent plot of Fe2+/heme molar ratio (C) during heme degradation for reaction mixtures as described above with the addition of 250 μM ferrozine. The amount of iron released
was estimated using an extinction coefficient of 27.9 mM−1 cm−1 at 562 nm.

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A. Soldano et al. / Biochimica et Biophysica Acta 1840 (2014) 3208–3217
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zyme uses NADPH cytochrome P450 reductase as its sole source of
electrons [26,72], whereas the plant, bacterial, algal and cyanobacterial
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[30]. In this work we found that L. interrogans FNR is the redox partner
of LepHO and the degradation of heme occurs efficiently in the absence
of an auxiliary electron donor (ascorbate or trolox). Moreover, we ob-
served that in vitro heme cleavage proceeds to iron and α-biliverdin
as the final products of the LepFNR-LepHO reaction; release of Fe2+
does not require the addition of acid or a chelating agent such as
desferroxiamine like was previously reported for the P. aeruginosa
FPR-HO system [30] and other HOs [28,75]. Our findings indicate that
LepFNR is capable to drive the heme degradation to completeness.
Consequently, it may be proposed that the LepFNR/LepHO system may
be sufficient to sustain the release of heme-iron in Leptospira.
5. Conclusions
In this work we have demonstrated that HO from Leptospira
interrogans is able to bind and efficiently catalyze the cleavage of
heme to free iron and biliverdin, using LepFNR as the sole electron
source. Due to existing evidence of LepHO participation in pathogenesis,
the understanding of this metabolic step may be relevant for the devel-
opment of future therapeutic intervention.
At present, the only recognized protein substrates of FNRs are ferre-
doxin and flavodoxin. Our results, in addition to those already published
for the P. aeruginosa HO, add a new substrate to this list, encouraging the
search for new redox partners of FNR. Moreover, this finding may ex-
tend the range of possible metabolic pathways where the flavoenzyme
could be involved.
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This work was supported by grants from PICT-2012-1841, ANCYPT,
Ministerio de Ciencia, Tecnología e Innovación Productiva, Argentina
(www.agencia.mincyt.gov.ar); PIP 252 and 114-200901-00337 from
CONICET Consejo Nacional de Investigaciones Científicas y Técnicas,
Argentina (www.conicet.gov.ar), BIO187 from the University of Rosario,
Argentina (www.unr.edu.ar), and National Science Foundation MCB
1158469 (to M.R.). We are grateful to Gustavo Millán for the technical
assistance with the LC-MS/MS experiments.
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Appendix A. Supplementary Data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbagen.2014.07.021.
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