In addition to the HxH motif, Irr binds heme at a second, lower-affinity site.. Spectroscopic studies of wild-type Irr and His variants show that His46 and probably His66 are involved in
Trang 1global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization
Gaye F White1,2, Chloe Singleton1, Jonathan D Todd2, Myles R Cheesman1, Andrew
W B Johnston2and Nick E Le Brun1
1 School of Chemistry, Centre for Molecular and Structural Biochemistry, University of East Anglia, Norwich Research Park, Norwich, UK
2 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK
Introduction
Iron is essential for almost all forms of life, fulfilling
functions ranging from respiration to DNA synthesis
The metal occurs in cells as a protein cofactor in
dif-ferent forms: as the bare metal or, more commonly, in
heme and iron-sulfur clusters Despite its abundance in
the biosphere, iron has poor bioavailability owing to its propensity, in the presence of oxygen and water, to form insoluble ferric oxy–hydroxide complexes [1], such that cells of all types employ complex mecha-nisms to recruit it from the environment In addition,
Keywords
a-proteobacteria; fur; heme; iron;
transcriptional regulation
Correspondence
N E Le Brun, School of Chemistry,
University of East Anglia, Norwich NR4 7TJ,
UK
Fax: +44 1603 592003
Tel: +44 1603 592699
E-mail: n.le-brun@uea.ac.uk
(Received 3 February 2011, revised 17
March 2011, accepted 1 April 2011)
doi:10.1111/j.1742-4658.2011.08117.x
The iron responsive regulator Irr is found in a wide range of a-proteobac-teria, where it regulates many genes in response to the essential but toxic metal iron Unlike Fur, the transcriptional regulator that is used for iron homeostasis by almost all other bacterial lineages, Irr does not sense Fe2+ directly, but, rather, interacts with a physiologically important form
of iron, namely heme Recent studies of Irr from the N2-fixing symbiont Rhizobium leguminosarum (IrrRl) showed that it binds heme with submi-cromolar affinity at a His-Xxx-His (HxH) motif This caused the protein to dissociate from its cognate DNA regulatory iron control element box sequences, thus allowing expression of its target genes under iron-replete conditions In the present study, we report new insights into the mecha-nisms and consequences of heme binding to Irr In addition to the HxH motif, Irr binds heme at a second, lower-affinity site Spectroscopic studies
of wild-type Irr and His variants show that His46 and probably His66 are involved in coordinating heme in a low-spin state at this second site By contrast to the well-studied Irr from Bradyrhizobium japonicum, neither heme site of IrrRl stabilizes ferrous heme Furthermore, we show that heme-free IrrRl exists as a mixture of dimeric and larger, likely hexameric, forms and that heme binding promotes IrrRloligomerization Bioanalytical studies of IrrRlvariants showed that this property is not dependent on the HxH motif but is associated with heme binding at the second site
Structured digital abstract
l Irr binds to irr by molecular sieving (View Interaction 1 , 2 )
l Irr binds to irr by cosedimentation in solution (View interaction)
Abbreviations
EPR, electron paramagnetic resonance; HRM, heme regulatory motif; ICE, iron control element.
Trang 2the very properties that make iron essential also make
it toxic in oversupply, such that the amount and form
of iron in the cell must be carefully regulated [1,2]
In bacteria, several different iron regulatory systems
are now known The best studied are the functionally
and structurally similar, but evolutionarily unrelated
global regulators, Fur and DtxR Under iron-sufficient
conditions, both of these bind Fe2+, causing a
confor-mational change that increases the affinity of the
pro-tein for operator sequences located 5¢ of the regulated
genes, resulting (usually) in transcriptional repression
[3,4] When iron is scarce, Fe2+ dissociates, releasing
the protein from DNA, thereby switching on genes
involved in, for example, iron recruitment and uptake
The subphylum a-proteobacteria contains important
pathogens of animals (e.g Brucella, Rickettsia) and
plants (Agrobacterium), symbionts (the N2-fixing
rhizo-bia), many of the most abundant bacteria in the
oceans (the Roseobacters and the SAR11 clade,
includ-ing Pelagibacter) and some laboratory model
organ-isms (e.g Paracoccus, Rhodobacter), as well as being
the source of mitochondria The relatively few studies
on iron-responsive gene regulation in members of this
important group have shown that these bacteria are
very different from those that use the Fur regulator
Although many a-proteobacteria contain a Fur
homo-logue [5], in those cases where this gene product was
studied directly, it was shown to have a minor
regula-tory role, repressing a few genes in response to
manga-nese (and not iron) availability; hence, it was renamed
‘Mur’ for mangenese uptake regulator [6,7]
Instead, many a-proteobacteria contain another
transcriptional repressor called Irr (iron responsive
reg-ulator) which, although a member of the Fur
super-family, has important features that distinguish it from
Fur sensu stricto (see below) Irr functions to repress a
wide range of genes under low iron availability by
binding to cis-acting regulatory sequences known
as iron control element (ICE) boxes that are 5¢ of the
target genes It was first discovered in
Bradyrhizobi-um japonicBradyrhizobi-um [8–12] and was also studied in
Rhizo-bium leguminosarum, where it represses a wide range of
genes in cells grown under iron-depleted conditions
[13,14], and in Brucella abortus, where it regulates
siderophore biosynthesis [15] In addition to Irr,
a-pro-teobacteria that are closely related to Rhizobium,
Agro-bacterium, Brucella and Bartonella contain another
wide-ranging iron-responsive regulator called RirA
[16–19] RirA belongs to the Rrf2 family of regulators,
which includes IscR and NsrR [20,21] and, similar to
them, is a predicted FeS cluster-binding protein RirA
represses many genes under iron-replete conditions,
recognizing the cis-acting iron regulatory sequences
(RirA-boxes) that precede genes similar to those that are commonly regulated by Fur in other organisms [5,16–19] Thus, RirA and Irr are ‘opposing’ regula-tors, which repress different portfolios of target genes
in cells grown in sufficient (RirA) or deficient (Irr) lev-els of iron Because these two regulators respond to the availability of iron in the form of iron-sulfur clus-ters and heme, respectively, and exhibit regulatory
‘cross-talk’ in response to iron availability [14], they may represent a combined regulatory system that senses the physiologically relevant status of iron and not just the concentration of the metal per se, as is the case in those organisms that use Fur or DtxR [13] Few Irr proteins have been studied in detail so far The first, from B japonicum (IrrBj), exhibits a highly unusual regulatory mechanism Under iron-replete conditions, IrrBj interacts with heme at two known sites: an N-terminal heme regulatory motif (HRM) and an internal, histidine-rich (HxH) motif [12], with the heme being normally delivered to IrrBj by ferroch-elatase [22] The heme-IrrBj complex is extremely unstable and is rapidly degraded The mechanism by which this remarkable response occurs is not known but may involve heme- and oxygen-mediated oxidative damage that acts as signal(s) for protease-mediated degradation [23] The end result is that, under iron-replete conditions, IrrBj is unavailable to act as a repressor
Another member of the rhizobia, R leguminosarum, which forms nodules on peas, clovers and beans, has
an Irr (IrrRl) that is 57% identical to IrrBj but lacks the N-terminal heme regulatory motif We recently reported that IrrRlfunctions by a very different mecha-nism that does not involve degradation in response to elevated iron Instead of being degraded, the interac-tion between IrrRl and heme causes an allosteric change in the protein, which prevents it from binding
to its cognate ICE box DNA sequences [24] In vitro studies showed that specific binding to DNA was abol-ished by the direct addition of heme, and spectroscopic studies of heme binding to wild-type IrrRl and mutant forms of the protein revealed two low-spin ferric heme-binding sites Substitution of the HxH motif abolished one of these sites, with a concomitant loss of DNA binding in vitro and regulatory function in vivo, demonstrating a key role of this motif [24] The prop-erties and location of the second, lower-affinity heme-binding site of IrrRl have not been fully investigated, although electron paramagnetic resonance (EPR) and magnetic CD spectroscopic studies showed that the heme bound at the second heme site is coordinated by two His residues [24] In the present study, we used spectroscopic and bioanalytical methods to gain
Trang 3further insight into heme binding by R leguminosarum
Irr, with a focus on the second heme-binding site
These studies highlight important new features of Irr
that have relevance to Irr proteins from a wide range
of a-proteobacteria
Results
Identification of the second heme-binding site of
IrrRl: importance of His46 and His66
It was previously shown that the second heme-binding
site of IrrRl is associated with low-spin S = ½ heme
with g-values of 2.95, 2.26 and 1.55, and a
near-infra-red magnetic CD band centnear-infra-red at approximately
1580 nm [24], demonstrating that the second heme iron
has bis-His ligation [25] However, these previous
stud-ies did not establish which His residues are involved
In total, IrrRl has seven His residues (Fig 1A), three
of which are at the HxH motif (His93, His94 and
His95) One or more of the remaining His residues
(His39, His46, His66 and His128) must therefore
supply the ligands for the second heme site A set of variants, in which each of these His residues was substituted with alanine, had been previously generated and their absolute heme spectra measured [24] Although the spectra were similar in form to the wild-type IrrRl spectrum, indicating no change in the ratio
of high- to low-spin bound heme, the amounts of heme bound differed according to which histidine had been substituted Thus, variants H39A and H128A were essentially identical to the wild-type IrrRl protein, whereas H46A and H66A exhibited spectra with signif-icantly lower heme intensity [24] Consistent with this, hemochromogen analyses revealed that, although H39A and H128A IrrRl proteins bound only margin-ally less heme than wild-type ( 1.4 compared to
1.5 heme per protein), H66A ( 1.1 heme per pro-tein) and H46A ( 0.9 heme per propro-tein) exhibited sig-nificantly lower heme binding (not shown)
EPR spectra of each of the single His variants were recorded Previous studies showed that the heme that binds at the HxH motif is EPR silent, very likely result-ing from the magnetic couplresult-ing of HxH hemes within a
B A
D C
Fig 1 Spectroscopic studies of Irr Rl His variants (A) Amino acid residue sequence of Irr Rl , with His residues highlighted (B) EPR spectra of wild-type (black), H39A (green), H46A (blue), H66A (purple) and H128A (orange) Irr variants (C) UV-visible absorbance and (D) EPR spectra
of H93 ⁄ 94 ⁄ 95A (red), His39 ⁄ 93 ⁄ 94 ⁄ 95Ala (olive green), His39 ⁄ 46 ⁄ 93 ⁄ 94 ⁄ 95Ala (dark cyan), His39 ⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95Ala (magenta) and His-free (grey) Irr variants Note that UV-visible and EPR spectra of wild-type and H93 ⁄ 94 ⁄ 95A Irr were reported previously [24] and are included here for reference Excess heme was added to each protein and non- or weakly-bound protein was removed by gel filtration and the protein concentrated as necessary Proteins (10 l M for absorbance, 100 l M for EPR) were in 50 m M Tris-HCl, 50 m M KCl (pH 8) EPR measurement conditions were: temperature, 10 K; microwave power, 2 mW; modulation amplitude, 10 G.
Trang 4proposed Irr dimer [24] Therefore, the low-spin heme
signals observed in the EPR spectrum are the result of a
heme bound at the second heme site We noted that the
low-spin heme signal intensity was decreased in both
the H46A and H66A variants, although it was
unaf-fected by substituting His39 or His128 (Fig 1B),
sug-gesting that His46 and His66 may be involved in
binding heme at the lower-affinity site This raised the
question of why EPR-active low-spin heme binding was
not completely abolished in one or more of these
vari-ants One explanation for this is that a minority of the
heme bound at the HxH site may not be magnetically
coupled, most likely as a result of incomplete
occupa-tion, which would result in a small component of the
HxH-bound heme being EPR-active Alternatively, loss
of the second heme site ligands could interfere with
cou-pling of heme at the HxH site In either case,
inactiva-tion of both heme-binding sites would be required to
abolish all low-spin heme binding
To test this model, a further set of site-directed
vari-ants was generated Beginning with the H93⁄ 94 ⁄ 95A
variant (in which the three His residues at the HxH
motif are substituted for Ala residues and previously
referred to as the HHH variant) [24], each of the
remaining His residues was substituted stepwise,
sequentially generating H39⁄ 93 ⁄ 94 ⁄ 95A, H39 ⁄ 46 ⁄ 93 ⁄
94⁄ 95A and H39 ⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95A, as well as an
entirely His-free IrrRl protein, H39⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95 ⁄
128A (His-free IrrRl) The far-UV CD spectrum of the
fully His-free variant indicated that the protein was
folded with a secondary structure content similar to
the wild-type protein (Fig S1) UV-visible absorbance
(Fig 1C) and EPR analysis (Fig 1D) of this His-free
variant after the addition of heme revealed only weak,
nonspecific (adventitious) binding (Fig S2) Having
established the baselines for the fully His-free form of
IrrRl, we used UV-visible absorbance to examine heme
binding to the IrrRl variants that retained at least one
histidine residue (Fig 1C) The data obtained showed
that, as additional His residues were substituted, the
form of the spectrum changed, with that of H39⁄ 93 ⁄
94⁄ 95A being similar to the H93 ⁄ 94 ⁄ 95A variant,
whereas that of H39⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95A more closely
resembled the His-free variant This reflects a decrease
in the proportion of heme binding in the low-spin
con-figuration and is consistent with a progression towards
only low-affinity adventitious, high-spin heme binding
in the absence of His residues (Fig S2) EPR spectra
of the multi-His variants were recorded (Fig 1D)
These showed that the ability to bind low-spin heme
was significantly diminished in the H39⁄ 93 ⁄ 94 ⁄ 95A
variant, and was lost entirely in H39⁄ 46 ⁄ 93 ⁄ 94 ⁄ 95A
IrrRl
IrrRlstabilizes heme in the ferric form Previous studies of heme binding to Irr from B japoni-cum indicated that the protein has both ferric and fer-rous heme-binding sites [12] To investigate whether IrrRlhas a specific ferrous heme-binding site, IrrRlwas titrated with heme at pH 7 using reduced hemin under anerobic conditions in the presence of a two-fold excess of sodium dithionite As shown by UV-visible absorbance spectra, heme was bound in a reduced state, almost all of which was in the low-spin form, with the Soret band at 426 nm and resolved a⁄ b bands
at 528 and 559 nm [24] (Fig 2A) Measurements of
A426 (converted to fractional saturation) fitted well to
a single site model, giving a Kd of 1 ± 0.2· 10)6m (Fig 2A, inset) This indicated that ferrous heme has a significantly lower affinity than ferric heme (Kd= 1· 10)7m) [24] (Fig S3), and that ferrous heme apparently binds at only one of the two heme-binding sites of IrrRl Upon removing dithionite under anerobic conditions, the UV-visible absorbance spec-trum revealed that heme was bound only in the oxi-dized state (not shown), consistent with a higher affinity binding of ferric heme
Heme binding to proteins is commonly pH-depen-dent [26–28] and thus heme binding in both the oxi-dized and reduced states was investigated at higher
pH In the oxidized state, although the affinity was found by absorbance and fluorescence titrations to decrease somewhat with increasing pH (Doc S1 and Fig S3A, B), the form of heme binding (as judged from difference and absolute UV-visible spectra) was unaltered (Doc S1 and Fig S3C) By contrast, at
pH 8, the reduced heme UV-visible difference spectrum was quite different from that at pH 7, indicating that only ferric heme was bound to the protein (Fig 2B) Even at a five-fold excess of dithionite, a mixture of reduced and oxidized heme was observed, which again was all converted to oxidized heme upon the anerobic removal of excess dithionite (data not shown) Although there is no indication that heme undergoes redox cycling in IrrRl, we attempted to determine the reduction potential of IrrRl-bound heme Spectropoten-tiometric titration experiments at pH 7 using dithionite
in the presence of mediators were unsuccessful because
a significant reduction of heme was not observed in the accessible potential range (data not shown), consis-tent with IrrRl-bound heme having a very low reduc-tion potential (i.e similar to, or lower than, that of the HSO3 )⁄ S2O4 ) couple of <)500 mV at pH 7) [29] Thus, IrrRl has a considerable preference for heme in the ferric state, and strongly promotes the oxidation of heme when it encounters this ligand in the ferrous
Trang 5form Therefore, it is clear that IrrRl does not contain
a ferrous heme-binding site
Heme binding to IrrRlpromotes oligomerization
The pathogen B abortus is a member of the
Rhizobi-ales that is more closely related to Rhizobium than it is
to Bradyrhizobium Its Irr protein (IrrBa) also more
clo-sely resembles IrrRl (67% identical) than IrrBj (56%
identical) and it lacks the N-terminal HRM Gel
filtra-tion studies on purified Irr (IrrBa) from this species
had shown that it is likely dimeric [30], as are other
members of the Fur superfamily [31,32] To analyse
the association state of IrrRl, analytical gel filtration
experiments were performed For the ‘as isolated’,
heme-free protein, two distinct peaks were detected in
the chromatograph (Fig 3A) Thus, the heme-free
pro-tein exists as an equilibrium mixture of two principal
association states, which must be in slow exchange,
such that they can be separated by gel filtration
Cali-bration of the column suggested that the lower
molec-ular weight species corresponds to a dimeric form,
consistent with our previous proposal that heme bound
at the HxH motif of IrrRlis EPR-silent as a result of
magnetic coupling between two closely located
HxH-bound hemes The higher molecular weight species
cor-responded to a much larger, oligomeric form, although
this could not be precisely defined by gel filtration
alone Interestingly, addition of heme to IrrRl resulted
in significant changes in the elution profile: only one
major band was observed, corresponding to the higher
molecular weight species (Fig 3A) Analysis of elution
data at different wavelengths confirmed that the
observed profile changes were the result of a change in the distribution between higher and lower molecular weight species and not the preferential binding of heme
to the larger species (Fig S4) Heme binding, however, did not cause major changes in the secondary structure because far-UV CD spectra of IrrRl before and after the addition of heme were very similar (Fig 3B)
To determine the mass of the oligomeric species more precisely, analytical ultracentrifugation experi-ments were run for heme-IrrRlat five different concen-trations, in the range 5–19 lm, at 15 000 and at
17 000 r.p.m Figure 3C shows the data obtained for one representative concentration of heme-IrrRl(10 lm) Fitting of the data, at both speeds and for all concen-trations, to a single component model gave a mass of 96.6 ± 8 kDa for the heme-bound IrrRlsample (resid-uals for the fits are shown in the inset to Fig 3C) The data thus indicate that the higher molecular weight species of IrrRlcorresponds to a hexamer
To examine the role (if any) of the HxH motif in the heme-dependent oligomerization, the association state of the H93⁄ 94 ⁄ 95A mutant IrrRlwas also investi-gated The gel filtration profile (Fig 3D) was very sim-ilar to that of the wild-type protein, and the addition
of heme to the mutant polypeptide caused a similar shift in equilibrium towards the larger (hexameric) spe-cies Therefore, the process of oligomerization is not directly connected to heme binding at the HxH motif and must be associated with heme binding at the lower-affinity site To test this proposal, IrrRlvariants containing single substitutions of His residues consid-ered to be involved in heme binding were examined by analytical gel filtration H46A and H66A IrrRlproteins
Fig 2 Binding of ferrous heme by IrrRl (A) UV-visible difference absorbance spectra recorded upon titration of ‘as isolated‘ (heme-free) Irr (17 l M ) in 50 m M Tris-HCl, 50 m M KCl (pH 7) with ferrous heme The trend of absorption changes are indicated by arrows The inset shows
a plot of fractional saturation (from DA426) as a function of reduced heme concentration A fit of the data to a single site model is drawn Pathlength, 1 cm; temperature, 20 C (B) UV-visible difference absorbance spectra as recorded in (A), except that the pH of the Irr (18 l M ) solution was 8 Note that the form of the difference titration was different from that observed for the oxidized heme titration (Fig S3A), although this is because hemin remained in the ferrous state in the reference cuvette, thereby imparting a different form on the difference spectrum.
Trang 6exhibited somewhat different behavior to the wild-type
and H93⁄ 94 ⁄ 95A proteins; oligomerization was still
observed upon heme binding, although to a
signifi-cantly lesser extent, as judged by the more prominent
peak due to the dimeric protein remaining after the
addition of heme (Fig 4A) A simple analysis of
absorbance changes indicated that oligomerization of
H46A and H66A IrrRl in the presence of heme was
approximately 60% of that observed for the wild-type
protein (Fig 4B, C) A decreased level of
oligomeriza-tion was also observed in the His-free variant,
although this was both before and after the addition
of heme By contrast, H39A, H93A, H94A and H95A
all behaved similarly to the wild-type and H93⁄ 94 ⁄ 95A
proteins (Fig 4)
Discussion
The transcriptional regulator Irr represents something
of a signature polypeptide for the a-proteobacteria,
being found in no other bacterial lineages [5]
How-ever, it has only been studied directly in a few species,
namely B japonicum, R leguminosarum and, to a lesser
extent, B abortus, all of which are in the same Order (the Rhizobiales) These studies have shown that Irr is
a remarkable protein, sensing iron in the form of heme, which, on binding to Irr, exerts unusual effects
on the protein that cause it to lose its repressive, DNA-binding ability It is clear that the Irr proteins of different bacteria have much in common because they recognize the same conserved ICE box sequences and these cis-acting elements are found in the operators of some genes (e.g mbfA in R leguminosarum corre-sponds to blr7895 in B japonicum strain 110) that are equivalent in different species [14] However, it is also apparent that there are significant differences in the behavior of Irr in different species Thus, in B japoni-cum, the interaction with heme results in a rapid and dramatic destruction of the IrrBj, but, as we recently found, this does not occur in Rhizobium Rather, when IrrRlinteracts with heme, this abolishes its DNA-bind-ing ability, although this does not destroy the polypep-tide [24]
This difference in behavior is at least partly a result
of the different ways in which the proteins interact with heme Although the proteins have a functionally
A
B
Fig 3 Bioanalytical studies of the association state of Irr Rl (A) Analytical gel filtration plots of A240as a function of elution volume for sam-ples of Irr Rl (17 l M ) in 50 m M Tris-HCl, 100 m M KCl, 10% (v ⁄ v) glycerol (pH 8) in the absence (apo) and presence of heme (two per protein),
as indicated (B) Far-UV CD spectra of wild-type IrrRlwith (grey line) heme (two per protein) The spectrum of Irr without heme (black line) [24] is shown to aid comparison Irr (10 l M ) was in 50 m M potassium phosphate (pH 8) Pathlength, 1 mm; temperature, 20 C (C) Analytical equilibrium ultracentrifugation plots of A280as a function of the radius after equilibration at 15 000 and 17 000 r.p.m at 25 C of Irr (10 l M in
50 m M Tris-HCl, 100 m M KCl, pH 8) containing two heme per protein, as indicated A fit to a single component model is drawn on each plot and the residuals for each are shown in the inset (D) Analytical gel filtration plots as in (A), except that H93 ⁄ 94 ⁄ 95A Irr was analyzed in the absence and presence of heme.
Trang 7important HxH motif in common that binds heme,
this analyte also binds elsewhere In the present study,
we have focussed on understanding the nature of the
second heme-binding site and its importance for the
properties of IrrRl The most significant changes in the
IrrRl-heme absorbance spectrum occurred on
substitu-tion of His46 or His66 [24], and the EPR low-spin
heme signal intensity was reduced in His46 and His66
variants Substitution of His39 or His128 (i.e the other
His residues present in IrrRloutside of the HxH motif)
had no effect on the UV-visible or EPR spectra,
clearly indicating that these residues are not directly
involved in heme binding Furthermore, low-spin heme
binding to IrrRl already lacking the HxH motif was
totally abolished when His46 was substituted
Unex-pectedly, substituting His39 in the H93⁄ 94 ⁄ 95A variant
background resulted in a significant decrease in
low-spin heme binding Figure S5 shows that, in the
previ-ously generated IrrRl model (based on the available
structures of Fur proteins) [24], His39 lies very close to
the HxH motif, and it is possible that the combination
of these substitutions, neither of which alone
signifi-cantly affects heme binding at the second site, causes a
conformational change that affects the second heme site, resulting in a loss of low-spin binding and an increase in high-spin heme (Fig 1C, D)
Taken together, the UV-visible and EPR data indi-cate that His46 is a key ligand at the second, lower-affinity heme site and that His66 is also important Although not involved directly in heme binding, His39
is likely to play an important, but as yet undefined, role in IrrRl because it is absolutely conserved among Irr proteins and in the wider Fur families, represented
by Fur itself, the Zur and Mur regulators, and PerR, which respond to zinc, manganese and peroxide stress, respectively [33,34] In these proteins, it serves as a ligand at a divalent metal ion binding site By contrast
to His39, His128 is not conserved in the Fur superfam-ily, nor in the Irr famsuperfam-ily, and therefore is unlikely to
be involved in heme binding in IrrRl We note that His46 and His66 are conserved in the Irr proteins of other members of the Rhizobiaceae family, including the closely related Sinorhizobium and Agrobacterium,
as well as in strains of Mesorhizobium (in the family Phyllobacteriaceae) Interestingly, all of these Irr pro-teins lack the heme-binding HRM found near the
A
C B
Fig 4 Analytical gel filtration studies of Irr Rl His variants (A) Analytical gel filtration plots of relative A280as a function of elution volume for samples of wild-type, H46A and H66A Irr (17 l M ), as indicated, in 50 m M Tris-HCl, 100 m M KCl, 10% (v ⁄ v) glycerol (pH 8) with no heme (apo) and after the addition of excess heme per protein and removal of unbound heme by passage down a PD10 column (indicated by ‘+ heme’) (B) Histogram plot of the ratio of absorbance as a result of the higher (hexameric) and lower (dimeric) molecular weight forms of Irr Rl , giving a quantitative indication of the extent of oligomerization in the apo- (grey bars) and heme-bound (black bars) forms Data were obtained from (A) and from equivalent experiments on additional IrrRlproteins, as indicated (C) Histogram plot of the ratio of ratios for apo-and heme-bound Irr Rl proteins [i.e the data in (B)], giving a direct quantitative indication of the extent of heme-induced oligomerization.
A value of 1 indicates no change in association state upon binding heme.
Trang 8N-termini in Irr proteins from Bradyrhizobium and
other members of the Bradyrhizobiaceae family, in
which His46 and His66 are not present
On the basis of the IrrRl model, we predict that
His46 and His66 are located in the DNA-binding
domain on consecutive a-helices that are connected by
a loop (Fig S5) In the model, the two His residues
are not sufficiently close to cooperate in binding a
sin-gle heme, although a conformational rearrangement of
the helices could potentially align them appropriately
Alternatively, the second, lower-affinity heme binding
may not be at an intrasubunit site but, instead, could
be at an intersubunit site involving His residues from
juxtaposed subunits
Even though the IrrBj and IrrRl proteins both
con-tain an HxH motif, the binding characteristics of the
motif in the two proteins are different IrrBj binds
fer-rous heme at its HxH motif [12], whereas, in the
pres-ent study, we have shown that both heme sites of IrrRl
have a very significant preference for ferric heme and
do not bind ferrous heme in the absence of an excess
of reductant It remains unclear why the IrrRland IrrBj
HxH motifs should exhibit different heme iron
oxida-tion state preferences
The propensity of IrrRlto oligomerize has also been
demonstrated in the present study, and this was
enhanced by heme binding at the lower-affinity heme
site associated with His46 and His66 Because IrrBjlacks
an equivalent site, we anticipate that it might exhibit
different behavior, although the association state
prop-erties of the Bradyrhizobium Irr have not yet been
inves-tigated IrrBawas found to be a dimer and no evidence
of oligomerization was reported [30] Irr is a member of
the Fur superfamily and it has long been known that
Fur itself can exist in oligomeric forms in solution, as
well as when bound to DNA [35,36] Furthermore, we
noted that high molecular weight forms of IrrRloccur in
whole cell extracts of R leguminosarum [24]
Currently, the functional roles of the second heme
site and of oligomerization remain unclear because
variants disrupted in heme binding at this site were not
affected in their ability to bind DNA, nor were they
significantly affected in their ability to function in vivo
[24] However, the conservation of the site ligands,
together with their importance for the properties of the
protein in vitro, in heme binding and in
oligomeriza-tion, suggests that this site has functional importance
Clearly, further studies are required to understand
bet-ter the role of the second heme site and the functional
consequences of heme-induced oligomerization For
example, it is not known whether the dimeric or
hexa-meric (or both) form of IrrRlcan bind ICE box DNA
sequences
Given the remarkable variation in the properties of the very few Irr proteins that have been studied directly, it will be of interest to examine the somewhat more distantly related versions of Irr in other Orders
of the a-proteobacteria, not least, members of the Rhodobacterales and the SAR 11 clade, which form the most abundant bacteria in the world’s oceans Despite this, we still know almost nothing about their iron-responsive biology
Materials and methods
Generation of sequential His variants of IrrRl
individually substitute H93, H94 and H95 with alanine resi-dues was carried out using the primers listed in Table S1 and a QuikChange XL mutagenic PCR kit (Stratagene, La Jolla, CA, USA) in accordance with the manufacturer’s
(H94A) and pBIO1841 (H95A) Successive rounds of
H94 and H95 residues are all substituted with the corre-sponding alanines; termed H93⁄ 94 ⁄ 95A IrrRl
) and deriva-tives were carried as described above, resulting in the stepwise substitution of all of the His residues in IrrRlwith Ala, generating pBIO1820 (H39⁄ 93 ⁄ 94 ⁄ 95A), pBIO1821 (H39⁄ 46 ⁄ 93 ⁄ 94 ⁄ 95A), pBIO1822 (H39 ⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95A)
(H39⁄ 46 ⁄ 66 ⁄ 93 ⁄ 94 ⁄ 95 ⁄ 128A) IrrRl
Verified mutated plas-mids (Table S2) were used to transform Escherichia coli BL21(DE3) for protein over-expression
Purification of wild-type and variant forms of IrrRl and in vitro heme additions
heme-free form as previously described [24] and exchanged into 50 mm Tris-HCl, 50 mm KCl (pH 7 or 8, as req-uired) Protein concentrations were determined using an
e280 nm of 5800 m)1Æcm)1 obtained from amino acid analy-sis (Alta Biosciences, Birmingham, UK) For spectroscopic and analytical studies, heme additions were made using a micropipette (Gilson Inc., Middleton, WI, USA) or a mi-crosyringe (Hamilton, Reno, NV, USA) For UV-visible difference absorption titration experiments, Irr was added
to the sample cuvette and heme additions were made to
( 1 mm) were freshly prepared as described previously [24] Bound heme concentrations were determined using a modified version of the hemochromogen method [37] as described previously [24] For ferrous heme titrations, hemin was reduced using a two-fold molar excess of sodium dithionite
Trang 9Spectroscopic and bioanalytical methods
UV-visible absorption spectra were recorded using a Hitachi
U-4010 or U-2900 spectrophotometer (Hitachi Corp.,
Tokyo, Japan) EPR spectra were measured using an
X-band spectrometer (Bruker ER200D; Bruker,
Rheinstet-ten, Germany) with an EPS 3220 computer system (Bruker)
fitted with an ESR9 liquid helium flow cryostat (Oxford
Instruments, Abingdon, UK) Spin intensities of
paramag-netic samples were estimated by double integration of EPR
EDTA as the standard Binding isotherms obtained from
spectroscopic titrations of IrrRl with heme were analyzed
using origin, version 7 (Microcal; OriginLab Corporation,
Northampton, MA, USA) employing a single binding site
model (where the free ligand concentration was unknown)
USA) for single-site and two-site binding models [38]
Sedi-mentation-equilibrium experiments were performed using a
Beckman XL-I analytical ultracentrifuge in an AN50Ti
12 mm charcoal-filled Epon double-sector cells with quartz
windows The sample volume was 110 lL and the reference
sector of the cell contained identical buffer Samples of IrrRl
containing heme at five different concentrations were spun
at 15 000 or at 17 000 r.p.m until equilibrium was reached,
as judged by cessation of changes in scans collected 4 h
apart Data were collected at 280 nm and analyzed using
ul-traspin, version 2.5 (http://ultraspin.mrc-cpe.cam.ac.uk/)
The density of the buffer was taken as 1.005 gÆmL)1and the
0.7421 mLÆg)1using the software sednterp [39] Analytical
gel filtration of samples of IrrRlutilized a Superdex 75
col-umn (GE Healthcare), equilibrated in 50 mm Tris-HCl,
50 mm KCl, 10% glycerol (v⁄ v) (pH 8.0) and operated at a
flow rate of 0.8 mLÆmin)1 The column was calibrated using
cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa),
bovine serum albumin (66 kDa) and alcohol dehydrogenase
(150 kDa)
Acknowledgements
This work was supported by the UK BBSRC through
the award of grant BB⁄ E003400 ⁄ 1, to A.W.B.J and
N.E.L.B., and the Wellcome Trust through an award
from the Joint Infra-structure Fund for equipment We
thank Dr Tom Clarke for assistance with the AUC
experiments
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