vinelandii, O2shows no effect on the transcriptional activity of VnfA but reactive oxy-gen species is reactive to cause disassembly of the Fe-S cluster and turns active VnfA inactive.. T
Trang 1nitrogenase transcriptional activator VnfA from
Azotobacter vinelandii
Hiroshi Nakajima1, Nobuyuki Takatani2, Kyohei Yoshimitsu1, Mitsuko Itoh1, Shigetoshi Aono3, Yasuhiro Takahashi4and Yoshihito Watanabe2
1 Department of Chemistry, Graduate School of Science, Nagoya University, Japan
2 Research Center of Materials Science, Nagoya University, Japan
3 Okazaki Institute for Integrative Biosciences, Japan
4 Division of Life Science, Graduate School of Science and Engineering, Saitama University, Japan
Keywords
Azotobactor vinelandii; iron-sulfur cluster;
nitrogen fixation; nitrogenase; transcriptional
regulator
Correspondence
H Nakajima, Department of Chemistry,
Graduate School of Science, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya
464-8602, Japan
Fax: +81 52 789 2953
Tel: +81 52 789 3557
E-mail: hnakajima@mbox.chem.
nagoya-u.ac.jp
Database
VnfA has been submitted to the Swiss-Prot
database under the accession number
C1DI41
(Received 12 October 2009, revised 28
November 2009, accepted 3 December
2009)
doi:10.1111/j.1742-4658.2009.07530.x
Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobac-ter vinelandii In the present study, we have purified full-length VnfA pro-duced in E coli as recombinant proteins (Strep-tag attached and tag-less proteins), enabling detailed characterization of VnfA for the first time The EPR spectra of whole cells producing tag-less VnfA (VnfA) show distinc-tive signals assignable to a 3Fe-4S cluster in the oxidized form ([Fe3S4]+) Although aerobically purified VnfA shows no vestiges of any Fe-S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe3S4]+ dominantly in the protein Additional spectroscopic evidence of [Fe3S4]+
in vitro is provided by anaerobically purified Strep-tag attached VnfA Thus, spectroscopic studies both in vivo and in vitro indicate the involve-ment of [Fe3S4]+ as a prosthetic group in VnfA Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe-S cluster Quantitative data of iron and acid-labile sulfur in reconsti-tuted VnfA are fitted with four 3Fe-4S clusters per a tetramer, suggesting that one subunit bears one cluster In vivo b-gal assays reveal that the Fe-S cluster which is presumably anchored in the GAF domain by the N-termi-nal cysteine residues is essential for VnfA to exert its transcription activity
on the target nitrogenase genes Unlike the NifAL system of A vinelandii,
O2shows no effect on the transcriptional activity of VnfA but reactive oxy-gen species is reactive to cause disassembly of the Fe-S cluster and turns active VnfA inactive
Structured digital abstract
l MINT-7311946 : VnfA (uniprotkb: C1DI41 ) and VnfA (uniprotkb: C1DI41 ) bind ( MI:0407 ) by molecular sieving ( MI:0071 )
l MINT-7311931 : VnfA (uniprotkb: C1DI41 ) and VnfA (uniprotkb: C1DI41 ) bind ( MI:0407 ) by blue native page ( MI:0276 )
Abbreviations
AAA+, ATPases associated with various cellular activities; AMP-PNP, 5¢-adenylyl-b,c-imidodiphosphate; BCA, bicinchoninic acid; b-gal, b-galactosidase; GPC, gel permeation chromatography; IscS, cysteine desulfurase; IPTG, isopropyl thio-b- D -galactoside; o-phen,
o-phenanthroline; PMS, phenazine methosulfate; ROS, reactive oxygen species; UAS, upstream activator sequence.
Trang 2The diazotroph Azotobacter vinelandii contains three
distinct nitrogenases Nitrogenase-1 is a conventional
molybdenum nitrogenase that bears a metal-sulfur
cluster with molybdenum and iron as the reactive site
By contrast, the active center of nitrogenase-2 consists
of vanadium and iron, and that of nitrogenase-3
con-tains only iron [1,2] The expression of each set of
structural genes is regulated by specific transcriptional
activator proteins, namely, NifA, VnfA and AnfA,
which regulate nifHDK (nitrogenase-1), vnfDGK
(nitro-genase-2) and anfHDGK (nitrogenase-3), respectively
[3] Gene analyses suggest that these activators belong
to rN-dependent regulatory proteins generally
consist-ing of three major domains [4], and the N-terminal
domain termed GAF (i.e cGMP-specific and
-stimu-lated phosphodiesterases, Anabaena adenylate cyclases
and Escherichia coli FhlA) is considered to be a
sen-sory domain [5] The primary structure of the GAF
domain is highly conserved in VnfA and AnfA,
whereas NifA shares little homology with them,
sug-gesting that the sensor structure of NifA is distinct
from that of VnfA and AnfA [3] Indeed, the GAF
domain of NifA forms a complex with another sensory
protein, NifL, which contains a flavin moiety that
serves as an oxygen sensor in the cytosol [6–8],
whereas VnfA and AnfA work independently and do
not have proteins corresponding to NifL [3,9–11]
Instead, there are characteristic rich motifs,
Cys-X-Cys-XXXX-Cys and Ser-Cys-X-Cys-XXXX-Cys,
preced-ing the GAF domains of VnfA and AnfA, respectively
[3] These motifs have been suggested to form active
centers in the sensory domains containing metal atoms
or clusters as prosthetic groups A previous study of
AnfA variants in vivo revealed that AnfA requires Cys
residues in the N-terminus and iron ions for
transcrip-tional function [12] Similar inferences have been
pro-posed for nitrogenase regulatory proteins isolated from
other diazotrophs, such as Herbaspirillum seropedicae
[13,14] and Bradyrhizobium japonicum [15,16] These
regulatory proteins also have Cys-rich motifs in their
central domains and have a specific requirement for
iron ions to allow activatation of the transcription of
nitrogenase genes in their host cells, whereas it is still
obscure whether the Cys-rich motifs are associated
with the requirement for iron
By contrast to a number of studies conducted in vivo
[9,12,17–21], there have been essentially no structural
and functional analyses of VnfA and AnfA conducted
in vitro because of the insolubility of the proteins as
well as difficulty in overexpressing their genes using
recombinant systems This has hampered their
isola-tion by convenisola-tional purificaisola-tion methods such as col-umn chromatography An exceptional success is the purification of an AnfA variant reported by Austin
et al [22] In their study, the N-terminal domain of AnfA was truncated to prevent the intrinsic aggrega-tion of the intact form during purificaaggrega-tion The obtained variant retained transcriptional activator activity and provided fundamental information about the function of AnfA, including the binding sequence
in the anfH promoter region and prerequisites for ren-dering AnfA transcriptionally active However, the sensing mechanism that may reside in the GAF domain and the environmental factors affecting AnfA activity remain unknown because of the absence of the N-terminal domain in this variant Because sensing is a principal function of regulatory proteins, the isolation
of VnfA and AnfA with their sensor (GAF) domains
is highly desirable
In the present study, we have succeeded in the pro-duction and purification of recombinant full-length VnfA in both Strep-tag attached and tag-less forms in
E coli Spectroscopic and biochemical characterization
of the recombinant VnfA both in vitro and in vivo show that VnfA function requires iron-sulfur (Fe-S) clusters as a prosthetic group We describe a functional form of VnfA including the number of subunits in the native form and the type and presumable locus of the Fe-S cluster, as well as the stoichiometry of the cluster Activity assays conducted in vivo allow discussion of a role for the Fe-S cluster in the transcriptional function
of VnfA as well as putative environmental factors reac-tive to the cluster
Results
Cell growth conditions and whole cell EPR spectra
The production of tag-less VnfA (VnfA) in E coli is sensitive to the cultivation temperature When induc-tion by isopropyl thio-b-d-galactoside (IPTG) was per-formed above 25 C, most of the produced protein was found in the insoluble fraction, whereas, below
25C, soluble VnfA can be obtained after cell lysis by sonication and subsequent centrifugation of the cellu-lar debris (data not shown) Therefore, we cultivated the cells for 16 h at 20C to allow efficient induction
of soluble VnfA The amount of oxygen in the culture had little effect on the production: VnfA was produced similarly under both aerobic and micro-aerobic growth conditions As described below, VnfA produced under
Trang 3these conditions could be purified through a
combina-tion of column chromatography and ammonium
sulfate fractionation
Having established the culture conditions that allow
the accumulation of VnfA in the cytosol of E coli,
EPR spectroscopy using whole E coli cells
overex-pressing vnfA was attempted to obtain information
regarding the metals present in the prosthetic group
The results obtained are shown in Fig 1 Regardless
of the aeration level of the culture, the cells produced
distinctive signals at g = 2.03 and 2.01 at 10K
(aero-bic cultures are shown Fig 1A; data not shown for
micro-aerobic cultures), and this is different from the
native signals of E coli, which are mainly the result of
high-spin Mn2+ species and free organic radicals [23]
(Fig 1B) Figure 1D shows the overall shape of the
signals obtained by subtraction of Fig 1B from
Fig 1A, which is consistent with an oxidized 3Fe-4S
cluster ([Fe3S4]+) found in metalloproteins, such as
inactive cytosolic aconitases, ferredoxin and enzymes
bearing [Fe3S4] [24] The temperature dependence of
the signal intensity also supports the presence of an
Fe-S cluster Weaker signals are observed at higher temperature and almost disappear at 50K (Fig 1C) Thus, the EPR results indicate the accumulation
of [Fe3S4]+ in E coli overexpressing vnfA (i.e the involvement of [Fe3S4]+in VnfA) However, the EPR data cannot exclude possible presence of other types of Fe-S clusters, such as 4Fe-4S ([Fe4S4]) and 2Fe-2S ([Fe2S2]), because the Fe-S clusters could be EPR-silent depending on their oxidation state To address this measurement problem encountered in vivo, we purified and characterized VnfA in vitro
Purification of recombinant VnfA VnfA produced in the cytosol of E coli was purified
by column chromatography and ammonium sulfate fractionation under aerobic conditions The addition
of 1 mm dithiothreitol throughout the procedure and 0.2% (v⁄ v) Triton X-100 after the final step (heparin Sepharose column chromatography) was, however, essential for suppressing aggregation of the protein
In the absence of dithiothreitol and Triton X-100, purified VnfA precipitated after several hours, even at
4C Complete elimination of E coli chromosomal DNA during the first pass through an anion exchange column was also crucial for the subsequent purification steps because VnfA cannot be resolublized once co-precipitated with DNA An almost homogeneous band was obtained after the final step, comprising heparin column chromatography on SDS-PAGE (Fig S1) The estimated molecular mass of the band was 58 kDa,
in agreement with the calculated value of VnfA (57 608 Da) based on the nucleotide sequence of vnfA [3] Conclusive confirmation was obtained by N-termi-nal amino acid sequence aN-termi-nalysis of the first ten resi-dues of the purified protein, providing the sequence MSSLPQYCEC, which is identical to the sequence of VnfA The yield of purified protein after the final step was approximately 3 mg if started with 20 g of cell pel-lets Thus, we have successfully purified a recombinant VnfA that is amenable to further investigation in vitro
Reconstitution of the Fe-S cluster in apo-VnfA
By contrast to the results of the EPR performed in vivo, the UV-visible spectrum of aerobically purified VnfA shows no features arising from any Fe-S clusters (Fig 2A, dotted line) other than an unidentified shoul-der band observed at 330 nm Because some Fe-S clusters in proteins are unstable in atmospheric oxy-gen, the vanishment of the Fe-S cluster from purified VnfA could be a result of the disassembly of the cluster during aerobic purification Fe-S clusters in
2.03 2.01
A
B
C
D
Magnetic field (mT) Fig 1 Whole cell EPR spectra of E coli JM109 strain cultured
under aerobic conditions: (A) overexpressing vnfA recorded at
10 K, (B) transformed with pKK223-3 carrying no structural gene
of VnfA and (C) overexpressing vnfA recorded at 50 K (D)
Differ-ence spectrum obtained from (A) – (B) Spectra were recorded at
2.5 mW microwave power and a field modulation of 0.8 mT The
intensities of the spectra were normalized with native signals of
Mn 2+ species from E coli.
Trang 4apo-proteins in vitro are commonly reconstituted to
regenerate their original structures and functions
[25–27] Therefore, we attempted the reconstitution of
purified VnfA under anaerobic conditions Enzymatic
production of S2)from l-cysteine by cysteine
desulfur-ase (IscS) from A vinelandii [28] was used rather than
Na2S to avoid coprecipitation of VnfA with a large
amount of Fe-S colloids formed during the reaction
After reconstitution and subsequent purification using
desalting columns, fractions containing VnfA showed
an apparent shoulder and broad bands at 310 and
420 nm, respectively (Fig 2A, solid line) The latter
band was bleached upon the addition of the reductant,
dithionite salt (Fig 2A, dashed line) These
character-istic properties indicate that apo-VnfA is reconstituted
with [Fe3S4]+ and⁄ or [Fe4S4]2+ EPR spectroscopy
provides further information on the nature of the Fe-S
cluster The reconstituted holo-VnfA gave a signal with
a g-value of 2.01, which disappeared upon the addition
of the reductant (Fig 2B) Although the rhombicity of
the spectrum found in the whole cell measurement
vanishes, the observed properties are common to
[Fe3S4]+ Quantification of the signals using
Cu(II)EDTA as a standard indicated that the
concen-tration of [Fe3S4]+ was approximately 34 lm, which
corresponds to approximately 70% of the VnfA
mono-mer concentration (50 lm) determined by the
bicinch-oninic acid (BCA) method The iron and sulfur contents in the reconstituted holo-VnfA were deter-mined by inductively coupled plasma–optical emission spectroscopy and acid labile sulfide analysis, respec-tively The reconstituted holo-VnfA was found to con-tain 2.8 ± 0.1 equivalents of iron and 3.5 ± 0.3 equivalents of sulfur per monomer (Table S1), corre-sponding to one monomer bearing one Fe-S cluster These quantitative results indicate that [Fe3S4]+ is a major species found in VnfA reconstituted under the present conditions No EPR signals assignable to [Fe4S4]2+ were observed, either before or after reduc-tion by dithionite salt
The lost rhombicity in the EPR spectrum was partially recovered by the addition of 5¢-adenylyl-b,c-imidodiphosphate (AMP-PNP) to the reconstituted holo-VnfA, although the signal at g = 2.03 in vivo was still shifted to 2.02 (Fig 2C) AMP-PNP is a nonhy-drolysable ATP analog that is used to trap an ATP binding state of ATP hydrolases Some ATPases asso-ciated with various cellular activities (AAA+) proteins are known to bind AMP-PNP and reproduce their conformational changes to exert the original functions
of the proteins [29,30] Although the ATPase activity has not been reported for VnfA so far, the central domain of VnfA is deduced to be an AAA+ domain based on high homology to the AAA+ domain of
0
0.5
1.0
1.5
Wavelength (nm)
2.0
D C
330
Magnetic field (mT)
Fig 2 (A) UV-visible spectra Dotted line, aerobically purified VnfA (apo-form); solid line, after reconstitution with an Fe-S clus-ter; dashed line, the reconstituted holo-VnfA after addition of the reductant, dithionite salt (B) EPR spectrum of the holo-VnfA with a g-value of 2.01 (solid line) that disap-peared following reduction with dithionite salt (dotted line) (C) EPR spectrum of the holo-VnfA after the addition of 1 m M AMP-PNP (D) EPR spectrum reproduced from Fig 1D for facile comparison with the spec-tra (B) and (C) The concenspec-tration of VnfA for both UV-visible and EPR measurements was 50 l M in 20 m M HGDT buffer (deter-mined by the BCA method) EPR spectra were recorded at 10 K using 2.5 mW microwave power and a field modulation of 0.8 mT.
Trang 5NifA [3] Consistently, our preliminary study of
N-terminally truncated VnfA constituted with the
cen-tral and C-terminal domains had exhibited ATPase
activity compatible with other rN-dependent
transcrip-tional activators, such as NorR [31] (N Takatani,
H Nakajima, Y Watanabe, unpublished data)
There-fore, it is likely that VnfA binds AMP-PNP in the
cen-tral domain to initiate a conformational change
required for the subsequent hydrolysis Indeed, limited
protease digestion assays with either apo- or
reconsti-tuted holo-VnfA have provided results that reveal
several conformations of VnfA corresponding to a
combination of the presence and absence of AMP-PNP
and the Fe-S cluster (vide infra) This could help to
solve the problem of why binding AMP-PNP has an
influence on the Fe-S cluster detected in the EPR
mea-surement This point will be discussed subsequently
The studies with the reconstitution of Fe-S clusters
in aerobically purified apo-VnfA support the presence
of [Fe3S4]+ in VnfA To obtain further evidence
demonstrating the involvement of the Fe-S cluster in
in vitro experiments, we attempted the anaerobic
purification of VnfA attached to a Strep-tag at the
C-terminus of the protein
Anaerobic purification of Strep-tag attached VnfA
Attempts to purify VnfA as a fusion protein to
gluta-thione S-transferase, thioredoxine or His-tag were
unsuccessful because the produced proteins were
insol-uble, despite manipulation of the aeration and
temper-ature in the culture conditions VnfA conjugated with
Strep-tag at the C-terminus (Strep-VnfA) yielded a
small amount of soluble protein in the cell-free lysate
(Fig S2) However, the solubility of Strep-VnfA was
markedly improved when the SUF proteins, which are
known to be involved in biological Fe-S cluster
assem-bly [32,33], were co-produced with Strep-VnfA After
single-step purification under anaerobic conditions
using streptavidin attached to an affinity column,
Strep-VnfA provided an almost homogeneous band on
SDS-PAGE
The UV-visible spectrum of anaerobically purified
Strep-VnfA showed bands at 330 and 420 nm
(Fig 3A, solid line), which diminished upon the
addi-tion of dithionite salt (dotted line) Featureless
absorp-tion observed at wavelengths longer than 500 nm
might indicate the participation of some [Fe2S2]2+
spe-cies However, the EPR measurement for Strep-VnfA
showed a single signal characteristic of [Fe3S4]+ at
g= 2.01 before the reduction (Fig 3B, solid line) and
no signal assignable to [Fe2S2]+ even after the
reduc-tion (dotted line) Although the rhombicity of the EPR
signal of Strep-VnfA is still unclear, the overall shape
is rather similar to that observed in the whole cell mea-surements Thus, Strep-VnfA purified under anaerobic conditions affords additional support for the involve-ment of [Fe3S4]+in VnfA as a prosthetic group
Limited protease digestion assay
To obtain experimental evidence for a conformational change of VnfA triggered by AMP-PNP binding, VnfA
of either the apo- or reconstituted holo-form was sub-jected to limited trypsin digestion in the presence and absence of AMP-PNP Figure 4 shows the time course
of proteolysis for VnfA under each set of conditions AMP-PNP afforded a higher resistance to the
Wavelength (nm)
0.0 0.4 0.8
A
B
1.2 1.6
2.01
Magnetic field (mT)
C
Fig 3 (A) UV-visible and (B) EPR spectra of anaerobic purified VnfA The solid lines represent the spectra of purified Strep-VnfA The dotted line represents the spectra following reduction with dithionite (C) EPR spectrum reproduced from Fig 1D for facile comparison with the spectra in (B) The EPR spectra were recorded at 10 K using 2.5 mW microwave power and a field modulation of 0.8 mT.
Trang 6proteolysis for both the apo- and holo-forms, as
dem-onstrated by a much slower digestion of the original
bands under +AMP-PNP conditions, whereas the
digestion patterns of both the apo- and holo-form
appeared to be little affected by the presence or
absence of AMP-PNP By contrast, an effect of the
Fe-S cluster on the proteolysis was not found in the
sensitivity to the digestion but was observed with
respect to the alteration of the digestion patterns (i.e
digestion sites in apo- and holo-VnfA) One particular
change in the digestion pattern was found between 31
and 45 kDa in which two major fragments in the
apo-form were not observed in the holo-apo-form, whereas the
fragment at 28 kDa in the holo-form was scarce in the
apo-form A fragment at 19 kDa in the holo-form is
the other major difference, although this was hardly
observed in the apo-form Regarding the effect of
AMP-PNP on the proteolysis of VnfA, a similar effect
of the nucleotide binding was reported in a study of
the limited trypsin digestion with NifA + MgADP, in
which binding MgADP to the central AAA+ domain
is ascribed to the trigger of a conformational change
of NifA to avoid further proteolysis [34,35] By
anal-ogy with the study on NifA, the observed
transforma-tion of VnfA to a more resistant form to proteolysis is
ascribed to a conformational change induced by
bind-ing AMP-PNP, presumably at the central domain of
VnfA Similarly, the changes in the fragmentation
depending on the Fe-S cluster can be accounted for by
a conformational change caused by the cluster
forma-tion in VnfA The variaforma-tion in the digesforma-tion patterns
corresponding to a combination of the presence and
absence of AMP-PNP and the Fe-S cluster suggests
that the conformational changes by the Fe-S cluster
and AMP-PNP are interdependent
Number of subunits in native VnfA
The molecular mass of native VnfA with and without
the Fe-S cluster was determined to characterize
the quaternary structure of VnfA Gel permeation
chromatography (GPC) of purified VnfA bearing no Fe-S cluster (apo-VnfA) eluted in a single and some-what broad peak that corresponds to a molecular mass
of 224 kDa (Fig S3) This value is 3.9-fold higher than that of the VnfA monomer (57 608 Da, calculated from the inferred amino acid sequence) Because of technical difficulties in performing GPC under fully anaerobic conditions, the mass of reconstituted VnfA could not be measured by GPC Instead, holo-VnfA was subjected to anaerobic blue native PAGE [36] using degassed electrophoresis buffers and an argon atmosphere Holo-VnfA provided a homoge-neous band with a molecular mass of 213 kDa, which corresponds to a 3.7-fold higher mass of the subunit (Fig S4) Thus, the mass analyses of VnfA confirm a tetrameric configuration both in the presence and absence of the Fe-S cluster As described for the recon-stitution of VnfA with the Fe-S cluster, quantitative analyses for iron and acid labile sulfur in the reconsti-tuted VnfA indicated one Fe-S cluster in each mono-mer, as well as the stoichiometry of four Fe-S clusters
in native VnfA
Functional analyses of the Fe-S cluster
To clarify the roles of the Fe-S cluster found in VnfA,
we performed in vivo assays under various growth conditions by using a heterogeneous reporter system carrying the lacZ gene preceded by the vnfH promoter
in the E coli JM109 strain With the view of immuno-logical detection of produced VnfA, we employed Strep-VnfA as a source of VnfA for the reporter sys-tem A similar heterogeneous reporter system has been reported and was shown to be valid for elucidating the biological properties of VnfA and NifAL [6,19]
To determine whether the Fe-S cluster is required for transcriptionally active VnfA, we employed o-phe-nanthroline (o-phen) as a metal chelater for the assay, which is expected to permeate cell membranes and restrict iron atoms available for Fe-S cluster assembly
in the cell [37,38] Activity was determined by the
66.2
45
KDa
Apo-VnfA
31
21.5
14.4
116.3
Holo-VnfA
0 5 10 30 60 0 5 10 30 60 min 0 5 10 30 60 0 5 10 30 60 min
VnfA
38 32
19
VnfA
38
19
Fig 4 Limited tryptic digestion assays with VnfA of either apo- or reconstituted holo-form in the presence or absence of AMP-PNP The reactions were analyzed on 15% polyacrylamide gels Digestion fragments were obtained by the reaction with trypsin (weight ratio 1 : 180) at 20 C for 60 min Details of the reaction conditions are pro-vided in the Materials and methods.
Trang 7transcript level of the lacZ gene immediately after the
addition of o-phen to minimize the effect of the growth
inhibition by o-phen on the transcriptional activity of
VnfA Figure 5 shows the time course of the VnfA
activity immediately after the addition of 150 lm
o-phen to the growth medium under micro-aerobic
conditions Five minutes after the addition of o-phen,
the activity began to decrease and reached 30% of the
initial level in 45 min, whereas a control assay under
same conditions without the addition of o-phen
showed virtually no alteration in the lacZ gene
tran-script Because a western blot analysis confirmed the
constant level of Strep-VnfA during the assays both in
the presence and absence of o-phen, it would be
rational to ascribe the drop in the lacZ transcript to
the repression of the transcriptional activity of
Strep-VnfA The specific EPR signals of [Fe3S4]+ observed
for E coli overexpressing vnfA disappeared after
o-phen treatment and, instead, a signal of free ferric
iron emerged at g = 4.3 (data not shown), indicating
that the reaction of o-phen brings about disassembly
of the Fe-S cluster in transcriptionally active VnfA
Thus, we conclude that the Fe-S cluster is essential for
transcriptionally active VnfA and disassembly and⁄ or
that deformation of the Fe-S cluster turns active VnfA
inactive
The transcript assay with o-phen under aerobic con-ditions provided virtually the same result as that obtained under micro-aerobic conditions (data not shown), implying that the transcriptional activity of VnfA is insensitive to the aeration conditions Then,
we inspected the effect of the aeration conditions on the transcriptional activity of VnfA (Fig 6) A shift of the micro-aerobically grown cells to the aerobic culture caused no significant change in the transcript level of lacZ A consistent result was also obtained by the b-galactosidase (b-gal activity) assay (Table S3) The accumulation of b-gal in the reporter strain was at the same level after the aerobic and micro-aerobic cultures This finding contrasts with previous studies on tran-scriptional regulation by NifAL As observed in the
in vivo activity assay using the homogeneous reporter strain, NifL produced in the E coli reporter strain also showed sensitivity to cytosolic O2 of the aerobic culture Consequently, the transcriptional activity of the NifAL system was affected by the aeration condi-tions of the growth media [7] Thus, the results obtained allow the inference that the 3Fe-4S cluster in VnfA is insensitive to O2 permeating living cells from the air, and therefore cannot serve as an O2 sensor
Time (min)
Strep-VnfA
5
Micro-aerobic culture
Aerobic culture
A
B
0
1.0
0.25 0
0.5 0.75
Fig 6 (A) Time course of the VnfA activity assessed by lacZ tran-script level at early exponential phase After culture under micro-anaerobic conditions, the cells was divided into aerobic ( ) and micro-aerobic ( ) cultures at 0 min for the subsequent assay Each plot presents the mean values from three independent experi-ments, normalized with the activity at 0 min (B) Western blot anal-yses for Strep-VnfA recorded at a time corresponding to the performed assays.
0
1.0
0.25
0
Time (min)
0.5
0.75
Strep-VnfA
5
– o-phen
+ o-phen
A
B
Fig 5 (A) Time course of the VnfA activity assessed by lacZ
tran-script level at early exponential phase After the addition of 150 l M
o-phen ( , +o-phen); no addition of o-phen ( , )o-phen) Each plot
presents the mean values from three independent experiments,
normalized with the activity at 0 min (B) Western blot analyses for
Strep-VnfA recorded at a time corresponding to the performed
assays.
Trang 8under physiological conditions Then, we screened the
effect of reactive oxygen species (ROS) on the
tran-scriptional function of VnfA
As shown in Fig 7, the level of the lacZ transcript
decreased upon the addition of phenazine methosulfate
(PMS), which is known to be an efficient superoxide
generator in aerobically grown cells [39] The initial
induction period immediate after the addition of PMS
was followed by a drop in the transcript level by 90%
in 60 min Because the level of VnfA was largely
unaf-fected by PMS during the assay, the observed decrease
in the transcript was not associated with growth
inhibi-tion of the strain but, instead, is ascribed to immediate
inactivation of VnfA by PMS The EPR spectrum
from E coli overexpressing vnfA under the same
con-ditions exhibits replacement of the signals from
[Fe3S4]+ with a strong signal at g = 2.00, which is
assignable to organic radicals generated by the reaction
of amino acid residues with ROS such as superoxide
and peroxide (Fig 8) [23] These findings indicate that
ROS formed in the cytosol are reactive with the Fe-S
cluster and turn active VnfA inactive VnfA Thus,
ROS could be considered as candidate environmental
factors However, further evidence is needed before
this conclusion can be made because ROS are known
to cause rearrangement and⁄ or disassembly of Fe-S
clusters in proteins regardless of the physiological sig-nificance of the reaction [40–42]
Transcriptional activity of cysteine variants of VnfA
The findings obtained in the present study indicate that VnfA bears the 3Fe-4S cluster as the prosthetic group The involvement of some metal ion as a prosthetic group was originally deduced from the characteristic cysteine-rich motif, 8-CXCXXXXC-15, in the N-termi-nal region of VnfA and a mutagenesis study for AnfA [3,12] Therefore, it is likely that these cysteine residues participate in binding the cluster However, VnfA has additional three cysteine residues, namely at position
107, at position 134 in the GAF domain and at posi-tion 267 in the possible AAA+ domain Accordingly,
to determine which Cys are associated with the binding
of the Fe-S cluster, we prepared six Cys variants of Strep-VnfA (C8A, C10A, C15A, C107A, C134A and C267A, in which each cysteine residue was replaced with alanine) and performed the in vivo b-gal activity assay for each variant (Fig 9) The result obtained apparently classifies the variants in two parts Three variants of the N-terminal Cys residues (C8A, C10A and C15A) showed significantly low transcriptional activities corresponding to 12%, 23% and 1% of that
of wild-type, respectively On the other hand, the remaining variants (C107A, C134A and C267A)
2.00
Magnetic field (mT) Fig 8 Effect of PMS on the whole cell EPR spectrum of aerobi-cally grown E coli JM109 overexpressing vnfA Addition of PMS to the NFDM medium (final concentration of 50 l M ) was followed by
60 min of further culture and then harvesting The spectrum was recorded at 10 K using 2.5 mW microwave power and a field mod-ulation of 0.8 mT.
Time (min)
Strep -VnfA
5 +PMS
A
B
–PMS
0
1.0
0.25
0.5
0.75
0
Fig 7 (A) Time course of the VnfA activity assessed by lacZ
tran-script level at early exponential phase after the addition of 50 l M
PMS ( , +PMS); no addition of PMS ( , )PMS) Each plot
presents the mean values from three independent experiments,
normalized with the activity at 0 min (B) Western blot analyses for
Strep-VnfA recorded at a time corresponding to the performed
assays.
Trang 9retained almost original or rather higher activities
(65%, 81% and 117% of that of wild-type,
respec-tively) Western blot analysis showed approximately
the same stability of the variants compared to that of
wild-type, confirming that the difference in activity of
the variants reflects the intrinsic ability of the variants
compared to the transcriptional activator The result
obtained indicates that the N-terminal cysteine-rich
motif serves to harbor the Fe-S cluster in VnfA
How-ever, it is still controversial whether all cysteine
residues in the N-terminal participate in binding the
same Fe-S cluster because the amino acid sequence,
Cys8Glu9Cys10, restricts the cysteine residues from
binding to the same cluster
Discussion
Prosthetic group of VnfA
The EPR data of the whole E coli cells overexpressing
vnfA suggested the involvement of the 3Fe-4S cluster
in VnfA, which was supported by the spectroscopic
analyses for the reconstituted VnfA and anaerobically
purified Strep-VnfA The quantitative analyses for the
reconstituted VnfA provide the estimate that
appro-ximately 70% of apo-VnfA is reconstituted with
[Fe3S4]+, indicating that [Fe3S4]+is a major species in
VnfA reconstituted under the present experimental
conditions However, the UV-visible spectrum
indi-cated the partial participation of some 2Fe-2S cluster
species in the purified Strep-VnfA, which offers the
possible involvement of other types of Fe-S clusters in VnfA Further identification of the Fe-S cluster in transcriptionally active VnfA, including its conforma-tion and oxidaconforma-tion state, is required Regarding a locus
of the Fe-S cluster, information pertinent to the pres-ent study was provided by a previous systematic muta-genesis study [12] of the N-terminal Ser and Cys residues of AnfA In that study, it was demonstrated that Cys21 and 26, corresponding to Cys10 and 15 in VnfA, respectively, are essential for the transcriptional activity of AnfA In agreement with such a finding, our in vivo b-gal activity assays for cysteine variants of Strep-VnfA indicate that the N-terminal cysteine resi-dues are plausible candidates for the ligands of the Fe-S cluster Thus, the locus of the Fe-S cluster should
be in the N-terminal GAF domain Because a single residue gap between Cys8 and Cys10 is unusual in ligands for a single Fe-S cluster, it is unlikely that all the N-terminal cysteine residues in the subunit of VnfA bind the single Fe-S cluster A possible scenario
is that two of three cysteine residues (Cys15 and Cys8
or Cys10) bind the Fe-S cluster and the remaining resi-due binds the neighboring Fe-S cluster Alternatively,
a non-cysteinyl residue such as histidine, aspirate or glutamate could comprise a third ligand Then, the reduction of the transcriptional activity for C8A or C10A is associated with the indirect influence of muta-genesis at the neighboring residue
The EPR spectrum of the reconstituted VnfA showed a signal (g = 2.01) of different rhombicity from those observed in the whole E coli cell measure-ment (g = 2.01 and 2.03) The addition of AMP-PNP
to the reconstituted VnfA served to recover the rhomb-icity Although the signal at g = 2.03 still shifted to 2.02 and a fully identical spectrum to that observed in the whole cell measurement has not been reproduced under the present reconstitution conditions, the partial recovery of the rhombicity implies that VnfA can bind
a nucleotide, and the whole cell EPR spectrum might reflect VnfA of the nucleotide binding form It has been reported that binding of ATP or ADP to NifA of
A vinelandii leads to rearrangement of interaction between the GAF and AAA+ domains (and thereby a conformational change in the protein), which are con-sidered to couple with transmission processes of the sensing events [34] Considering the functional and structural analogies to NifA, it is presumably rational
to expect that VnfA also causes a conformational change in a similar manner to NifA; the binding of the ATP analog induces the rearrangement of the GAF and possible AAA+ (the central domain) domains in VnfA Indeed, the limited protease assays confirmed that the conformational changes are dependent on a
0
500
Wild type
C15A C107A C134A C267A –VnfA
Wild typeC8A C10A C15A C107A C134A C267A
1000
1500
2000
2500
Fig 9 Conventional in vivo b-gal activity assays for the Strep-VnfA
wild-type and Cys variants under aerobic conditions The upper
panel shows the stability of the wild-type and variants as monitored
by western blot analysis )VnfA, the kpvnfH strain transformed with
the plasmid, pASK-IBA3 plus , carrying no structural gene of VnfA.
Trang 10combination of the presence and absence of
AMP-PNP and the Fe-S cluster As described above,
N-ter-minal cysteine residues located immediately upstream
of the GAF domain are the potential ligands of the
Fe-S cluster Consequently, the binding of the ATP
analog and the subsequent rearrangement of the
inter-domain interaction affects the electronic condition of
the Fe-S cluster through the protein scaffold, resulting
in an alteration of the signal rhombicity of the EPR
spectrum The divergence of the g-value from that of
the whole cell spectrum remains to be solved The
dif-fering conditions during biosynthetic assembly in vivo
and artificial reconstitution in vitro may affect the
spectra For example, the signal intensity ratio of
the EPR spectra of [Fe3S4]+ changes in response to
the buffer composition, such as the concentration of
glycerol [43] In ferredoxin II from Desulfovibrio gigas,
differing purification conditions cause variation in the
shape of the EPR spectrum of [Fe3S4]+ [44] Further
modification of the reconstitution procedure is still in
progress, aiming to obtain an EPR spectrum identical
to that observed in the whole cell measurement
Native molecular mass analyses by native PAGE
and GPC show that VnfA remains tetrameric both in
the presence and absence of the Fe-S cluster A similar
oligomeric configuration has been reported for
trun-cated AnfA, which is in equilibrium between the
dimeric and tetrameric forms, whereas NifA of A
vine-landii is known to exist as a dimer [22] A previous
investigation of the vnfH promoter revealed that the
binding site of VnfA consists of two dyad upstream
activator sequence (UAS) motifs
(5¢-GTAC-N6-GAAC-3¢ and 5¢-GTAC-N6-GTAC-3¢) that lie on top
of each other on the same face of the DNA helix
[11,17,19] Similar features are commonly required for
promoters of rM-dependent transcriptional regulators,
although there are several variations with respect to
the number and distance of the dyad UAS motifs In
most cases, the regulators in a dimeric form bind to
each dyad UAS motif cooperatively to associate with
the target promoters [45] However, such a binding
mode is unlikely for tetrameric VnfA because it has
four DNA binding parts Simultaneous binding to all
four UAS motifs on the vnfH promoter is therefore
the most plausible association mode for single native
VnfA
Native VnfA takes the tetrameric form irrespective
of the presence or absence of the Fe-S cluster, raising
the problem of how the Fe-S cluster regulates the
tran-scriptional activity of VnfA To address this, we
attempted an in vitro DNA binding assay using the
flu-orescence polarization technique for apo- and
reconsti-tuted VnfA with an oligo nucleotide containing one of
the cognate promoters (i.e the vnfH promoter) The reconstituted VnfA provided a dissociation constant of
87 nm (Fig S5), whereas, as a result of the propensity for facile aggregation with the oligo-nucleotide, quanti-tative analysis for apo-VnA has not succeeded to date Further modification of the fluorescence polarization technique is ongoing aiming to avoid the aggregation
of apo-VnfA during measurement
Candidates for an environmental factor for VnfA Previous studies have reported that neither molybde-num (Mo) nor vanadium (V) show a direct effect on the transcriptional function of VnfA [9,20,21,46]
We also obtained results consistent with these findings
in the b-gal activity assays regarding Mo and V (Table S4) Previous studies on the expression from promoters of vnfA, vnfH and vnfDGK demonstrated that V is not required for the transcription of each promoter [20,21], but is for the translation of the vnfDGK transcript [46], and that the repressive effect
of Mo on the vnfH and vnfDGK promoters is mediated through the repression of vnfA transcription [9] On the basis of these considerations, we conclude that both Mo and V are excluded from being candidates for the VnfA environmental factor O2 is a well-known environmental factor for nitrogenase transcriptional regulators This is also true for the NifAL system in
A vinelandii, in which a prosthetic molecule in NifL, flavin, undergoes a redox reaction with O2 to control the transcriptional activity of NifA [7,8] Recent kinetic studies on Fnr, a well-studied O2 responsive transcriptional regulator bearing a 4Fe-4S cluster, have proposed the transient formation of [Fe3S4]+ in Fnr upon reaction with O2, followed by self-disassembly to [Fe2S2]2+ and a complete loss of the Fe-S cluster [47,48] In accordance with this mechanism, it could be considered that [Fe3S4]+ observed for VnfA in the EPR measurement is a stable intermediate generated from EPR silent [Fe4S4]+in the process of O2 sensing However, our in vivo assays performed under aerobic and micro-aerobic conditions provided no supportive data for O2 sensing and revealed that VnfA is sensitive
to ROS generated in the cytosol, which represses its transcriptional activity Because the lacZ transcript assay with o-phen confirms that the Fe-S cluster is an essential component of transcriptionally active VnfA and that disassembly of the cluster turns active VnfA inactive, the observed inactivation of VnfA by ROS could be associated with disassembly of the 3Fe-4S cluster upon reaction with ROS
The production of ROS by nitrogenases has been proposed as an initial reaction of a possible protection