Isolation and biochemical characterization of two soluble ironIIIJirˇı´ Mazoch1, Radek Tesarˇı´k2, Vojteˇch Sedla´cˇek1, Igor Kucˇera1and Jaroslav Tura´nek2 1 Department of Biochemistry,
Trang 1Isolation and biochemical characterization of two soluble iron(III)
Jirˇı´ Mazoch1, Radek Tesarˇı´k2, Vojteˇch Sedla´cˇek1, Igor Kucˇera1and Jaroslav Tura´nek2
1
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic;2Department of Immunology, Veterinary Research Institute, Brno, Czech Republic
Two soluble enzymes (FerA and FerB) catalyzing the
reduction of a number of iron(III) complexes by NADH,
were purified to near homogeneity from the aerobically
grown iron-limited culture of Paracoccus denitrificans using
a combination of anion-exchange chromatography
(Seph-arose Q), chromatofocusing (Mono P), and gel permeation
chromatography (Superose 12) FerA is a monomer with
a molecular mass of 19 kDa, whereas FerB exhibited a
molecular mass of about 55 kDa and consists of probably
two identical subunits FerA can be classified as an
NADH:flavin oxidoreductase with a sequential reaction
mechanism It requires the addition of FMNor riboflavin
for activity on Fe(III) substrates In these reactions, the
apparent substrate specificity of FerA seems to stem
exclu-sively from different chemical reactivities of Fe(III)
com-pounds with the free reduced flavin produced by the enzyme
Observations on reducibility of Fe(III) chelated by vicinal
dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthes-ized by P denitrificans Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coen-zyme, can utilize NADPH in place of NADH, does not reduce free FMNat an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilo-triacetate as substrates FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homo-logy to the previously described chromate reductase from Pseudomonas putida Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzo-quinone
Keywords: iron(III) reductase; NADH:flavin oxidoreduc-tase; Paracoccus denitrificans
Ferric iron [Fe(III)] reductases, which catalyze the reduction
of various complexed forms of Fe(III), are of ubiquitous
occurrence in nature [1] Conversion of Fe(III) to more
soluble Fe(II) generally increases the bioavailability of iron
and occurs either outside the cell [in conjunction with an
upward Fe(II) transport] or intracellularly (e.g iron release
from the accumulated ferrisiderophores) [2] In bacteria,
environmental Fe(III) can serve as a hydrogen sink,
allowing the regeneration of NAD+ [3], or even as a
terminal electron acceptor in an anaerobic respiration
associated with translocation of H+and generation of a
transmembrane potential difference [4,5]
Fe(III) reductases constitute a heterogeneous group of
enzymes in respect to their biochemical properties The
majority of the known bacterial Fe(III) reductases resides
in the cytoplasm, consists of one small polypeptide chain lacking any distinguishable prosthetic group, reduces Fe(III) at the expense of NADH or NADPH and requires flavin as an electron transfer mediator [6–13] On the contrary, Fe(III) reductases of Fe(III) respirers are c-type cytochromes excreted into the medium or exposed at the cell surface where they can make direct contact with the solid metal oxides [14,15] A representative of eukaryotic type enzymes is the FRE1 Fe(III) reductase of Saccharomyces cerevisiae, a transmembrane flavocytochrome b with prop-erties similar to those of the NADPH oxidase of human neutrophils [16] In some instances the observed reduction
of Fe(III) possibly represents a side activity of an enzyme that may catalyze a different reaction in vivo, as exemplified
by the enzymes flavin reductases [12], sulfite reductase [17]
or dihydropteridine reductase [18]
The facultative anaerobe Paracoccus denitrificans posses-ses a flexible respiratory chain able to incorporate consid-erable amounts of iron-containing proteins in response to the changed growth conditions [19,20] This ability depends
in part upon a capacity of the microbe to mobilize iron from external and internal sources Evidence for a high-affinity iron-scavenging system involving low molecular weight, Fe(III)-specific siderophores was first presented by Tait [21] who isolated three iron-binding catechol compounds from the culture supernatant of P denitrificans and provided initial data on their structure, metal-binding properties and enzymes needed for their biosynthesis Since then, several
Correspondence to I Kucˇera, Department of Biochemistry,
Faculty of Science, Masaryk University, Kotla´rˇska´ 2,
CZ-61137 Brno, Czech Republic.
Fax: + 420 541129623, Tel.: + 420 541129351,
E-mail: ikucera@chemi.muni.cz
Abbreviations: Bis/Tris,
bis(2-hydroxyethyl)amino-tris(hydroxy-methyl)methane; DHB, 2,3-dihydroxybenzoate; FNR, fumarate and
nitrate reductase regulator (Escherichia coli); FnrP, FNR homologue
from P denitrificans; NfsB, oxygen-insensitive nitroreductase of
E coli; PVDF, poly(vinylidene difluoride); pyrocat, pyrocatechol;
QR, quinone reductase.
(Received 30 September 2003, revised 27 November 2003,
accepted 5 December 2003)
Trang 2thorough investigations into the mechanism,
stereospecific-ity and kinetics of siderophore-mediated iron transport in
P denitrificanshave been carried out [22–24], but
informa-tion on the enzymology of Fe(III) reducinforma-tion still remains
scarce and incomplete Tait [21] described formation of
Fe(II) from the Fe(III)–siderophore complex upon
incuba-tion with NADH, succinate and an ultracentrifuged and
dialyzed cell-free extract while Dailey and Lascelles [25]
noticed reduction of Fe(III) citrate by NADH or succinate
as electron donors in the presence of crude cell extracts
containing membranes More recently, Kucera and
Match-ova [26] succeeded in discriminating between the reduction
of Fe(III) sulfate by NADH catalyzed by cytosolic and
membrane fractions The former reaction differed markedly
from the latter by about 10-times lower apparent Kmvalue
for NADH, by a discernible stimulatory effect of FMN, and
by the inability of succinate to substitute for NADH
The present report describes the results of studies leading
to the conclusion that at least two distinct soluble enzymes of
P denitrificansexhibit an activity of Fe(III) reductase These
enzymes were purified and their molecular and catalytic
properties were analyzed The data obtained indicate that
one of them can be assigned to the NADH-dependent flavin
reductases while the other carries a bound flavin prosthetic
group and bears a resemblance to a previously described
enzyme capable of reducing chromate [27]
Materials and methods
Organism and cultivation
Paracoccus denitrificans, strain CCM 982 was obtained in
freeze-dried form from the Czech Collection of
Micro-organisms (CCM), Masaryk University, Brno, Czech
Republic It was maintained on agar plates and stored at
4C Growth was achieved via batch cultivation using
minimal succinate medium, containing (in mM) sodium
succinate, 50; KH2PO4, 33; Na2HPO4, 17; N H4Cl, 50;
MgSO4, 1.1 and Fe(III) citrate, 0.5 lM The final pH was
adjusted to 7.3 by dropwise addition of 0.1 M NaOH
Separate 750 mL aliquots were autoclaved in 3-L
Erlenmeyer flasks, inoculated with 20 mL of a pregrown
culture and incubated for 10–12 h at 30C on a reciprocal
shaker at 180 r.p.m Cells were sedimented by
centrifuga-tion (6200 g, 20 min at 4C), washed twice with 50 mM
Tris/HCl buffer, pH 7.4, and then taken up in this buffer
Preparation of the enzymes
For a typical purification 10 g wet cells harvested from
3-L culture were used Chromatography was run on a
Pharmacia FPLC apparatus
Step 1: Preparation of cell-free extract.Cell-free extracts
were made by passing the cell suspension twice through a
33-mL X-Press cell (AB Biox, Sweden), treating with 4 lg of
deoxyribonuclease (Sigma) per mL for 30 min at room
temperature to reduce the viscosity caused by DNA and
removing the cellular debris by centrifugation at 109 000 g
for 40 min in a rotor-type 45 TI (Beckman) The Fe(III)
reductase-containing extracts were stored at)15 C until
commencement of purification
Step 2: Ion exchange chromatography About 40 mL of cell-free extract, prepared as described above, were loaded onto an HP Sepharose Q (Pharmacia) column (XK26:
10 cm· 2.6 cm) pre-equilibrated with 20 mM Tris/HCl,
pH 7.4, and washed by the same buffer The proteins were eluted in two phases with 300 mL of linear gradient of 0–0.6 M NaCl and 50 mL of linear gradient of 0.6–1M NaCl at a flow rate of 0.8 mLÆmin)1 Fractions of 5 mL were collected and those displaying Fe(III) complex reduc-tase activity were pooled (each activity peak separately), concentrated in an ultrafiltration cell (Amicon) through YM-3 membrane (Millipore) and then dialyzed against
20 mMTris/HCl, pH 7.4
Step 3: Chromatofocusing Two buffer systems in this chromatography on MonoP HR 5/20 (Pharmacia) column (20· 0.5 cm) for two distinct enzymes were employed First, starting with 0.075M Tris/acetate, pH 9.3 used Polybuffer 96, second one started with 0.025M Bis-Tris/ iminodiacetic acid, pH 7.1, and Polybuffer 74 (Pharmacia) was applied here, according to instructions of the manu-facturer The flow rate was 0.3 mLÆmin)1 during sample loading and 0.5 mLÆmin)1 during focusing Fractions of 0.5 mL were collected Pooled active fractions were desalted
on PD-10 columns (Supelco) equilibrated by the start buffer for the next step
Step 4 (optional): Chromatofocusing The same condi-tions as in the previous step with Polybuffer 74 were used Peak fractions were collected
Step 5: Gel permeation chromatography Maximum 0.5 mL of sample was loaded onto Superose 12 (Pharmacia) analytical column (30 cm· 1 cm) equilibrated by 50 mM Tris/HCl, pH 7.4 containing 150 mMNaCl The flow rate after sample application was 0.5 mLÆmin)1 For determin-ation of molecular masses, the same column was calibrated
by marker proteins including alcohol dehydrogenase (141 kDa), albumin (67 kDa), ovalbumin (43 kDa), chy-motrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) Step 6 (optional): Preparative native electrophoresis.In some instances (e.g for N-terminal sequenation), final purification of the enzymes was performed from the activity bands on native gels (see below) The proteins were recovered by electroelution using electroelution tubes and
a Mini Protean Cell (Bio-Rad) with Tris/glycine buffer,
pH 8.3 [no sodium dodecyl sulfate (SDS)] at constant current 8 mA per one electroelution tube for 5.5 h Determination of protein concentration
Protein concentration was determined by the method of Lowry et al [28] using bovine serum albumin as the standard Protein elution profiles from chromatographic columns were also monitored by measuring fractional absorbance at 280 nm
Enzyme assays Fe(III) reductase activity was determined according to
a variant of the method by Dailey and Lascelles [25] The
Trang 3assay measures the formation of the Fe(II)(ferrozine)3
complex at 562 nm (absorption coefficient¼ 28 mM )1Æ
cm)1) in 25 mM Tris/HCl buffer, pH 7.4, 30C, with
0.8 mM ferrozine, 0.05 mM FMNand 0.2 mM Fe(III)
complex The reaction was started by the addition of
0.15 mMNADH
Chromate reductase activity was assayed at 30C in
0.5-mL reaction mixtures containing 25 mM Tris/HCl
buffer, pH 7.4, 0.045 mMK2CrO4, 0.15 mMNADH, and
5–30 lL of enzyme preparation Chromate concentration
was quantified by adding samples to the reagent consisting
of 0.1M H2SO4 and 0.01% 1,5-diphenyl carbazide and
measuring the absorbance at 540 nm [27]
Quinone reductase and flavin reductase activities were
monitored by following the disappearance of the NADH
absorbance at 340 nm in 25 mMTris/HCl buffer, pH 7.4,
30C Concentrations of electron acceptors were 100 lM
for quinones when measuring quinone reductase and
50 lMfor FMNin flavin reductase assay Stock solutions
of quinones were prepared as 5 or 10 mMin 20% acetone
(v/v)
Stopped-flow experiments
A JASCO J-810 stopped-flow spectrophotometer with a
dead time of 5.2 ms and a cell path length of 1.5 mm was
used to measure redox reactions of Fe(III) complexes with
chemically reduced FMN A solution of 1.25 mMFMNin
25 mMTris/HCl, pH 7.4, containing PtO2(0.1 mgÆmL)1) as
a catalyst [29] was made anaerobic with argon bubbling and
then a stream of hydrogen gas was introduced to convert
FMNinto FMNH2 Following the sedimentation of the
catalyst, the yellowish supernatant was placed in one
syringe, and the second syringe was filled with a buffered
solution of 1.6 mMferrozine and 0.4 mMFe(III) complexed
with an appropriate ligand After 1 : 1 mixing, changes in
absorbance at 562 nm were recorded Data from at least six
repetitions were averaged and processed with the software
package supplied with the instrument, giving the desired
initial velocity values
Mass spectrometry
The mass spectra of proteins were recorded on a Bruker
Reflex IV mass spectrometer Prior analysis, the samples
were dialyzed against 10 mM Tris/HCl, pH 7.4, and then
concentrated to a final value of about 1–10 lg of protein per
lL using a Speed-Vac concentrator (Savant, Holbrook,
USA) One microliter each of sample was mixed with 2 lL of
matrix solution (ferulic acid, saturated in 50% acetonitrile,
1% trifluoroacetic acid) Aliquots (0.6 lL) were placed on
the target and left to dry The spectra were taken in
positive-ion linear mode External mass calibratpositive-ion was performed
using the molecular ions from the bovine insulin at
5734.5 Da and the horse myoglobin at 16952.6 Da 100
single shot spectra were accumulated in each mass spectrum
Gel electrophoresis
One-dimensional SDS gel electrophoresis was carried out in
10–15% polyacrylamide gels [30], containing 0.1% SDS,
using a Mini Protean II (Bio-Rad) vertical electrophoretic
system Staining was carried out using Coomassie brilliant blue R-250 Standard molecular mass markers were from Fluka Non-denaturing electrophoresis was carried out in the same manner, but with omission of SDS from the gel running and loading buffers, and the sample was not pretreated under denaturing conditions Staining was car-ried out using Coomassie blue R-250 or by zymogram analysis The latter entailed overlay of the gel with 0.15 mM NADH and 0.8 mM ferrozine in 50 mM Tris/HCl buffer,
pH 7.4, containing 0.05 mM FMN Fe(III)nitrilotriacetic acid solution in the same buffer was added to the resulting concentration of 0.2 mM Bands of Fe(III) reductase activity were detected as red zones against a slightly reddish background
Spectroscopic measurements Absorption spectra of the enzymes were taken with an Ultrospec 2000 UV/visible spectrophotometer (Pharmacia) using a 1-cm light path cell Fluorescence spectra were obtained using an LS 50B spectrofluorimeter (Perkin Elmer) Thin-layer chromatography analysis of flavins
About 0.4 mg of the purified flavoprotein, dissolved in 0.1 mL of buffer, was mixed with 0.9 mL of methanol and the mixture was heated at 95C for 15 min After cooling
on ice and centrifugation at 12 000 g for 5 min, the supernatant was dried with a Speed-Vac concentrator Water (10 lL) and methanol (10 lL) were added to the residue, and the solution was analyzed by TLC employing a cellulose gel plate (DC-Alufolien Cellulose, 0.1 mm thick-ness; Merck AG, Darmstadt, Germany) and the upper layer
of an n-butyl alcohol/glacial acetic acid/water (4 : 1 : 5) mixture as the solvent phase Methanol/water (1 : 1) solution of riboflavin, FMNand FAD were spotted as reference and the migration of the compounds were visualized with UV light
N-Terminal sequence analysis The highly purified enzymes were subjected to SDS/PAGE (15% polyacrylamide) and then electroblotted onto a poly(vinylidene difluoride) membrane (Bio-Rad) Selected bands were cut out of the Coomassie Blue-stained blots and analyzed by the Edman method with a 491 protein sequencer (Perkin-Elmer Applied Biosystems) according to the standard program (PL PVDF Protein)
Homology searching and sequence alignments Homology searching against the databases was performed with the Basic Logical Alignment Search Tool (BLAST) on NCBI Server (http://www.ncbi.nih.nlm.gov) Alignments of the sequences were produced using CLUSTAL W Multiple Sequence Alignment software on NPS@Server (http:// npsa-pbil.ibcp.fr)
Kinetic data analysis Input data represented the mean values from at least three replicates Nonlinear regression analysis was performed
Trang 4with the kinetic software package EZ-FIT developed by
Perrella [31]
Results
Choice of a Fe(III) substrate for the enzyme assay
Most of Fe(III) reductases have no known native substrates,
but they exhibit a fairly weak specificity and can reduce
many artificial Fe(III) complexes Therefore, we explored
a series of five compounds (Fe(III)EDTA, Fe(III)EGTA,
Fe(III)nitrilotriacetic acid, Fe(maltol)3and FeCl3) as
poten-tial artificial substrates for measuring the activity in cell-free
extracts by the ferrozine assay When no flavin was added
exogenously, we found the following specific activities [nmol
Fe(II)(ferrozine)3Æs)1Æg protein)1, means of three replicates]:
2, 151, 146, 16, 2, whereas with 50 lMFMNthe respective
values were: 5, 210, 392, 323, 83 These results told us that
the Fe(III)nitrilotriacetic acid substrate together with FMN
would best serve the purpose of activity measuring during
enzymes purification Contrary to some [8,13], but in
accordance with other [10] previous research, our assay
for the enzymes from P denitrificans was not significantly
sensitive to oxygen, as air removal from the reaction mixture
with a stream of argon increased the rate of
Fe(II)(ferro-zine)3production by 10% maximum Based on this finding,
all further measurements could be performed under ambient
oxygen pressure
Purification of the Fe(III) reductases
Preliminary experiments established that cultivation of
P denitrificans in a growth medium with limiting initial
levels of Fe(III) citrate (0.5 lMinstead of the normally used
concentration of 30 lM) increased the specific activity of
Fe(III) reductase in the cell-free extract about 1.5-fold
Accordingly, the enzyme was isolated from the
iron-deficient cells that had reached the stationary phase after
aerobic growth A summary of the data of the purification
is given in Table 1 for a typical preparation Aspects
deserving comment are as follows:
(i) Assay of the fractions in the HP-Sepharose Q elution
profile identified two peaks of Fe(III) reductase activity,
eluting at approximately 50–150 mM and 250–400 mM NaCl They were termed FerA and FerB, respectively Peak fractions were pooled separately and purification was continued for each pooled peak of activity
(ii) The presence of two Fe(III) reductases in cell-free exacts could also be directly demonstrated by native gel electrophoresis of the cell-free extracts followed by activity staining, which produced two bands with the intensity ratio
of about 10 : 1 The more intense band corresponded to the enzyme with a higher electrophoretic mobility towards the anode
(iii) Preparative chromatofocusing proved to be an efficient purification step, which could be used twice for FerB, but not for FerA due to less resistance to pH changes Chromatofocusing enabled us to determine isoelectric points of FerA and FerB as 6.9 and 5.5, respectively These values are in accord with the observed chromatographic behavior of the enzymes at the Sepharose Q step
(iv) During optimization of the purification protocol the possibility of employing a hydrophobic interaction chro-matography was also noticed After loading the test sample
on a Phenyl Superose column (Pharmacia) or on a Hema-Bio 1000 Phenyl column (Tessek, Prague) and applying a 1.5–0M ammonium sulfate gradient, FerA was typically eluted at 80–90% of the gradient compared to FerB eluted
at 45–50% FerA thus seems to be considerably more hydrophobic than FerB
Determination of molecular mass SDS/PAGE analysis gave single Coomassie blue staining bands at a position of an approximate Mrof 18 000 for both the purified FerA and FerB (Fig 1) These values were further refined by applying a MALDI-MS analysis, which revealed peaks at m/z 18 814 and 18 917 for FerA and at m/z 20 196 for FerB We are currently unable to identify unequivocally the reason for the presence of two peaks in the spectrum of FerA
As the enzymes could be separated in a column of Superose 12, the same column was used to estimate their molecular sizes By calibration of the column with standard proteins of known molecular masses, the molecular masses
of native FerA and FerB were estimated as 25 kDa and
Table 1 Purification of P denitrificans Fe(III) reductases The activity was determined following absorbance of Fe(II)(ferrozine) 3 complex at
562 nm Specific activity is expressed as enzyme activity per mg of total protein.
Treatment
Volume (mL)
Total protein (mg)
Total activity (nkat)
Specific activity (nkatÆmg)1)
Purification factor (-fold)
Yield (%) FerA
FerB
Trang 555 kDa, respectively From these results, we concluded that
FerA exists in a monomeric form in the native state and that
FerB has an oligomeric structure (a homodimer or, less
probable, a homotrimer)
N-Terminal sequence comparison
The N-terminal amino acid sequences of FerA and FerB
were determined and are presented in Fig 2(A) A BLAST
search revealed sequence similarity of 73% and 65%
(sequence identity of 50% in both cases) of this region in
FerB with chromate reductase from Pseudomonas putida
and flavin reductase form Pseudomonas syringae (Fig 2B),
respectively On the contrary, no significant similarity was
found between the N-terminus of FerA and any known oxidoreductase
Spectral properties Measurement of the UV-visible absorption spectrum of FerA (about 95% pure, 0.04 mgÆmL)1) showed no chro-mophore uniquely attributable to this enzyme On the other hand, the solution of FerB was yellowish in color and exhibited absorption bands at 380 and 448 nm (Fig 3), diagnostic for the presence of a flavin group When 0.4 mM NADH was added, the absorption peak at 448 nm disappeared, indicating a reducibility of the bound flavin
by the physiological electron donor The possession by FerB
of a flavin cofactor was also apparent from the fluorescence
Fig 1 SDS/PAGEfollowed by silver staining Lane 1, 10 lg protein of
the starting cell-free extract; lane 2, 0.3 lg of purified FerA; lane 3,
1 lg of purified FerB; lane 4, molecular mass markers indicated (in
kDa) on the right.
Fig 2 N-Terminal amino acid sequences of P denitrificans FerA and FerB (A) and comparison of the N-terminal amino acid sequence of FerB with the sequences of other oxidoreductases (B) N-Terminal sequences were determined by Edman degradation Multiple alignment of chosen sequences was performed using CLUSTAL W programme Fre, flavin reductases of Pseudomonas syringae pv syringae B728a (gi:23469150) and of P syringae pv tomato DC3000 (gi:28870863); CR, chromate reductase of Pseudomonas putida (gi:14209680); ?, a hypothetical protein (sequence derived from the known DNA sequence) Identical (w), strongly similar (:) and weakly similar (.) residues are indicated.
Fig 3 Absorption spectra of as isolated (—) and NADH reduced (- - -) FerB The spectrum of FerB, 0.2 mg proteinÆmL)1, were measured under aerobic conditions in 25 m M Tris/HCl buffer (pH 7.4), employing the buffer solution as a reference, with a light path of 1 cm The reduced spectrum was obtained 5 min after the addition of 0.4 m M NADH.
Trang 6spectrum where the enzyme excited at 446 nm shows a
fluorescence emission with a maximum at 506 nm
Identification of the flavin moiety of FerB
Exposure of FerB to heated methanol released a yellow
soluble compound from the holoenzyme Thin-layer
chro-matography of the methanol extract gave a single
fluores-cent spot with a Rfof 0.075 Under identical conditions,
riboflavin, FMNand FAD exhibited Rfvalues of 0.40, 0.19
and 0.075, respectively Thus, we concluded that FerB
contains a noncovalently bound FAD
Substrate specificity
In order to investigate the catalytic abilities of the isolated
enzymes, a specificity study was undertaken with
Fe(III)ni-trilotriacetic acid and NADH as reference substrates The
rate of reduction of different Fe(III) compounds, under the
conditions specified, is shown in Table 2 FerA has an
absolute requirement for the flavin and cannot effectively use
NADPH as an electron donor in place of NADH A
substi-tution of riboflavin for FMNled to a decrease by 73% in the
reaction rate (not shown in Table 2) Unlike FerA, FerB
does not require exogenous flavin and utilizes both NADH
and NADPH Probably the most striking difference between
FerA and FerB lies in the inability of the latter enzyme to
reduce Fe(III) complexed by vicinal dihydroxy groups, as
present in maltol, pyrocatechol and 2,3-dihydroxybenzoic
acid In line with this, we also observed that FerA, but not
FerB, exhibited a reducing activity towards the complex
of Fe(III) with a crude isolate of parabactin, the natural
catecholic siderophore of P denitrificans (data not shown)
By monitoring the absorbance at 340 nm, we observed
that the addition of FMNto FerA resulted in a significant
rate of NADH oxidation also in the absence of Fe(III),
which suggested the presence of an NADH : FMN
oxido-reductase activity Its value typically amounted to
330 nmol NADHÆs)1Æmg protein)1 For this reason we
tested whether the reduction of Fe(III) could be due to the FMNH2formed by FerA This possibility gained support from the stopped-flow experiments in which we compared the rates of a direct nonenzymatic reduction of Fe(III) complexes by the chemically prepared FMNH2 The relative rate values presented in the last column of Table 2 match more closely those of FerA (r¼ 0.92) rather than those for FerB (r¼ 0.77), demonstrating that FerA indeed may act primarily as a flavin reductase, and, per se, not mediate the subsequent redox reactions of Fe(III)
The identification of a sequence homology between FerB and chromate reductases (Fig 2B) prompted an investigation of whether or not the Paracoccus enzyme can reduce chromate The positive answer is conveyed
by the results in Fig 4, showing a significant speed-up in
Table 2 Substrate specificity of Fe(III) reductases from P denitrificans, compared with the chemical reactivity of FMNH 2 Experimental details are
described in the Material and methods section The Fe(III) complexes were used at a final concentration of 0.2 m M The relative reaction velocity for
NADPH was related to that of NADH as 100% The relative rates for the different Fe(III) complexes were related to that of Fe(III)nitrilotriacetic
acid as 100% The relative rate zero means a relative reaction velocity of > 0.1%.
Substrate
Relative rate of Fe(II)(ferrozine) 3 complex formation
a 100% ¼ 2.04 · 10)7molÆs)1Æmg protein)1; b 100% ¼ 8.43 · 10)9molÆs)1Æmg protein)1; c 100% ¼ 3.73 · 10)5 Æs)1.
Fig 4 Chromate reductase activity of FerB Reaction mixture (0.5 mL) containing 0.045 m M chromate, 0.15 m M NADH and 25 m M
Tris/HCl buffer, pH 7.4, with (d) or without (s) 5 lg of FerB were incubated at 30 C Aliquots were withdrawn at the indicated time intervals and analyzed for the concentration of chromate.
Trang 7the reduction of chromate by NADH, caused by the
presence of FerB FerB also functioned as an efficient
quinone reductase For a series of four quinones, the relative
activities were found as follows:
2,3-dimethoxy-5-methyl-1,4-benzoquinone (ubiquinone-0), 100%; p-benzoquinone,
77%; menadione, 6%; duroquinone, 0.01% (100%¼
2.42 lmol NADHÆs)1Æmg protein)1) The enzyme did not
catalyze the oxidation of NADH by FMN at an
appreci-able rate (specific activity less than 1 nmol NADHÆs)1Æ
mg protein)1)
Kinetic studies of reactions catalyzed by FerA and FerB
When studying the Fe(III) reductase reaction of FerA, there
are effectively three compounds to be considered in a kinetic
analysis: NADH, FMN and complexed Fe(III) Initial
velocity studies were carried out by varying the
concentra-tions of NADH in the presence of a series of concentraconcentra-tions
of Fe(III)nitrilotriacetic acid while maintaining FMNat a constant concentration As illustrated in Fig 5(A), recipro-cal plots are patterns of lines intersecting to the left of the vertical axis The FMNreductase reaction of FerA was analyzed with NADH and FMN in the absence of Fe(III) Under these conditions, kinetic patterns similar to those found above were obtained (Fig 5B) This behavior indi-cates a sequential mechanism with NADH and FMN as substrates
The results obtained on studying the Fe(III) reductase reaction of FerB are in Fig 6 It can be seen that the reciprocal plot for NADH as variable substrate at a series of concentrations of Fe(III)nitrilotriacetic acid consists of a series of parallel lines Kinetic patterns of this type occur in cases of ping-pong mechanisms At the highest concentra-tion of NADH the initial velocity of the enzyme turned out
to be slower than that predicted by the hyperbolic law Similar substrate inhibition was also observed in the 1/v vs 1/[Fe(III)nitrilotriacetic acid] plot; this issue was, however, not pursued further at this stage
Table 3 lists values of the kinetic parameters for individ-ual substrates, as estimated by computer fitting of velocity data of Figs 5 and 6 to the appropriate kinetic equations Besides the data for Fe(III)nitrilotriacetic acid in Table 3,
we also determined the kinetic parameters for some other Fe(III) complexes reducible by FerB In the presence of a saturation concentration (0.15 mM) of N ADH, the values
of Kmand kcatare: (0.4 ± 0.2) mMand (0.91 ± 0.02) s)1 for Fe(III)EGTA (1.6 ± 0.8) mMand (0.16 ± 0.03) s)1for Fe(III)EDTA (0.4 ± 0.2) mM and (0.08 ± 0.01) s)1 for Fe(III)citrate, and (0.4 ± 0.2) mMand (0.015 ± 0.003) s)1 for Fe(III) chloride The kinetics of the reduction of Fe(III) ligated with maltol could not be measured reliably due to the difficulty caused by an intensive coloration of the complex The fact that all the obtained values Kmare higher than 0.2 mM confirms that the measurements reported in Table 2 were performed under conditions of first-order
Fig 5 Initial-velocity kinetics of the reactions catalyzed by FerA.
Measurements were performed with 0.2 lg FerA proteinÆmL)1 in
25 m M Tris/HCl buffer, pH 7.4 at 30 C (A) Fe(III) reductase
reac-tion velocity as a funcreac-tion of NADH concentrareac-tion in the presence of
eight (s), 12 (d), 20 (h), 40 (j) and 67 (n) lM Fe(III)nitrilotriacetic
acid at constant concentrations of FMN(50 l M ) and ferrozine
(0.8 m M ) (B) Flavin reductase reaction velocity as a function of FMN
concentration in the presence of 5 (s), 10 (d), 20 (h) and 30 (j) l M
NADH In this case both ferrozine and Fe(III)nitrilotriacetic acid were
absent Data are presented as double-reciprocal plots.
Fig 6 Initial-velocity kinetics of the Fe(III) reductase reaction cata-lyzed by FerB Measurements were performed at 30 C with
3 lg FerB proteinÆmL)1in 25 m M Tris/HCl buffer, pH 7.4, contain-ing 0.8 m M ferrozine Reciprocal initial velocity is plotted against the reciprocals of NADH concentration at a series of fixed concentrations
of Fe(III)nitrilotriacetic acid equal to 40 (s), 66.7 (d), 100 (n) and 200 (m) lM.
Trang 8kinetics and hence the relative rates listed here, being
proportional to the kcat/Km ratios, correctly express the
substrate specificity of FerB
Discussion
In this study, we have demonstrated a coexistence of two
types of soluble Fe(III) reductases, FerA and FerB, in the
soil bacterium Paracoccus denitrificans Both enzymes are
flavin-dependent, but in different ways: a flavin serves as a
dissociable cosubstrate for FerA (a flavin reductase-type
enzyme) and as a tightly bound redox-active cofactor of
FerB (a flavoprotein-type enzyme)
Flavin reductases are present in a variety of organisms
[12] Extensive studies have been conducted, for instance,
with enzymes from E coli [7,32] or luminous bacteria
Vibrio harveyi[33–40] and Vibrio fischeri [41–44] Although
these enzymes catalyze similar reactions, they differ in
respect to substrate specificities and reaction mechanisms
Accordingly, they can be classified into D, P and G types
based on whether NADH, NADPH or both NADH and
NADPH are utilized Obviously, FerA falls into theD-type
Another classification criterion distinguishes between flavin
reductases lacking a detectable prosthetic group and those
having a noncovalently bound flavin cofactor Considering
the negative result of the spectroscopic search for a bound
flavin group and the sequential kinetic pattern obtained
(Fig 5), we conclude that the first possibility applies,
namely that FerA accelerates a direct redox reaction
between NADH and a flavin In this respect, it seems to
be similar to theD-type flavin reductase of V harveyi [35]
The observed substrate specificity of FerA (Table 2) adds to
the previous appreciation of the biological role of a free
reduced flavin as a reducing agent for Fe(III) [2,12,13,32]
and broadens it to account for the reduction of the
catechol-type Fe(III) complexes The high effectiveness of the redox
process mediated by free flavin is reflected by a low value of
the apparent Michaelis constant for the Fe(III) complex
with nitrilotriacetic acid (Table 3)
In the case of FerB, the distinctive spectral features
(Fig 3) and the ping-pong kinetic patterns (Fig 6) make it
likely that the enzyme shuttles between the forms containing
oxidized and reduced bound flavin during the catalytic
cycle The fact that FerB appears rather inefficient in reducing some substrates which are otherwise prone to uncatalyzed reactions with free FMNH2(Table 2) suggests that the protein moiety contributes significantly to the observed substrate specificity Although more direct infor-mation on the three-dimensional structure of FerB is obviously required before any firm conclusion can be drawn
as to how this specificity is achieved, we can tentatively imagine two situations In the first, the reduced flavin binds more tightly than the oxidized one, so that the cofactor becomes poorer electron donor and reactions of electron acceptors with low standard redox potentials are hampered Our voltammetric measurements (unpublished results) have revealed that the Fe(III)/Fe(II) pairs complexed with maltol, pyrocatechol and dihydroxybenzoate, which do not accept electrons from FerB, have indeed the lowest standard redox potentials among the iron compounds listed in Table 2 Alternatively, FerB may carry a buried substrate binding cavity whose size allows for interaction with smaller electron acceptors like 1 : 1 Fe(III) complexes, chromate or qui-nones, but not with the rather bulky 1 : 3 Fe(III) complexes
or exogenous free flavins
A survey of the literature indicates that FerB shares some biochemical properties with other enzymes Its relatedness
to the earlier described chromate reductase of Pseudomonas putida [27] is apparent from a match in the N-terminal sequence (Fig 2B) and the ability to reduce chromate at
a comparably high rate (Fig 4) The subunit and native masses of FerB found here agree favorably with the values
of 20 and 50 kDa reported before for Pseudomonas enzyme [27] However, there is a marked difference in isoelectric points While FerB is an acidic protein (pI 5.5 by chromatofocusing), pI of chromate reductase was claimed
to be higher than 7.0 from chromatographic behavior; our own calculation based on the known amino acid sequence (gi:14209680) and exploiting the pI/Mw tool [45] on http:// www.expasy.org gave the value of 8.55 Unfortunately, the Pseudomonasenzyme was not tested for the presence of flavin nor the activity towards electron acceptors other than chromate, and hence a more detailed comparison with FerB cannot be presented at the moment It may also be of interest to mention here the existence of the FAD-contain-ing DT-diaphorase [46] and the FMN-containFAD-contain-ing
flavin-Table 3 Michaelis constants (± SE) for the substrates of FerA and FerB Values were calculated from the data in Figs 5 and 6 The best fitting kinetic models, selected according to the Akaike’s information criterion, correspond to a sequential bi–bi mechanism for FerA and to a ping-pong mechanism for FerB Non-linear regression values of k cat are (14 ± 1) s)1and (28 ± 6) s)1for the Fe(III) reductase and FMNreductase activity of FerA, respectively, and (3.1 ± 0.5) s)1for FerB.
Substrate
(variable concentration)
Second substrate (fixed concentrations)
Third substrate (constant concentration)
K m
(l M ) FerA
NADH Fe(III)nitrilotriacetic acid FMN (50 l M ) 5.5 ± 0.7 Fe(III)nitrilotriacetic acid NADH FMN (50 l M ) 17 ± 2
FerB
a
Fe(II)(ferrozine) 3 production was used to quantify reaction progress except in these cases where NADH oxidation by flavin was measured.
Trang 9reductase [43,44] and nitroreductase [47] that resemble FerB
in being homodimers of comparable molecular size, utilizing
both NADH and NADPH as electron donors, exhibiting
ping-pong kinetics and having a number of activities
including that of quinone reductase Indications exist that
a quite small number of amino acid residues determine
electron acceptor specificity of these enzymes For example,
a single point mutation was enough to convert NfsB, one of
the oxygen-insensitive nitroreductases of Escherichia coli, to
a highly active flavin reductase [48] Constrains of similar
type as in NfsB may exist in FerB and preclude in this way
its reaction with free flavin We hope that future
compar-ative studies will clarify relationships (if any) between FerB
and the other mentioned proteins on a molecular level A
part of this effort is currently aimed at the cloning of the
gene for FerB for further characterization
Acknowledgements
We thank Dr Z Vobu˚rka (Institute of Organic Chemistry and
Biochemistry, AS CR, Prague) for performing N-terminal amino acid
sequencing, Dr Z Zdra´hal (Laboratory of Mass Spectrometry of
Biomolecules, Faculty of Science, MU, Brno) for MALDI-MS analyses
and Dr Z Prokop (National Centre for Biomolecular Research,
Faculty of Science, MU, Brno) for assisting us with the stopped-flow
measurements This work was supported by a grant from the Grant
Agency of the Czech Republic to I K (203/01/1589) and partly by a
contract from Ministry of Agriculture of the Czech Republic to J.T.
(M-3-99-01).
References
1 Schro¨der, I., Johnson, E & de Vries, S (2003) Microbial ferric
reductases FEMS Microbiol Rev 27, 427–447.
2 Cowart, R.E (2002) Reduction of iron by extracellular iron
reductases: implications for microbial iron acquisition Arch.
Biochem Biophys 400, 273–281.
3 Jones, J.G., Gardner, S & Simon, B.M (1983) Bacterial reduction
of ferric iron in a stratified eutrophic lake J Gen Microbiol 129,
131–139.
4 Short, K.A & Blakemore, R.P (1986) Iron respiration-driven
proton translocation in aerobic bacteria J Bacteriol 167, 729–
731.
5 Dobbin, P.S., Warren, L.H., Cook, N.J., McEwan, A.G., Powell,
A.K & Richardson, D.J (1996) Dissimilatory iron(III) reduction
by Rhodobacter capsulatus Microbiology 142, 765–774.
6 Moody, M.D & Dailey, H.A (1985) Ferric iron reductase of
Rhodopseudomonas sphaeroides J Bacteriol 163, 1120–1125.
7 Fontecave, M., Eliasson, R & Reichard, P (1987) NAD(P)H:
flavin oxidoreductase of Escherichia coli A ferric iron reductase
participating in the generation of the free radical of ribonucleotide
reductase J Biol Chem 262, 12325–12331.
8 Halle, F & Meyer, J.-M (1992) Ferrisiderophore reductases of
Pseudomonas: purification, properties and cellular location of the
Pseudomonas aeruginosa ferripyoverdine reductase Eur J
Bio-chem 209, 613–620.
9 Mazoy, R & Lemos, M.L (1996) Ferric-reductase activities in
whole cells and cell fractions of Vibrio (Listonella) anguillarum.
Microbiol 142, 3187–3193.
10 Mazoy, R., Lopez, E.M., Fouz, B., Amaro, C & Lemos, M.L.
(1999) Ferric-reductase activities in Vibrio vulnificus biotypes 1 and
2 FEMS Microbiol Lett 172, 205–211.
11 Vadas, A., Monbouquette, H.G., Johnson, E & Schro¨der, I.
(1999) Identification and characterization of a novel ferric
reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus J Biol Chem 274, 36715–36721.
12 Fontecave, M., Coves, J & Pierre, J.-L (1994) Ferric reductases or flavin reductases? Biometals 7, 3–8.
13 Coves, J & Fontecave, M (1993) Reduction and mobilization of iron by a NAD(P)H: flavin oxidoreductase from Escherichia coli Eur J Biochem 211, 635–641.
14 Myers, C.R & Myers, J.M (1997) Outer membrane cytochromes
of Shewanella putrefaciens MR-1: spectral analysis, and purifica-tion of the 83-kDa c-type cytochrome Biochim Biophys Acta
1326, 307–318.
15 Seeliger, S., Cord-Ruwisch, R & Schink, B (1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreductans acts as a ferric ion reductase and as an electron carrier to other acceptors or to partner bacteria J Bacteriol 180, 3686–3691.
16 Shatwell, K.P., Dancis, A., Cross, A.R., Klausner, R.D & Segal, A.W (1996) The FRE1 ferric reductase of Saccharomyces cere-visiae is a cytochrome b similar to that of NADPH oxidase J Biol Chem 271, 14240–14244.
17 Coves, J., Eschenbrenner, M & Fontecave, M (1993) Sulfite reductase of Escherichia coli is a ferrisiderophore reductase Biochem Biophys Res Commun 192, 1403–1408.
18 Lee, P.L., Halloran, C., Cross, A.R & Beutler, E (2000) NADH-ferric reductase activity associated with dihydropteridine reductase Biochem Biophys Res Commun 271, 788–795.
19 Sapshead, L.M & Wimpenny, J.W.T (1972) The influence of oxygen and nitrate on the formation of the cytochrome pigments
of the aerobic and the anaerobic respiratory chain of Micrococcus denitrificans Biochim Biophys Acta 267, 388–397.
20 Bosma, G., Braster, M., Stouthamer, A.H & van Verseveld, H.W (1987) Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat Eur J Biochem 165, 665–670.
21 Tait, G.H (1975) The identification and biosynthesis of sidero-chromes formed by Micrococcus denitrificans Biochem J 146, 191–204.
22 Bergeron, R.J., Dionis, J.B., Elliott, G.T & Kline, S.J (1985) Mechanism and stereospecificity of the parabactin-mediated iron-transport system in Paracoccus denitrificans J Biol Chem 260, 7936–7944.
23 Bergeron, R.J., Weimar, W.R & Dionis, J.B (1988) Demon-stration of ferric 1-parabactin-binding activity in the outer mem-brane of Paracoccus denitrificans J Bacteriol 170, 3711–3717.
24 Bergeron, R.J & Weimar, W.R (1990) Kinetics of iron acquisi-tion from ferric siderophores by Paracoccus denitrificans J Bac-teriol 172, 2650–2657.
25 Dailey, H.A Jr & Lascelles, J (1977) Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.
J Bacteriol 129, 815–820.
26 Kucera, I & Matchova, I (1997) Iron as a possible mediator of the oxic-to-anoxic transition in Paracoccus denitrificans Biochem Mol Biol Int 43, 305–310.
27 Park, C.H., Keyhan, M., Wielinga, B., Fendorf, S & Matin, A (2000) Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase Appl Environ Microbiol.
66, 1788–1795.
28 Lowry, O.H., Rosebrough, N.J., Farr, A.L & Randall, R.J (1951) Protein measurement with the folin-phenol reagents.
J Biol Chem 193, 265–275.
29 Halle, F & Meyer, J.-M (1992) Iron release from ferrisider-ophores A multi-step mechanism involving a NADH/FMN oxidoreductase and a chemical reduction by FMNH 2 Eur.
J Biochem 209, 621–627.
30 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.
Trang 1031 Perrella, F.W (1988) EZ-FIT: a practical curve-fitting
micro-computer program for the analysis of enzyme kinetic data on
IBM-PC compatible computers Anal Biochem 174, 437–447.
32 Fieschi, F., Niviere, V., Frier, C., Decout, J.-L & Fontecave, M.
(1995) The mechanism and substrate specificity of the NADPH:
flavin oxidoreductase from Escherichia coli J Biol Chem 270,
30392–30400.
33 Michaliszyn, G.A., Wing, S.S & Meighen, E.A (1977)
Purifica-tion and properties of a NAD(P)H: flavin oxidoreductase from
the luminous bacterium, Beneckea harveyi J Biol Chem 252,
7495–7498.
34 Jablonski, E & DeLuca, M (1977) Purification and properties of
the NADH and NADPH specific FMN oxidoreductases from
Beneckea harveyi Biochemistry 16, 2932–2936.
35 Jablonski, E & DeLuca, M (1978) Studies of the control of
luminiscence in Beneckea harveyi: properties of the NADH and
NADPH: flavin oxidoreductases Biochemistry 17, 672–678.
36 Lei, B., Liu, M., Huang, S & Tu, S.-C (1994) Vibrio harveyi
NADPH-flavin oxidoreductase: Cloning, sequencing and
over-expression of the gene and purification and characterization of the
cloned enzyme J Bacteriol 176, 3552–3558.
37 Tanner, J.J., Lei, B., Tu, S.-C & Krause, K.L (1996) Flavin
reductase P: Structure of a dimeric enzyme that reduces flavin.
Biochemistry 35, 13531–13539.
38 Liu, M., Lei, B., Ding, Q., Lee, J.C & Tu, S.-C (1997) Vibrio
harveyi NADPH: FMN oxidoreductase: preparation and
char-acterization of the apoenzyme and monomer-dimer equilibrium.
Arch Biochem Biophys 337, 89–95.
39 Lei, B & Tu, S.-C (1998) Mechanism of reduced flavin transfer
from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase.
Biochemistry 37, 14623–14629.
40 Wang, H., Lei, B & Tu, S.-C (2000) Vibrio harveyi
NADPH-FMNoxidoreductase Arg203 as a critical residue for NADPH
recognition and binding Biochemistry 39, 7813–7819.
41 Tu, S.-C., Becvar, J.E & Hastings, J.W (1979) Kinetic studies on the mechanism of bacterial NAD(P)H: flavin oxidoreductase Arch Biochem Biophys 198, 110–116.
42 Zenno, S., Saigo, K., Kanoh, H & Inouye, S (1994) Identification
of the gene encoding the major NAD(P)H-flavin oxidoreductase
of the bioluminescent bacterium Vibrio fischeri ATCC 7744.
J Bacteriol 176, 3536–3543.
43 Inouye, S (1994) NAD(P)H-flavin oxidoreductase from the bio-luminiscent bacterium, Vibrio fischeri ATCC 7744, is a flavopro-tein FEBS Lett 347, 163–168.
44 Koike, H., Sasaki, H., Kobori, T., Zenno, S., Saigo, K., Murphy, M.E.P., Adman, E.T & Tanokura, M (1998) 1.8 A˚ crystal structure of the major NAD (P) H: FMN oxidoreductase of a bioluminiscent bacterium, Vibrio fischeri: overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins J Mol Biol 280, 259–273.
45 Bjellqvist, B., Hughes, G.J., Pasquali, Ch, Paquet, N., Ravier, F., Sanchez, J.-Ch, Frutiger, S & Hochstrasser, D.F (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences Electrophoresis
14, 1023–1031.
46 Ernster, L (1987) DT-diaphorase: a historical review Chem Scripta 27A, 1–13.
47 Zenno, S., Koike, H., Tonokura, M & Saigo, K (1996) Gene cloning, purification, and characterization of NfsB, a minor oxy-gen-insensitive nitroreductase from Escherichia coli, similar in properties to FRase I, the major flavin reductase of Vibrio fischeri.
J Biochem (Tokyo) 120, 736–744.
48 Zenno, S., Koike, H., Tonokura, M & Saigo, K (1996) Con-version of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid sub-stitution J Bacteriol 178, 4731–4733.