Binding of the apoflavodoxin to the reductase was sufficient to over-come the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partne
Trang 1Inhibition of pea ferredoxin–NADP(H) reductase by Zn-ferrocyanide
Daniela L Catalano Dupuy, Daniela V Rial and Eduardo A Ceccarelli
Molecular Biology Division, IBR (Instituto de Biologı´a Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Argentina
Ferredoxin–NADP(H) reductases (FNRs) represent a
pro-totype of enzymes involved in numerous metabolic
path-ways We found that pea FNR ferricyanide diaphorase
activity was inhibited by Zn2+ (Ki 1.57 lM)
Dichloro-phenolindophenol diaphorase activity was also inhibited by
Zn2+(Ki 1.80 lM), but the addition of ferrocyanide was
required, indicating that the inhibitor is an arrangement of
both ions Escherichia coli FNR was also inhibited by
Zn-ferrocyanide, suggesting that inhibition is a consequence
of common structural features of these flavoenzymes The
inhibitor behaves in a noncompetitive manner for NADPH
and for artificial electron acceptors Analysis of the
oxida-tion state of the flavin during catalysis in the presence of the
inhibitor suggests that the electron-transfer process between
NADPH and the flavin is not significantly altered, and that
the transfer between the flavin and the second substrate is
mainly affected Zn-ferrocyanide interacts with the reduc-tase, probably increasing the accessibility of the prosthetic group to the solvent Ferredoxin reduction was also inhib-ited by Zn-ferrocyanide in a noncompetitive manner, but the observed Kiwas about nine times higher than those for the diaphorase reactions The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide Binding of the apoflavodoxin to the reductase was sufficient to over-come the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect
Keywords: ferredoxin; ferredoxin–NADP(H) reductase; flavodoxin; flavoproteins; zinc
Ferredoxin–NADP(H) reductases (FNRs) constitute a
family of hydrophilic and monomeric enzymes that contain
noncovanlently bound FAD [1,2] One of the exceptional
features of FNR is its ability to split electrons between
obligatory one-electron and two-electron carriers, as a
consequence of the biochemical properties of its prosthetic
group Flavoproteins with FNR activity have been found in
phototrophic and heterotrophic bacteria, animal and yeast
mitochondria, and apicoplasts of obligate intracellular
parasites They operate as general electronic switches at
the bifurcation steps of many different electron-transfer
pathways (for review see References [1–3])
In chloroplasts, they catalyze the final step of
photosyn-thetic electron transport, which involves electron transfer
from the iron-sulfur protein ferredoxin (Fd), reduced by
photosystem I, to NADP+ At the molecular level, the
reaction proceeds to the reduction of NADP+via hydride
transfer from the N5 atom of the flavin prosthetic group
This reaction provides the NADPH necessary for CO2 assimilation in plants and cyanobacteria
Some bacteria and algae possess an FMN-containing protein, flavodoxin (Fld), which is able to efficiently replace
Fd as the electron partner of FNR in different metabolic processes, including photosynthesis Fld expression is induced under conditions of iron deficit, when the [2Fe-2S] cluster of Fd cannot be assembled [4–6]
FNR displays a strong preference for NADP(H) and is a very poor NAD(H) oxidoreductase At variance, the reduced flavin can donate electrons to a remarkable variety of oxidants of very different structure and properties through a largely irreversible reaction named NADPH diaphorase [7] The list of acceptors includes ferricyanide and other transition metal complexes, substituted phenols such
as 2,6-dichlorophenolindophenol (DCPIP), nitroderivatives, tetrazolium salts, NAD+(transhydrogenase activity), vio-logens, quinones, and cytochromes (reviewed in [8]) Some
of these artificial reactions may have technological relevance for bioremediation and the pharmaceutical industry [9,10] Plant FNRs ( 35 kDa) comprise two structural domains, each containing 150 amino acids [11] The C-terminal region includes most of the residues involved in NADP(H) binding, and the large cleft between the two domains accommodates the FAD group A large portion of the isoalloxazine moiety is shielded from the bulk solution, but the edge of the dimethyl benzyl ring that participates in electron transfer remains exposed to solvent in the native holoenzyme
The structural determinants involved in the electron-transfer process, substrate recognition, and the likely
Correspondence to E A Ceccarelli, IBR, Facultad de Ciencias
Bio-quı´micas y Farmace´uticas, Universidad Nacional de Rosario,
Suipa-cha 531 (S2002LRK) Rosario, Argentina Fax: +54 341 4390465,
Tel.: +54 341 4351235, E-mail: cecca@arnet.com.ar
Abbreviations: FNR, ferredoxin–NADP(H) reductase; Fd, ferredoxin;
Fld, flavodoxin; DCPIP, 2,6-dichlorophenolindophenol; FPR,
Escherichia coli FNR; GST, glutathione S-transferase; DNT,
2,4-dinitrotoluene.
(Received 13 August 2004, revised 6 October 2004,
accepted 11 October 2004)
Trang 2catalytic mechanism have been intensely analysed and
debated but aspects of the subject remain to be revealed
Here, we report the use of Zn2+, in conjunction with
ferrocyanide, as a specific inhibitor to analyze the catalytic
process and electron transfer in pea FNR Zn2+ has
catalytic, cocatalytic, and/or structural roles in a myriad of
metalloenzymes [12] In addition, it inhibits some enzymes
that are not necessarily zinc ones [13–19] Until now, no
metal-binding site of high affinity has been identified in
FNRs even though there is some evidence of metal-binding
sites in FNR-like enzymes (e.g NO synthase) [13,20], other
flavoproteins [14,15,21,22] and a number of different
enzymes [18,19,23,24] Our results may also have some
environmental significance in the light of the enormous
amount of metal cyanides released as industrial waste and
recent evidence of ferrocyanide transport by plants [25]
FNRs can efficiently interact with and accommodate two
completely different protein partners, i.e Fd and Fld
Contacts between Fd and FNR occur through ionic
interactions including acidic and basic residues present in
each protein, respectively These interactions determine the
initial relative orientation between both proteins, which is
finally tuned for electron exchange [3,26,27] We found that
pea FNR diaphorase activities were inhibited in a
noncom-petitive manner by Zn2+when equimolar concentrations of
this metal and ferrocyanide were present Escherichia coli
FNR (FPR) behaves similarly to the pea enzyme with
respect to the inhibitor In contrast, to obtain a comparable
inhibition of the Fd reduction catalyzed by pea FNR,9
times higher inhibitor concentration is needed We observed
that Fld from Anabaena is able to accept electrons from
pea FNR, and that this reaction was not affected by
Zn-ferrocyanide Moreover, the addition of apoFld was
sufficient to avoid enzyme inhibition by Zn-ferrocyanide,
indicating that a conformational change is probably
produced in the reductase upon binding of Fld In addition,
our data show that electron transfer from the reduced flavin
to an oxidant can be inhibited without affecting the electron
transfer between the NADPH and the prosthetic group
These results provide insights into enzyme catalysis and are
discussed in the light of current knowledge
Experimental procedures
Protein expression and purification
Pea FNR, Y308 [28] and C266 FNR mutants were
overexpressed in E coli as reported [28] using vector
pGF205+ [29] Vector pGF205+ [29] was obtained by
inserting an adapter formed from oligonucleotides 1
(TTGGTTCCGCGTGGATCCCGAGCT) and 2 (AGTT
CCAGTTCCCAACATGATGATGACAGTAGC) at the
SacI site of plasmid pGF105 [30] The insertion generates a
fusion protein GST-FNR+ containing the amino acid
sequence LVPRGSRA, which includes a thrombin
recog-nition site between the C-terminus of glutathione
S-transferase (GST) and the first amino acid of the mature
FNR Purification of pea FNR from E coli JM109 was
carried out as described [30], except that the gel-filtration
step was replaced by anion-exchange chromatography using
a DEAE Macroprep column (1.5· 15 cm; Bio-Rad,
Hercules, CA, USA) equilibrated in 50 m Tris/HCl,
pH 8 (buffer A) The resin was extensively washed with the same buffer, and FNR eluted using a linear gradient from 0 to 0.3MNaCl in buffer A The fractions containing the enzyme were dialyzed against 50 mMTris/HCl, pH 8, and concentrated on a DEAE Macroprep column (1· 3 cm, equilibrated in 50 mM Tris/HCl) eluted with
250 mMNaCl in buffer A
Recombinant pea Fd was obtained by expression in
E coli Briefly, a pET28-Fd expression vector was con-structed by inserting the cDNA corresponding to the mature pea Fd into the pET28a vector (Novagen Inc., Madison, WI, USA) The coding sequence for the mature
Fd was amplified by PCR using as primers the oligonucle-otides Fdup 5¢-GCAACACCATGGCTTCTTACAAAG TGAAA-3¢ and Fdlw 5¢-CCACAAGCTTGATATCATA TCATAGCATAGCAGT-3¢ and the full length pea Fd precursor cDNA as template To facilitate the cloning process, NcoI and HindIII restriction sites were introduced
in primers Fdup and Fdlw, respectively After amplification, the product was digested with NcoI and HindIII, and the fragment (350 bp) was ligated to the pET28a vector digested with the same enzymes, obtaining the plasmid pET28-Fd This vector allows the expression of pea Fd in
E colias a soluble protein with high yield Fd purification was performed essentially as described [31]
The E coli Fd-NADP+reductase was purified according
to published procedures [32]
Fld from Anabaena was kindly provided by M Medina (University of Zaragoza, Zaragoza, Spain) ApoFld from Anabaena Fld was obtained by treatment with trichloro-acetic acid [33]
Spectral analyses Absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer To study the inhibition by Zn-ferro-cyanide of the flavin reduction, the FNR samples were diluted in 50 mM HEPES, pH 7.5 (at 25C) to a final concentration of 20 lM Absorption spectra were recor-ded both before and after the addition of 2.5 mMNADPH (donor electron substrate), in either the absence or presence
of 20 lMZn-ferrocyanide This procedure was also carried out with 1 mM potassium ferricyanide (electron acceptor substrate) in the solution
Protein and flavin fluorescence was monitored using a Kontron SFM 25A spectrofluorimeter (Zu¨rich, Switzer-land) interfaced with a personal computer Solution for fluorescence measurements contained 1 lM protein in
50 mM HEPES, pH 8 Assays were performed in either the absence or presence of 15 lM Zn-ferrocyanide, at
25C
Activity measurements FNR-dependent diaphorase activity was determined by a published method [34] The reaction mixture (1 mL) contained 50 mMHEPES, pH 7.5, 3 mM glucose 6-phos-phate, 0.3 mM NADP+, 1 U glucose-6-phosphate dehy-drogenase, and either 1 mM potassium ferricyanide or 0.033 mMDCPIP After the addition of 20 nMpea FNR (or 150 nME coli FPR), reactions were monitored spec-trophotometrically by following ferricyanide reduction at
Trang 3420 nm (e420Ử 1 mM )1ẳcm)1) or DCPIP reduction at
600 nm (e600Ử 21 mM )1ẳcm)1)
Ferredoxin reductase activity of FNR was assayed
in reaction medium (0.5 mL) containing 50 mM HEPES,
pH 7.5, 0.3 mM NADPH and 25 lM Fd After addition
of 75 nM FNR, the reaction was monitored
spectro-photometrically by following the decrease in A340(e340Ử
6.22 mM )1ẳcm)1) Different FNR and Fd concentrations
were tested to ensure linearity of the reaction
Fld-dependent oxidase activity of FNR was determined
using Anabaena Fld The reaction mixture (0.6 mL)
contained 50 mM HEPES, pH 7.5, 0.25 mM NADPH,
12.5 lMFld and 100 nMFNR The reaction was monitored
by following NADPH oxidation at 340 nm This activity
was also assayed in the presence of 0.25 mM
2,4-dinitro-toluene (DNT) All kinetic experiments were performed at
30C
Inhibition assays
Inhibition studies were performed by adding equimolar
quantities of ZnSO4and potassium ferrocyanide (0Ờ20 lM)
to the reaction medium, except for the ferricyanide
diapho-rase reactions, in which only ZnSO4 was added The
inhibition reversibility was studied by adding 1 mMEDTA,
pH 8.0, to the reaction medium
The inhibition by Zn-ferrocyanide of different FNR
variants was studied by assaying ferricyanide diaphorase
activity in the absence and presence of 5 lMZnSO4in the
reaction medium after the addition of FNR as follows:
wild-type, 0.021 lM; Y308G, 0.27 lM; Y308F, 0.026 lM; Y308S,
0.10 lM; Y308W, 0.06 lM; or C266A, 0.12 lM The data
obtained are presented as the percentage of the remaining
activity observed in the presence of Zn-ferrocyanide in each
case
Determination of kinetic parameters
To determine the kinetic parameters of the diaphorase and
Fd reduction reactions, measurements were carried out at
different NADPH, potassium ferricyanide, DCPIP or Fd
concentrations, at a fixed saturating concentration of the
other substrate, in both the absence and presence of
inhibitor (0, 1.5, 3 and 5 lMZn-ferrocyanide) Steady-state
kinetic data were fitted to the theoretical curves using
SIGMAPLOT software (Jandel Scientific, San Rafael, CA,
USA) Inhibition constants (Ki) for the different substrates
were determined by Dixon plots of 1/v plotted against
inhibitor concentration (0, 1.5, 3 and 5 lM), at different
concentrations of NADPH (5, 10, 25, 50, 100 and 300 lM),
DCPIP (5, 10, 15, 25, 35 and 50 lM), ferricyanide (100, 250,
500, 750 and 1000 lM) and Fd (10, 15, 25, 40 and 50 lM)
Determination of the dissociation constants of the
FNRẳNADP+, FNRẳFd and FNRẳFld complexes
To determine the Kdvalues of the complexes between FNR
and NADP+or Fd, 0.6 lMflavoprotein in 50 mMHEPES,
pH 8, was titrated at 25C with the corresponding
substrate After each addition, the flavin fluorescence
(excitation at 456 nm; emission at 526 nm in the case of
NADP+) or the flavoprotein fluorescence quenching
(excitation at 270 nm; emission at 340 nm for the FNRẳFd complex) were monitored using a Kontron SFM 25A spectrofluorimeter The fluorescence data were fitted to a theoretical equation as described in [35] for a 1 : 1 complex using the nonlinear regression program included in the SIGMAPLOTsoftware package (Jandel Scientific) to optimize the value of Kd[36] The effect of Zn-ferrocyanide on the
Kd values of these complexes was also determined The experimental setup was as above, except that 15 lM Zn-ferrocyanide was included in the solution
Difference absorption spectroscopy was used to evaluate the dissociation constant of the FNRẳFld complex [37] The experiment was performed essentially as described [37] on a solution containing 6.55 lMFNR in 50 mMHEPES, pH 8,
at room temperature, to which aliquots of Anabaena Fld were added The absorbance differences (DA) at 465 nm were registered and fitted to the theoretical equation [38]:
DAỬ 0:5DefđơPtợ ơLtợ Kdỡ đđơPtợ ơLtợ Kdỡ2
4ơPtơLtỡ0:5g for a 1 : 1 complex using a nonlinear regression, where [P]t and [L]t are the total concentration of FNR and Fld, respectively, and, De the molar absortivity of the complex [37] This procedure does not require the determination of the titration endpoint [38]
Protection against inhibition by Fld The inhibition of DCPIP diaphorase activity was assayed in the presence of Fld, apoFld or Fld previously treated with
1 : 100 (w/w) trypsin (15 h, at 24C) After the addition of
20 nMFNR, the reaction was monitored spectrophoto-metrically by following DCPIP reduction at 600 nm The inhibition protection profile was made in the presence of
20 lM Zn-ferrocyanide and varying either Fld or apoFld concentrations (0Ờ8 lM)
Inactivation of FNR by Zn-ferrocyanide The DCPIP diaphorase activity of FNR was assayed using FNR samples previously treated with 3 lMZn-ferrocyanide during different periods of time (0Ờ80 min)
Results
Inhibition of FNR activities by Zn-ferrocyanide The effect of the presence of Zn2+during FNR catalysis is shown in Fig 1A Ferricyanide diaphorase activity was inhibited by the addition of increasing concentrations of ZnSO4, with an I0.5, the concentration that produces 50% inhibition, of about 1 lMZn2+ In contrast, no effect was observed by Zn2+ addition on the DCPIP diaphorase activity (Fig 1A) NADPH oxidation catalyzed by FNR in the presence of Fd or Fld was not affected by the presence of
up to 1 mMZnSO4 The effect of equimolar concentrations of Zn2+ and ferrocyanide on diaphorase activities with different sub-strates and Fd reduction were then investigated In all cases, the addition of metal ion and ferrocyanide to the reaction medium produced strong enzyme inhibition (Fig 1B)
Trang 4Zn-ferrocyanide was about six times more effective at
inhibiting diaphorase activities than Fd reduction However,
in all cases total inhibition was obtained At pH 7.5, a 50%
inhibition was observed for the ferricyanide diaphorase
activity with 1 lMZn2+, meanwhile 6 lMZn-ferrocyanide
was necessary to obtain the same inhibition of the Fd
reduction Likewise, when the FPR from E coli was
investigated, inhibition of diaphorase activity was obtained
with Zn-ferrocyanide (Fig 1C) Neither FNR nor FPR
activity was inhibited by sodium sulfate or ferrocyanide alone
using DCPIP or ferricyanide as electron acceptors Similarly,
no enzyme inhibition was detected using ferrous sulfate
Co2+, which is able to replace Zn2+in metaloenzymes,
also inhibited the diaphorase reaction of pea FNR only if
ferrocyanide was added with a I0.5of 25 lM Cu2+and
Ni2+were also tested, and no inhibition was observed up to
100 lMfor the ferricyanide diaphorase activity using any of the metals on pea reductase Higher concentrations had some effect on the pea enzyme, but, in all cases, much lower inhibition was observed (not shown) In all cases, the addition of 1 mM EDTA final concentration after 2 min
of reaction reversed the enzyme inhibition instantly and completely (not shown) This observation suggests that the Zn-ferrocyanide is accessible to the solvent Incubation of the enzyme with Zn-ferrocyanide for longer periods of time resulted in inactivation of 57% in 60 min without the release of the prosthetic group (not shown)
To evaluate this inhibitor in more detail, the steady-state kinetics of the FNR for the different substrates were examined at pH 7.5 Plots for the inhibitions of FNR diaphorase activities with increasing concentration of Zn-ferrocyanide (range 0–5 lM of inhibitor) showed that
Fig 1 Inhibition of FNR activities by
Zn-fer-rocyanide Residual FNR activity as a
func-tion of ZnSO 4 concentration (A) or ZnSO 4
and potassium ferrocyanide equimolar
con-centrations (B) using ferricyanide (d), DCPIP
(s) and ferredoxin (m) as electron acceptors.
(C) Inhibition of ferricyanide (d) and
DCPIP (s) diaphorase activities of E coli
FPR as a function of Zn-ferrocyanide
con-centration In all cases, activity measurements
were performed at pH 7.5 (D) A typical
steady-state kinetics experiment of the FNR
diaphorase activity for different DCPIP
con-centrations at a fixed NADPH concentration
of 300 l M , performed at increasing
concen-trations of inhibitor [0 (s), 1.5 (d), 3 (m) and
5 (j) l M ] Inset: a typical K i determination by
Dixon plot of 1/v (B) vs inhibitor
concentra-tion (A) at different DCPIP concentraconcentra-tions.
Table 1 Kinetic, inhibition and binding parameters for various activities of FNR The kinetics parameters were determined as describe in Experi-mental procedures Each parameter value represents the average of three independent experiments K i values were calculated from Dixon plots of Zn-ferrocyanide noncompetitive inhibition with respect to the indicated substrate at a fixed saturating concentration of the other substrate ND, Not determined.
Substrate
K m (l M )
(no inhibitor)
k cat (s)1) (no inhibitor) K i (l M )
K d
(l M , NADP + ) (no inhibitor)
K d
(l M , NADP + ) (15 l M Zn-ferrocyanide) Type of inhibition
a,b NADPH-ferricyanide diaphorase activity c NADPH-DCPIP diaphorase activity d NADPH-Fd reduction.
Trang 5the compound was a noncompetitive inhibitor of the
enzyme for NADPH and ferricyanide Similar results were
obtained when DCPIP diaphorase activity and Fd
reduc-tion were analysed for the substrates DCPIP and Fd,
respectively (Table 1) In all cases a linear noncompetitive
inhibition was observed, indicating that the inhibitor
binding to the enzyme produces a nonproductive enzyme–
substrate–inhibitor complex Dixon plots were used to
calculate the Kivalues (Table 1), which were consistent with
the I0,5values extracted from Fig 1 Calculation of enzyme
activity at infinitive inhibitor concentration showed that
total inhibition was obtained in all cases
The dissociation constants of the FNRÆNADP+ and
FNRÆFd complexes were estimated in the absence or
presence of 15 lM inhibitor by measuring flavin
fluores-cence and flavoprotein fluoresfluores-cence quenching, respectively,
after addition of each substrate As shown in Table 1, the
presence of the inhibitor did not change the enzyme affinity
for its substrates This is in agreement with the inhibition
kinetic data presented above
Zn-ferrocyanide inhibition of the reduction and oxidation
of the flavin
We have analyzed the spectral properties of FNR, and no
differences were observed on addition of Zn-ferrocyanide
(Fig 2A, compare thick and thin solid lines) Then, we
studied the spectral changes of the enzyme by addition of an
excess amount of NADPH The oxidation state of flavins
can be distinguished by spectrophotometric means They
can exist in three different redox states: oxidized,
one-electron reduced (semiquinone) radical, and fully reduced
hydroquinone The isolated FNR in solution contains
mostly oxidized FAD The neutral flavin radical absorbs
light of long wavelength with a maximum at 570 nm, which
is only detectable in FNR when the enzyme is anaerobically
reduced In aerobic conditions, when 2.5 mMNADPH was
added to the enzyme solution and the spectral changes were
recorded after 5 s, a decrease in absorbance was observed at
459 nm with a concomitant increase with a maximum at
590 nm Similar results were obtained when reduction of
the enzyme by NADPH was performed in the presence of
Zn-ferrocyanide (Fig 2A, thick and thin dashed lines)
These results indicate that, in both cases under aerobic
conditions and with an excess of NADPH, the neutral
semiquinone of FAD appeared, with its typical absorption
band which usually expands from 520 to 680 nm
The same experiment was then performed in the presence
of the electron acceptor potassium ferricyanide in the
absence of the inhibitor, recording the spectral changes 5 s
after the addition of the substrates Under these conditions,
the enzyme containing a reduced flavin form was
spectro-photometrically undetectable (Fig 2B) The addition of
20 lM Zn-ferrocyanide in the reaction medium from the
beginning of the measurement leads to the appearance of
the reduced form of the enzyme, even in the presence of
the electron acceptor (Fig 2B) These results allow us to
conclude that the electron-transfer process between
NADPH and the flavin was not significantly altered by
the presence of the inhibitor, and disrupting the electron
transfer between the flavin and the second substrate mainly
causes enzyme inhibition by Zn-ferrocyanide
Effect of Fld on the inhibition of FNR activities
by Zn-ferrocyanide
In some photosynthetic systems, such as that of certain algae and cyanobacteria, the FMN-containing protein Fld
Fig 2 Reduction and oxidation of the flavin Optical spectra of FNR FAD reduction, 5 s after mixing, measured as the decrease in absorbance at 459 nm, and the increase at 550 nm to 650 nm range All reactions were performed under aerobic conditions and contain
50 m M HEPES, pH 7.5, 20 l M FNR and the following additions (A)
In the absence of an electron acceptor: thick solid line, no addition; thick dashed line, 2.5 m M NADPH; thin solid line, 20 l M Zn-ferro-cyanide; thin dashed line, 20 l M Zn-ferrocyanide and 2.5 m M
NADPH (B) In the presence of an electron acceptor: thick solid line,
no addition; thick dotted line, 1 m M potassium ferricyanide; thick dashed line, 1 m M potassium ferricyanide and 2.5 m M NADPH; thin solid line, 20 l M Zn-ferrocyanide; thin dotted line, 20 l M Zn-ferro-cyanide and 1 m M potassium ferricyanide; thin dashed line, 20 l M
Zn-ferrocyanide, 1 m M potassium ferricyanide and 2.5 m M NADPH Insets: amplified view of the region between 500 and 700 nm of the corresponding figures.
Trang 6can efficiently replace Fd in the protein–protein
electron-transfer process catalyzed by FNR Fld and Fd bind to the
same FNR site for catalysis, and, despite the difference in
size, they seem to be equally oriented during binding to
FNR and electron transfer [27] Although there is no Fld in
plants, Fld is able to efficiently accept electrons from plant
FNR ([39] and this work)
Surprisingly, Zn-ferrocyanide was unable to inhibit the
electron transfer from NADPH to Fld catalyzed by pea
FNR We tested concentrations up to 20 lM
Zn-ferro-cyanide without any apparent loss of enzyme activity
NADPH oxidation by FNR using Fld as electron
acceptor proceeds at a low rate This rate can be enhanced
by the addition of the electron acceptor DNT The
interpretation of this observation is that Fld mediates the
electron transfer between the reductase and DNT, as FNRs
catalyze the reduction of DNT very slowly This system can
be used to better estimate the electron-transfer rate between
the reductase and the Fld Flavodoxin oxidase activity both
in the absence and presence of DNT as artificial electron
acceptor was insensitive to the addition of the metal
ferrocyanide (Fig 3A) Addition of Fld at saturating
concentrations produced an increase of about 100% in the
NADPH oxidation (Fig 3A), which was not obtained
by the addition of apoFld (not shown) Interestingly,
Zn-ferrocyanide did not inhibit the reduction of DNT
mediated by Fld, but completely prevented the direct
transfer to DNT (Fig 3A)
We also investigated the oxidase activity of FNR in the
absence of added electron acceptors and, unexpectedly we
found that it was completely insensitive to Zn-ferrocyanide
(v¼ 0.28 lmolÆmg)1Æmin)1 in the presence of 15 lM
Zn-ferrocyanide vs v¼ 0.29 lmolÆmg)1Æmin)1 in the
absence of the inhibitor) (Fig 3A)
We then decided to investigate if Fld protects FNR
against Zn-ferrocyanide inhibition When the FNR DCPIP
diaphorase activity was measured in the presence of 20 lM
Zn-ferrocyanide and 12.5 lM Fld, no inhibition was
observed (Fig 3A,B) Under identical conditions, the
reduc-tion of DCPIP was inhibited 98% by Zn-ferrocyanide
Two possible explanations for the unexpected protection
displayed can be envisaged Fld may bypass the pathway
that is inhibited by Zn-ferrocyanide, transferring the
electron to DCPIP or, the binding of the carrier protein to
the FNR directly affects the interaction of the inhibitor with
the enzyme As shown in Fig 3B the apoprotein protects
FNR against Zn-ferrocyanide inhibition As a control, a
sample containing Fld previously treated with trypsin did
not display any protection (Fig 3B), indicating that the
effect was a result of the presence of the polypeptide itself
Figure 4 shows a protection assay of the inhibition by
Zn-ferrocyanide of the DCPIP diaphorase activity at different
Fld concentrations It can be observed that flavoprotein and
its apoform displayed similar abilities to protect the enzyme
Moreover, the protection profile obtained can be correlated
with the affinity of the FNRÆFld complex (13.4 lM) as
obtained from the binding experiment depicted in Fig 5
Interaction of Zn-ferrocyanide with the FNR reductase
To further investigate the interaction of Zn-ferrocyanide
with FNR, the prosthetic group environment was analyzed
by fluorescence spectroscopy Figure 6 shows that Zn-ferrocyanide interacts with the enzyme in the absence
of its substrates Addition of the metal complex induces an increase in FAD fluorescence with a concomitant shift of emission maximum to a lower wavelength resembling the one obtained with FAD in solution This observation can be considered to indicate that the prosthetic group undergoes a
Fig 3 Inhibition by Zn-ferrocyanide in the presence of Fld (A) The inhibition of flavodoxin oxidase activity was assayed in a DNT inde-pendent or deinde-pendent manner The inhibition of DCPIP diaphorase activity was measured in the absence or presence of 12.5 l M Fld Pure oxidase and DNT oxidase activities of FNR were assayed as controls Activity was measured in the absence (hatched bars) or presence (solid bars) of Zn-ferrocyanide Reactions were monitored by following NADPH oxidation at 340 nm (B) The inhibition of DCPIP diapho-rase activity was measured in the absence or presence of 12.5 l M Fld, apoFld, or Fld digested with trypsin DCPIP reduction was followed at
600 nm.
Trang 7rearrangement or that its exposure to the environment is
increased
A putative Zn2+-binding site within the FNR structure
The crystal structure of pea FNR (PDB entry 1QG0 [40]),
was analyzed searching for structures that could be able to
bind Zn2+ All residues potentially able to co-ordinate
Zn2+were identified, and distances and geometries within
the surrounding residues were determined using the
SWISS-PDBVIEWER 3.7 The FAD was also took into account in
the analysis because it has long been known that flavins
interact specifically with metals [41,42] We found a serine, a
glutamic acid, a cysteine and a tyrosine residue near the
isoalloxazine in a spatial orientation suitable for the
interaction with metals (Fig 7A) We also observed that
the space available to accommodate Zn2+is enough for
appropriate binding of the metal ion, which remains
accessible from the exterior (Fig 7B) Distances between
the N5 and O4 of the flavin, O of Ser90, S of Cys266, O of
Glu306 and O of Tyr308 indicate that almost all of them are
at bond distances between each other and nearly oriented
correctly to participate in Zn2+ co-ordination (Fig 7C)
This amino acid arrangement around FAD is conserved in
FPR and in the neuronal NO synthase (Fig 7D,E)
To obtain supporting evidence for the proposed binding
site and to investigate the participation of the above amino
acids, several FNR mutants were analyzed Table 2 shows
the FNR inhibition by Zn-ferrocyanide obtained under
identical experimental conditions with FNR mutants of
Cys266 or Tyr308 Cysteine is one of the amino acids most
commonly observed after histidine as a Zn2+ ligand in
metalloproteins [12] However, Cys266 does not appear to
have a central role in the interaction with Zn-ferrocyanide as
its replacement by alanine generates an enzyme that is still
affected by the inhibitor (Table 2) Similarly, replacing Tyr308 with other aromatic amino acids only slightly affects the inhibition by Zn-ferrocyanide on the enzyme In contrast, replacing Tyr308 with glycine or serine consider-ably reduced the inhibition Table 2 also shows the degree
of nicotinamide ring occupancy of the binding site of Tyr mutants, as calculated by Piubelli et al [43] We found an inverse correlation between the extent of Zn2+inhibition and nicotinamide ring occupancy in the FNR variants These observations indicate that the binding of NADP+to the enzyme either reduces the accessibility of the isoallox-azine itself to Zn2+and/or ferrocyanide or partially impairs the entry of the inhibitor to the proposed binding site Although a binding site for ferrocyanide, an octahedral
Fig 4 Protection of DCPIP diaphorase activity by Fld DCPIP
diaphorase activity was assayed in the presence of 20 l M
Zn-ferrocy-anide and different concentrations of either Anabaena Fld (solid bars)
or apoFld (hatched bars).
Fig 5 Determination of the dissociation constant of FNRÆFld complex (A) Difference absorption spectra obtained during the titration of pea FNR (6.55 l M ) with Anabaena Fld (B) Absorbance difference data at
465 nm fitted to the theoretical equation for a 1 : 1 stoichiometric complex by means of nonlinear regression The K d value obtained was 13.4 l M
Trang 8complex anion with a diameter of about 6 A˚, has never been
mapped within the structure of FNR, it could be possible
that this anion collides with the Zn2+-containing protein
structure, interacting strongly and filling the open space
near the isoalloxazine
Discussion
The data presented in this work clearly show that the pea
Fd-NADP(H) reductase is inhibited by Zn-ferrocyanide as
a result of a specific combined interaction of both ions with
the enzyme The inhibition was also observed on the E coli
enzyme, a member of the same protein family, even though
FPR is structurally distanced from the plant flavoprotein
[44] Inhibition by Zn2+has been reported for other
flavin-containing enzymes Cu2+and Zn2+inhibit all
NADPH-dependent reactions catalyzed by the neuronal NO synthase
[13] The authors of this work have concluded that
inhibition is produced by the interaction of the metal with
a unique site present in the reductase domain of the enzyme
[13] This domain binds one equivalent of FMN and one of
FAD and, members of the Fd–NADP(H) reductase family
share its structural features [45] Similarly, it has been
observed that Zn2+ inhibits the isolated a-oxoglutarate
dehydrogenase mitochondrial complex [14] A more
de-tailed study has shown that the dihydrolipoyl
dehydro-genase component of the complex is responsible for the
observed Zn2+ inhibition [15] This enzyme is a
homo-dimeric molecule which contains FAD and belongs to the
NAD-disulphide oxidoreductases class I group, which is led
by the glutathione reductase as the model protein [46] The
latter group does not contain either the well-defined
conserved sequences or displayed sequence similarity with
the chloroplast-type FNRs Taken together, these results
may support the idea that the flavin itself may be involved in
the interaction of the flavoproteins with the metal
The kinetic analysis of all FNR activities inhibited by Zn-ferrocyanide revealed noncompetitive behavior for NADPH, for artificial electron acceptors and, for Fd The FAD fluorescence of FNR showed a slight increase due to the addition of the inhibitor (10–15%), together with a shift
of the maximum emission wavelength to 526 nm, closer to that of the free flavin These rather small perturbations could be caused by changes in the microenvironment of the isoalloxazine ring, which is probably more exposed to the solvent after binding of the metal ferrocyanide Changes may also be produced by the interaction of the metal itself with the isoalloxazine However, the effect was only observed when Zn2+and ferrocyanide were added, indica-ting that the combination of the two ions, and not each one separately, was responsible for the observed change Another hypothesis to explain the observed inhibition proposes that Zn2+and ferrocyanide interact directly with the prosthetic group and/or with amino acid residues involved in the electron-transfer process Searching for structures that may be able to bind Zn2+on the crystal structure of FNR, we found a serine, a glutamic acid, a cysteine, and a tyrosine residue near the isoalloxazine in a spatial orientation suitable for the interaction with metals (Fig 7A), although no definite sites were identified It was also observed that the space available to accommodate
Zn2+is enough for appropriate binding of the metal ion Interestingly, these amino acids are conserved in FPR and in the neuronal NO synthase (Fig 7D,E) The residue homo-logous to FNR Tyr308 is a Phe in the NO synthase Consequently, it may be suggested that Tyr308 may not be directly involved in the Zn-ferrocyanide inhibition of the reductase Cysteine, histidine and glutamic acid are com-mon Zn2+ligands in metalloproteins [12] Although serine and tyrosine are less common Zn2+ ligands, they can interact with ions such as Zn2+especially in proteins with more than one metal center such as alkaline phosphatase from E coli [47]
We suggest that a partially or totally co-ordinated Zn2+ interacting with the bulky ferrocyanide, which can also interact with other amino acids, represents the true inhib-itor The binding of ferricyanide and ferrocyanide has been detected in some enzymes [48,49] Moreover, this interaction was proposed to occur via positively charged amino acids [49] Indeed, the solubility of salts is a consequence of a large energy gain during hydration of ions that is surplus to the lattice energy At the concentration of Zn-ferrocyanide used (0–30 lM), the salt is soluble but near its solubility product Thus, a small change in the availability of water, as would occur with the inclusion of both ions in a protein hydrophobic pocket, may induce a stable ionic interaction between Zn2+and ferrocyanide We are not able to give an explanation for the participation of ferrocyanide as an obligate partner in the interaction of Zn2+with the enzyme However, it may be suggested that binding of ferrocyanide,
an octahedral complex anion with a diameter of about 6 A˚, throughout the interaction with the bound Zn2+near the isoalloxazine could produce the observed inhibitory effect
on FNR activity This hypothesis is also supported by the finding that mutants with a catalytic site greatly occupied by the NADP+nicotinamide displayed a reduced susceptibility
to Zn-ferrocyanide inhibition (Table 2) It is worth men-tioning that the crystal structures of these mutants have
Fig 6 Interaction of Zn-ferrocyanide with FNR Fluorescence
emis-sion spectra of FNR (k exc ¼ 459 nm) in the absence (thick solid line)
or presence of 15 l M Zn-ferrocyanide (thin solid line) Fluorescence
emission of free FAD (thin dashed line).
Trang 9been obtained and that the overall conformations are equivalent to those of wild-type spinach and pea leaf FNRs, with no significant changes in the relative orientation of amino acids, the FAD or the conformation and binding of the 2¢-P-AMP portion of NADP+[40]
It has been observed that A-type monoamine oxidase
is inhibited by the zinc benzoate salt [16] Similarly, a-chymotrypsin may be inhibited by a substrate analog that interacts with a Zn2+ion that is partially co-ordinated
at the active site [17]
The Ki for the inhibition of Fd reduction is 8.8 times higher than those for diaphorase activity inhibition (Table 1) These results may be explained by the observa-tion that binding of Fd to FNR leads to structural changes
in the reductase After complex formation, the entire NADP(H) domain is displaced slightly as a single unit, and Glu306, which is located near the isoalloxazine, moves
to within hydrogen-bonding distance of the hydroxy group
of Ser90, as observed by Kurisu et al [50] in crystals of the
Table 2 Inhibition of wild-type and mutant FNR diaphorase activity by
Zn-ferrocyanide ND, Not determined.
FNR variant
Remaining activity (% of control)a
Nicotinamide ring occupancy of the binding site (%)b
a Remaining ferricyanide diaphorase activity in the presence of
5 l M Zn-ferrocyanide with respect to the control.bTaken from Ref
[43], calculated from the absorption coefficients at the peak near
510 nm of the difference spectra elicited by nicotinamide nucleotide
binding to the various pea FNR forms; 100% refers to NADP+
occupancy of FNR-Y308S.
Fig 7 Putative Zn2+-binding site in FNR-like enzymes Detail view of the spatial distribution of residues putatively involved in the interaction with
Zn 2+ in (A) pea FNR, (D) E coli FPR and (E) rat neuronal NO synthase Nitrogen 5 (N5) and oxygen 4 (O4) from FAD isoalloxazine are indicated (B) Ribbon diagram of the putative Zn2+-binding site in pea FNR (C) Distances between the atoms of pea FNR probably involved in the interaction with Zn2+ were measured in A˚ The schemes were drawn using SWISS - PDBVIEWER 3.7 and rendered with POV - RAY from the tridimensional structures as determined by X-ray diffraction (Protein Data Bank entries 1QG0, 1FDR, 1TLL) [40].
Trang 10FNRÆFd complex from maize This glutamic acid is
sufficiently exposed and readily available for the interaction
with Zn2+(Fig 7A,B) In the Anabaena FdÆFNR
crystal-lographic association resolved by Morales et al [26] the
carboxy group of the homologous Glu301 is no more
exposed to solvent but is hydrogen-bonded to the hydroxy
group of Fd Ser64
The role of these residues has been thoroughly
inves-tigated by site-directed mutagenesis When the homolog
Glu301 from Anabaena FNR was mutated to Ala, the
altered enzyme obtained was only 1% as active as the
wild-type enzyme in electron transfer to Fd [51] As
the photoreduction of NADP+ was not affected to the
same degree as the Fd reduction, the authors suggested
that the rate-determining step during catalysis involves
other processes in addition to the electron-transfer process
between the two prosthetic groups [51] The semiquinone
state of FAD was significantly destabilized in the FNR
mutant in which Glu301 was changed to Ala, and this was
probably the main reason for the electron-transfer
alter-ation observed in this mutant Similarly, four different
spinach FNR mutants of the equivalent Glu312 were
obtained and analyzed [52] The authors concluded that
this residue is directly involved in the electron transfer
between FNR and Fd They also hypothesized that the
residue may contribute to the tuning of the redox potential
of the flavin semiquinone to enhance efficient electron
transfer and/or may be acting as a proton donor/acceptor
to FAD [51,52]
The O of Ser90 and the S of Cys266 of pea FNR are close
to N5 and O4 of the isoalloxazine, which are involved in
hydride transfer The hydroxy group of Ser90 could accept a
hydrogen bond and thus help to stabilize the reduced flavin
Meanwhile its interaction probably affects the transition
state of hydride transfer The Ser96Val mutant of FNR
displayed a kcatnearly 2000 times lower than that of the
wild-type enzyme [53] Analysis of the crystal structure of
wild-type pea FNR shows that the Zn2+ ion can easily
access Ser90 Moreover, serine is among the amino acids
that could, although infrequently, co-ordinate Zn2+[12]
Thus, it may be one of the amino acid residues involved in
the inhibition of FNR by Zn-ferrocyanide
Our results (Fig 2) allow us to suggest that
Zn-ferro-cyanide mainly causes an interruption of the oxidative half
reaction in the diaphorase activity and the electron transfer
between FNRred and Fd It has been observed that
electrophilic metal ions such as Zn2+prefer co-ordination
with the one-electron reduced semiquinone state of flavin
[42] Thus, Zn-ferrocyanide may interact after reduction of
the enzyme by NADPH with the semiquinone state of the
flavoprotein, producing the observed inactive form or
altering the proton exchange between the flavin and the
surrounding amino acid residues, in particular Glu306 and
Ser90 Both residues have been proposed to participate in
the proton-transfer pathway between the exterior and
isoalloxazine [26,45,51–55]
It is interesting that Zn-ferrocyanide was unable to inhibit
the electron transfer from NADPH to Fld catalyzed by pea
FNR At present, no crystal structures of the complex
between FNR and Fld have been obtained Using several
charge-reversal mutants, it has been possible to infer that
FNR uses the same site for the interaction with both
electron partners, Fd and Fld [27] Moreover, it has been shown that Fd and Fld could be completely overlapped on the basis of their surface electrostatic potentials [56], but the interaction with Fld has been proposed to involve a larger FNR surface [57] Although the interaction of FNR with its substrates exhibits co-operativity [58,59], modifications of the structure that should lead to the observed effects have remained elusive or hard to detect [11,40,60]
Changes in hydrophobic patches of Anabaena FNR influenced the rates of electron transfer to and from Fld and
Fd However, the observed effects were more dramatic in the processes involving Fld than those involving Fd, suggesting that these Anabaena FNR residues do not participate to the same extent in the processes for the two proteins [61] Recently, electron transfer was obtained with the hybrid system bovine adrenodoxin reductase/Anabaena Fld, indicating that a highly specific interaction is not essential and that the process may proceed through multiple weak interactions So far no residue on the Fld surface has been identified to be critical for the interaction and the electron-transfer processes between Fld and FNR [62] It has therefore been suggested that there is a lower specificity for the FNR–Fld interaction than for the FNR–Fd one [62] Therefore a dynamic assembly of the former complex in which multiple orientations may exist can be proposed The fact that Zn-ferrocyanide was unable to inhibit the pea FNR electron transfer to Fld may not only be related to the protein size or a specific residue but also to the mechanisms
of interaction between the reductase and Fld The very short distance predicted between the two redox centers [62] may also account for our observations
On the other hand, interaction of FNR with Fd does lead
to structural changes in both electron carriers relative to the free protein conformations [26,50] The protein–protein interaction also affects the microenvironments of the two prosthetic groups In the case of the Fd and FNR, their redox potentials (Em) were shifted by )25 mV and +20 mV, respectively, reflecting theses changes [63]
We have observed that both Fld and its apoprotein are able to impede the inhibition of FNR by Zn-ferrocyanide More remarkable, the solely polypeptide interaction between FNR and apoFld is sufficient to prevent the inhibition, indicating that no participation of the Fld electronic transfer is involved in the observed protection
In addition, we observed that the Fld concentration needed
to protect FNR from Zn-ferrocyanide inhibition is similar
to the Kd for the pea FNRÆFld complex (13.4 lM) It is worth mentioning that it has been determined that the structure of apoFld is virtually equivalent to that of the holoprotein, the only exception being that the isoalloxazine-binding site closed [64]
In summary, our results indicate that determinants on the FNR polypeptide are essential for electron transfer between the reduced flavin and the substrate and, that this process can be completely inhibited by Zn2+ in the presence of ferrocyanide We have obtained evidence that isoalloxazine and the surrounding amino acids are the binding site of the inhibitor Clearly, the observation that binding of Fld or apoFld to the reductase was sufficient to overcome the inhibition may be taken as evidence for a conformational change produced in the reductase on interaction with this electron partner, modifying either the FAD environment or