Plant Cell Physiol. 39(8): 821-829(1998)
JSPP © 1998
The FAD-Enzyme Monodehydroascorbate Radical Reductase Mediates
Photoproduction of Superoxide Radicals in Spinach Thylakoid Membranes
Chikahiro Miyake 15 , Ulrich Schreiber2, Henning Hormann 2 , Satoshi Sano3 and Kozi Asada4
1
2
3
4
Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan
JuliuS'Von-Sachs-Institut fur Biov/issenschaften mit Botanischen Garten der Universitdt Wiirzburg, Lehrstuhl fur Botanik I, Mittlerer
Dallenbergweg 64, D-97082 Wiirzburg, Germany
Research Institute of Innovative Technology for the Earth (RITE), Kizu, Kyoto, 619-0225 Japan
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Gakuencho-1, Fukuyama, 729-0292 Japan
The photoreduction of dioxygen in spinach thylakoid
membranes was enhanced about 10-fold by the FAD-enzyme monodehydroascorbate radical (MDA) reductase at
1 fM. The primary photoreduced product of dioxygen catalyzed by MDA reductase was the superoxide radical, as
evidenced by the inhibition of photoreduction of Cytc
by superoxide dismutase. The apparent Km for dioxygen of
the MDA reductase-dependent photoreduction of dioxygen
was 100 /JM, higher by one order of magnitude than that
observed with thylakoid membranes only. Glutathione reductase, ferredoxin-NADP+ reductase, and glycolate oxidase also mediated the photoproduction of superoxide radicals in thylakoid membranes at rates similar to those with
MDA reductase. Among these flavoenzymes, MDA reductase is the most likely mediator stimulating the photoreduction of dioxygen in chloroplasts; its function in the protection from photoinhibition under excess light is discussed.
or A/B of the PSI complex (Asada et al. 1974, 1977), and
its photoreduction rate is 30 fimol (mg Chl)~' h" 1 at a maximum. Recently, it has been shown that, under bright light,
the photoreduction of dioxygen amounts to 20-30% of
total electron flux in intact leaves, much higher than the
maximum photoreduction observed in isolated thylakoids
(Osmond and Grace 1995). This suggests that stromal components may enhance the photoreduction of dioxygen in the
chloroplasts of intact leaves. Ferredoxin (Fd) on thylakoid
membranes (Misra and Fridovich 1971, Telfer et al. 1970),
FAD released from ferredoxin-NADP+-oxidoreductase
(FNR) (Firl et al. 1981), and FNR itself (Goetze and Carpentier 1994) have been previously shown to enhance
the photoreduction of dioxygen in thylakoids, but little systematic evaluation of these reactions has been done so far.
We report here an observation that monodehydroascorbate reductase (MDAR) is an effective mediator in the
photoreduction of dioxygen to superoxide radical in chloroplasts.
MDAR is a FAD enzyme (Hossain et al. 1984, HosKey words: Active species of oxygen — Monodehydrosain
and
Asada 1985) and the first known enzyme that uses
ascorbate radical — Monodehydroascorbate radical reducan
organic
radical as the substrate. It catalyzes the reductase (EC 1.6.5.4) — Photosystem I — Superoxide radical —
tion
of
MDA
to ascorbate using NAD(P)H as the electron
Thylakoid membranes.
donor:
NAD(P)H + 2 MDA => NAD(P) + +2 ascorbate
Plant leaves are frequently exposed to photon fluxes in
excess of their capacity to convert photon energy to chemical energy for reduction of CO2 to carbohydrate during
photosynthesis. Such conditions occur, for example, whenever the supply of CO2 is limiting, e.g. under water stress
when the stomata close or under bright light. Then, instead of CO2, dioxygen is reduced, resulting in the production of superoxide radicals. The photoreduction site of dioxygen in thylakoids has been assumed to be FeS center X
In the reaction mechanism of MDAR, the FAD of MDAR
is reduced by NAD(P)H with a rate constant of 1.8 x 108
M~'s~' (Sano et al. 1995), and the reduced FAD of
MDAR is oxidized by MDA with a rate constant of 2.6 x
108 M~' s"1 (Kobayashi et al. 1995). Thus, the interactions
between MDAR and the substrates proceed at diffusionlimited rates. MDAR has been found in the chloroplasts
and the cytosol; the chloroplastic MDAR attaches to thylakoid membranes (Hossain et al. personal communication).
The present findings indicate that MDAR could have
two physiological functions in chloroplasts; the first is the
regeneration of ascorbate from MDA, and the second is
the photoreduction of dioxygen to superoxide radical when
the substrate MDA is absent, as described in the text. We
found that glutathione reductase (GR), glycolate oxidase
(GlyOx) and FNR in principle may also serve this function.
The physiological role of the flavoenzyme-mediated photo-
Abbreviations: APX, ascorbate peroxidase; DBMIB, dibromothymoquinone; Fd, ferredoxin; F o , dark-level of Chi fluorescence yield; F M , maximal yield of Chi fluorescence; FNR, FdNADP + reductase; GlyOx, glycolate oxidase; GOX, glucose
oxidase; GR, glutathione reductase; MDA, monodehydroascorbate radical; MDAR, MDA reductase; qp, photochemical quenching of Chi fluorescence; SOD, superoxide dismutase.
5
Corresponding author. FAX: 07437-2-5569, e-mail: cmiyake®
bs.aist-nara.ac.jp
821
822
Photoproduction of superoxide by MDA reductase
reduction of dioxygen is discussed with special reference to
protection from photoinhibition. Part of this work has
been presented in a preliminary form (Miyake et al. 1996).
Materials and Methods
Isolation of thylakoid membranes from spinach chloroplasts
—Intact chloroplasts were isolated from spinach leaves and purified by Percoll density centrifugation as described previously
(Asada et al. 1990). The isolated chloroplasts were osmotically
shocked by 10-fold dilution with 50 mM potassium phosphate
(pH 7.5), 10 mM NaCl, and 2 mM MgCl2 and then centrifuged at
5,000 xg for lOmin. The sedimented thylakoid membranes were
suspended in the same medium and centrifuged again under the
same conditions. The pellets were suspended in the reaction medium (50 mM potassium phosphate (pH 7.5), 10 mM NaCl, 2 mM
MgCl2 and 400 mM sucrose). Chi concentration was determined
by the method of Arnon (1949).
Measurement of oxygen exchange—Uptake of dioxygen was
followed using a Hansatech oxygen electrode. After incubation in
the dark for 5 min, the reaction mixture (1 ml) was illuminated by
a iodine lamp projector at 1,000/zmol photon m~2 s" 1 at 25°C.
Measurement of Chi fluorescence—Modulated Chi fluorescence was measured with a PAM Chlorophyll Fluorometer (Walz,
Germany) with fiberoptics-cuvette geometry, using the emitterdetector unit ED101 (Walz, Germany). A thermostated (25CC)
cuvette (KS 101; Walz, Germany) was used, and the reaction mixture was stirred during the measurement. Chi fluorescence was
emitted upon excitation with a weak, modulated measuring beam;
its yield varied between 1 relative unit (dark-level, Fo) and somewhat more than 4 relative units (maximal level, FM) upon application of 900-ms putse of saturating light (5,000/imol photon m~ 2
s~'). Red light (>640 nm) was used as an actinic source at an intensity of 520//mol photon m~ 2 s~'. Quenching analysis of Chi
fluorescence by the saturation pulse method was carried out as described by Schreiber et al. (1986), Neubauer and Schreiber (1989)
and Miyake and Asada (1992a). The coefficient of photochemical
quenching, qp, was determined by saturation pulse quenching analysis (Schreiber et al. 1986). Such quenching analysis involves measurements of minimal and maximal fluorescence yields of darkadapted sample (F o and FM, respectively) and the measurements of
momentary and maximal yield in a given light state (F and FM', respectively). The quenching coefficeint, qp, was calculated on the
basis of the following equation:
qp=(FM'-F)/(FM'-Fo)
Measurement of superoxide radical production—Superoxide
radical-dependent reduction of Cytc by illuminated thylakoid
membranes was measured with a dual-wavelength double beam
spectrophotometer (Hitachi-356, Tokyo, Japan) with cross-illumination (>640nm). The standard reaction mixture (1.5 ml) contained 50 mM potassium phosphate (pH 7.5), 10 mM NaCl, 2 mM
MgCl2, 400 mM sucrose, 40 //M ferri-Cyt c, 1 mM potassium
cyanide, and the thylakoid membranes (0.5-5 fig Chi). The photoreduction of Cyt c was monitored by an increase in absorbance at
550 nm with reference to that at 540 nm. The differential absorption coefficient of ferro-/ferri-Cyt c at 550 nm was assumed to be
19 mM" 1 cm" 1 (Davis and San Pietro 1977). Reaction rates were
determined from the initial absorbance change for 30 s after onset
of illumination. A low, 1.0 ftM Mn-SOD-uninhibited rate was subtracted in order to obtain the photoproduction rate of superoxide.
Assay of MDAR—The activity of MDAR was assayed by
following a decrease in absorbance at 340 nm due to the oxidation
of NADH using an absorbance coefficient of 6.2 mM" 1 cm" 1 at
25°C. In this assay, MDA was generated by ascorbate oxidase
(Yamazaki and Piette 1961) using a reaction mixture (1 ml) containing 50 mM potassium phosphate (pH7.5), 10 mM NaCl, 2
mM MgCl2, 400 mM sucrose, 0.1 mM NADH, 2.5 mM ascorbate,
ascorbate oxidase (0.14 unit, 1 /imol ascorbate oxidized min" 1 being 1 unit), and enzyme. Under these conditions, the steady-state
concentration of MDA was 2.1 //M; 1 unit of MDAR is defined as
the amount of enzyme which oxidizes 1 /imol NADH min" 1 .
Enzymes—Chloroplastic MDAR was purified from spinach
chloroplasts to give a single protein band in native PAGE and a
specific activity of 19.4 units (mg protein)" 1 (Hossain et al. unpublished). Recombinant cucumber cytosolic MDAR overexpressed
in Eschericia coli was purified to a homogeneous state according
to Sano et al. (1995); the purified enzyme showed a specific activity
of 207 units (mg protein)" 1 . The chloroplastic and cytosolic isoforms of MDAR share similar enzymatic properties, but the chloroplastic MDAR has an additional domain (8 kDa) in the carboxyl
terminus (Hossain et al., unpublished). In the present work, most
experiments were done using the cytosolic MDAR unless otherwise specified. FNR was purified from spinach leaves (Asada and
Takahashi 1971). GR from wheat, Mn-SOD from Bacillus sp.,
Cyt c from horse heart, and catalase from bovine liver were purchased from Sigma. Glycolate oxidase (GlyOx) from spinach was
obtained from Sigma and gel-filtered through a PD-10 column
(Pharmacia) to remove free FMN before its use. Glucose oxidase
(GOX) from Aspergillus sp. and ascorbate oxidase from cucumber were obtained from Toyobo. The concentrations of MDAR,
FNR, GR, GlyOx, and glucose oxidase were determined assuming
their absorption coefficients at 450 nm of 10 mM" 1 cm" 1 , which is
the value for MDAR (Hossain and Asada 1985).
Results
MDAR induces quenching of Chi fluorescence in thylakoid membranes—Thylakoid membranes from spinach
showed the typical response of light-induced Chi fluorescence changes in the absence of any electron acceptor other
than dioxygen. On illumination by actinic light, the yield of
Chi fluorescence increased, but the photochemical quenching (qp) was low (Fig. 1), reflecting a slow electron flow
from water to dioxygen. On addition of 0.5 ^M MDAR,
surprisingly, a drastic increase in qp was observed, indicating the induction of rapid electron flow. The MDARinduced increase of qp was maintained for at least 3 min.
As the concentration of MDAR (0.5/^M) was too low to
serve as the electron acceptor for several minutes, the
present observation suggests that MDAR is not only an electron acceptor but also a mediator of electron flow to dioxygen. On removal of dioxygen by bubbling with argon gas
from the reaction mixture, not only the oxygen-dependent
quenching but also the MDAR-induced quenching of Chi
fluorescence were almost completely suppressed (Fig. 1).
Furthermore, the MDAR-induced quenching of Chi fluorescence was inhibited by DCMU, which inhibits the electron
flow between QA and Q B , and also by DBMIB, which inhibits the electron flow between the plastoquinone-pool
Photoproduction of superoxide by MDA reductase
+ DCMU
.. ..J..;..;..i..:.i... + DBMIB
+O
Fig. 1 Effect of MDAR on Chi fluorescence of thylakoid membranes from intact spinach chloroplasts. The reaction mixture
(1.0 ml) contained 50 mM potassium phosphate, pH7.5, 10 mM
NaCl, 2 mM MgCl2, 400 mM sucrose, and thylakoid membranes
(60//g Chi). The fluorescence yield was monitored with a weak,
modulated measuring light (ML), and the maximum yield (FM)
was induced by a 900-ms pulse of saturating white light (SP). Illumination by actinic light (AL, red light, >640 nm, 520 /imol photon m~ 2 s"') was started at the AL arrow. MDAR (0.5/^M) was
added 60 s after the actinic light was turned on. Where indicated,
dioxygen was removed by bubbling the reaction mixture with
argon gas (-O 2 ), and either 10//M DCMU or 0.5 /xM DBMIB was
added prior to the measurement.
and Cyt b/f complex (Fig. 1). These results indicate that
MDAR mediates the electron flow from PSII to dioxygen,
presumably at the acceptor side of PSI.
MDAR induces oxygen uptake in illuminated thylakoid membranes—Oxygen uptake was observed upon illumination of the thylakoid membranes (Fig. 2A), indicating
photoreduction of dioxygen, as first observed by Mehler
(1951). The rate of oxygen uptake depended on the concentration of oxygen, with an apparent Km for dioxygen of 10
fiM (Fig. 2B curve 1), which is consistent with the results
of Heber and French (1968), Asada and Nakano (1978),
and Takahashi and Asada (1982). As expected from the
Chi fluorescence data, MDAR stimulated this oxygen uptake, confirming that MDAR actually mediates the photore-
823
duction of dioxygen (Fig. 2A). In the presence of MDAR,
the dependency of the photoreduction rate of dioxygen on
its concentration showed two phases (Fig. 2B curve 2). The
apparent Km value for dioxygen of the MDAR-mediated
photoreduction was about \O0fiM, one order of magnitude
higher than that in the absence of MDAR as deduced by
subtraction of curve 1 from curve 2 (Fig. 2B curve 3).
Superoxide radicals are produced by MDAR-mediated
photoreduction of dioxygen—In thylakoid membranes, dioxygen is univalently photoreduced at PSI producing superoxide radicals (Asada et al. 1974). The photoreduction of
Cyt c in thylakoid membranes and its inhibition by MnSOD indicate the generation of superoxide radicals at a
rate of 30/miol (mg Chi)"1 h ~ \ as observed previously
(Asada et al. 1974) (Fig. 3). In the presence of 1 (iM
MDAR, however, the photoreduction of Cyt c was stimulated about 10-fold, up to 300^mol (mgChl)" 1 h" 1 . The
MDAR-mediated photoreduction of Cyt c was completely
suppressed by Mn-SOD, showing that MDAR mediates the
photoproduction of superoxide radicals and that Cyt c is
not directly reduced by the photoreduced MDAR (Fig. 3).
Further, it was observed that MDAR-dependent and -independent photoreductions of dioxygen to superoxide radicals were completely inhibited by DCMU, indicating that
the superoxide radicals are photoproduced at PSI of thylakoid membranes (Fig. 3). Thus, the MDAR-catalyzed, primary photoreduced product of dioxygen in thylakoid membranes was identified as the superoxide radical. In the
above experiments cytosolic MDAR from cucumber was
used to demonstrate the photoproduction of superoxide
radicals. The enhanced photoproduction of superoxide radicals was observed also with the chloroplastic MDAR from
spinach (60/umol Of (mgChl)" 1 h~") at 10 nM. Unfortunately, we could not test it at micromolar levels because of
its limited availability, but the photoproduction rate of superoxide radicals by 10 nM chloroplastic MDAR is similar
to that by cytosolic MDAR at the same concentration.
Dependencies of qp of Chi fluorescence and photoreduction rate of Cyt c on concentration of MDAR—The
MDAR-induced qp of Chi fluorescence of thylakoid membranes (Fig. 1) increased with the increasing concentration
of MDAR (Fig. 4A). The concentration of MDAR required
for half-maximum qp was around 150 nM. The rate of
MDAR-mediated photoreduction of Cyt c also increased
with an increase in the concentration of MDAR, with a
half maximum rate at 100 nM (Fig. 4B). The maximum rate
of MDAR-mediated photoproduction of superoxide radical was 300 /miol (mgChl)" 1 h" 1 at \ nM MDAR, which
also induced maximum qp of Chi fluorescence. The apparent difference between the concentrations of MDAR for
half-maximum qp and photoreduction rate of Cyt c is due
to the non-linear relationship between qp and electron flux
rate (Joliot and Joliot 1964).
Dependence of MDAR-mediated photoproduction of
824
Photoproduction of superoxide by MDA reductase
[B]
0.08
[A]
Thylakoids
•
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0.07
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0.06 •
o
0.05
Q-
0.04
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0.03
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0.02
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2. [Thylakoldi + MDAR]'
b
y&
a'
3. [2] " [1]
1. [Thylakoids]
Zatrtr
1
50
100
150
200
1 "
250
Thylakoids + MDAR
Fig. 2 Stimulation of uptake of dioxygen by MDAR in illuminated thylakoid membranes from spinach. (A) Effect of MDAR on photoreduction rate of dioxygen. The reaction mixture (1.0 ml) contained 50 mM potassium phosphate, pH 7.5, 10 mM NaCl, 2 mM MgCl2,
400 mM sucrose, thylakoid membranes (10/ig Chi), and 1 mM potassium cyanide. Illumination by continuous actinic light (AL, red
light >640 nm; 520^mol photon m~2 s"1) was started at the AL-on arrow. Where indicated (Thylakoids+MDAR), 1 /iM MDAR was
added to the reaction mixture prior to the measurement. (B) Dependencies of the uptake of dioxygen by illuminated thylakoids on the
concentration of dioxygen. The uptake rates of dioxygen by illuminated thylakoids in the absence and presence of MDAR are estimated
from the slope of the uptake of dioxygen at its respective concentrations. The uptake rates of dioxygen in the absence and presence of
MDAR (at 1 /*M) are plotted against the concentration of dioxygen (Thylakoids; closed circle (curve 1)) and Thylakoids+MDAR; closed square (curve 2), respectively. Differences between the uptake rates of dioxygen [(Thylakoids+MDAR)-Thylakoids](triangle (curve
3)), corresponding to the MDAR-induced uptake of dioxygen, are also plotted against the concentration of dioxygen in the reaction mixture.
superoxide radicals on light intensity—The photoproduction rate of superoxide radicals by thylakoid membranes
only was saturated at low light intensities (12^mol photon
(2)-
(1) * MDAR
(1). Thylaitoidi
<»)• (1) or (2) • SOD
( 4 ) . (1) + DCMU
or (2) • OCMU
m 2 s '), which agrees with previous observations (Heber
and French 1968). The rate of MDAR-mediated photoproduction of superoxide radicals, however, saturated at 500
photon m" 2 s~', i.e. over a 40-fold higher intensity
than that of thylakoid membranes only (Fig. 5).
Photoreduction of MDAR in thylakoids and autooxidation of reduced MDAR—All the presented results support the hypothesis that superoxide radicals are generated
via autooxidation of photoreduced MDAR at PSI. Under
anaerobic conditions, the photoreduction of MDAR-FAD
in thylakoids could be directly demonstrated as a decrease
in absorbance of the FAD (Fig. 6). The photoreduced FAD
of MDAR was rapidly autooxidized upon introducing dioxygen, as evidenced by the recovery of absorbance of the
FAD (Fig. 6). These results give evidence for the photoreduction of MDAR in thylakoids and for the autooxidation
of its reduced FAD. Thus, the superoxide is generated via
the following catalytic cycle:
OFF
Fig. 3 MDAR-induced photoreduction of Cyt c in thylakoid
membranes from spinach. The photoreduction of Cyt c by thylakoid membranes was followed by tracking the increase in the absorbance at 550 nm with respect to that at 540 nm. The reaction
was started by actinic light illumination (AL, red light;
' The reaction mixture (2.0 ml) contained 50 mM
photon m" 2 s~').
potassium phosphate, pH 7.5, 10 mM NaCl, 2 mM MgCl2, 400
mM sucrose, 40 /iM Cyt c, 1 mM potassium cyanide and thylakoid
membranes (0.28//g Chi) (1). Where indicated, 1 pM MDAR (2),
1 juM Mn-SOD (3), or 10/iM DCMU (4) was added to the reaction
mixture prior to the assay.
2[e~] + MDAR-FAD - • MDAR-FADH 2
(PSI) (1)
MDAR-FADH2 + 2 0 2 - > 2 O 2 " " + MDAR-FAD
(2)
where MDAR-FAD and MDAR-FADH2 represent the oxidized and reduced enzymes, respectively. At present, the
photogeneration of the semiquinone form of MDAR-FAD
in PSI and its participation in the generation of superoxide
cannot be excluded. Details of the reaction mechanisms
and kinetics of the photoreduction of MDAR and of the
production of superoxide will be reported elsewhere.
Photoproduction of superoxide by MDA reductase
•
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1
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GR
FAD
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Concentration
Concentration
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+
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[B]
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(pM)
Fig. 4 Effects of the concentrations of MDAR, GR, GlyOx, FNR, GOX, and FAD on photochemical quenching of Chi fluorescence
(qp) (A) and the production rate of superoxide radical as estimated by the reduction of Cyt c (B). Conditions for determinations of the
production rate of superoxide radical and qp were the same as in Fig. 3 and 1, respectively, except for the addition of either FAD (cross)
or flavoproteins (GR, closed circle; GlyOx, triangle; FNR, diamond; MDAR, reverse triangle; GOX, plus) to the reaction mixture at the
indicated concentrations prior to the assay. The insert shows the results at an expanded scale at low concentrations of either flavoproteins or FAD.
FAD and flavoprotein-dependent photoproductions
of superoxide radical in thylakoid membranes—In addition to MDAR, we also investigated whether free FAD and
other flavoenzymes can mediate the photoproduction of
superoxide radical in thylakoid membranes. The flavoenzymes tested were GR, which is localized in chloroplast stroma, FNR, which is bound to thylakoid membranes, GlyOx, which is localized in peroxisomes, and GOX from
Aspergillus sp. The effects of FAD and the other flavoenzymes on the SOD-inhibited photoreduction of Cyt c and
the qp of Chi fluorescence are summarized in Fig. 4. Free
FAD, GR, GlyOx, and FNR enhance the photoproduction
of superoxide radicals and the qp of Chi fluorescence in a
Photoreduction of
FAD of MDAR
400
500
Wavelength (nm)
500
400
Wavelength (nm)
350
o .— 300
o '? 250
tc
:r
+ MDAR
+ MDAR
200
150
100
/
• MDAR
'•
50
• MDAR
0
0
200
400
600
800 10001200 1400 1600
Llglit Intensity (jimol photon m*' 8'')
Fig. 5 Effect of light intensity on the production rate of superoxide radical estimated from the reduction rate of Cyt c. Conditions
for determination of the production rate of superoxide radicals
were the same as in Fig. 3, except for illumination by actinic light
of thylakoid membranes (—MDAR) and MDAR-containing thylakoid membranes ( + MDAR) at the indicated intensities. The insert shows the results at an expanded scale at low light intensities.
Fig. 6 Photoreduction of the FAD of MDA reductase by PSI of
thylakoids and its re-oxidation by dioxygen. The reaction mixture
contained 50 mM HEPES-KOH, pH 7.6, 2 mM MgCl2, 10 mM
NaCl, 7 n% (Chi ml)" 1 of thylakoid membranes, 26 (iM MDAR, 1
//M catalase, and 10 mM glucose. Anaerobic conditions were maintained by the addition of 2 (iM glucose oxidase (GOX) to the reaction mixture to remove dioxygen. (A) The absorption spectrum of
oxidized MDAR was recorded 10 min after the addition of GOX,
by measuring against a reference sample consisting of the above
anaerobic reaction mixture without MDAR. The top absorption
spectrum corresponds to the oxidized form of MDAR. Subsequently, illumination by continuous actinic light (red light >640
nm; 520/*mol photon m~ 2 s~') was started, and the absorption
spectra of the photoreduced FAD of MDAR were repetitively recorded, as indicated by the dotted arrow reaching toward the bottom spectrum. The numbers in parentheses show the times after
start of illumination at which the absorption at 450 nm was recorded. The scanning rate was 7.1 nm s~*. (B) The photoreduced FAD
of MDAR was re-oxidized by the addition of 1 mM H2O2 to the
reaction mixture to produce dioxygen by catalase, as shown by the
arrow (+O 2 ).
826
Photoproduction of superoxide by MDA reductase
pattern similar to that of MDAR. The concentrations of
GR, GlyOx, and free FAD required for half-maximum increases in the photoproduction of superoxide radicals and
the qp of Chi fluorescence were 0.2 /iM, almost the same
as that found for MDAR. FNR, however, required a distinctly higher concentration for half-maximum increases
in the superoxide radical production and the qp of Chi
fluorescence, namely 1.5//M. The observation that FNR
stimulates the photoreduction of dioxygen at PSI of thylakoid membranes is consistent with earlier findings of
Goetze and Carpentier (1994). In the present work, the primary product of photoreduction of dioxygen is unequivocally identified as the superoxide radical. On the other hand,
GOX from Aspergillus sp. did not stimulate the photoreduction of Cyt c or the qp of Chi fluorescence.
In all assays, red light (>640 nm) was used as actinic
light and, therefore, it is very unlikely that either FAD,
and the enzyme-bound FAD was directly reduced or the superoxide radicals were produced via photosensitized reactions. Their photoreduction must have involved PSI, because DCMU and DBMIB inhibited the MDAR-dependent
quenching of Chi fluorescence (Fig. 1), and DCMU inhibited the MDAR-dependent photoreduction of Cyt c
(Fig. 3). FAD has been shown to induce pseudocyclic electron flow, i.e. linear electron flow to dioxygen (Firl et al.
1981). Its concentration dependency was similar to those of
MDAR and GR (Fig. 4), but release of FAD from the flavoenzymes during the reaction and the photogeneration of superoxide radical by the released FAD appear highly unlikely. The enzymatic activity of MDAR did not change between before and after the photoreaction in thylakoid membranes, indicating no release of FAD from MDAR.
Discussion
Photoreduction of flavoenzymes in thylakoids and
autooxidation of the photoreduced enzymes—The present
data give evidence for the photoproduction of superoxide
in the presence of catalytic amounts of the flavoenzymes
chloroplastic and cytosolic MDARs, GR, FNR, and GlyOx
in thylakoid membranes at increased rates of up to 300
/miol (mgChl)" 1 h" 1 . This is an unexpected finding, because the autooxidation rates of the NAD(P)H-reduced dehydrogenases MDAR-, GR- and FNR-FADH 2 -NAD(P) +
(charge transfer complexes)(Hossain and Asada 1985, Sano
et al. 1995) are extremely slow. Though glucose oxidase
does not catalyze the photoproduction of superoxide,
GlyOx can do so, indicating that this reation is not limited
to the dehydrogenase type flavoenzymes.
We assume that the flavoenzymes MDAR, GR, FNR,
and GlyOx are photoreduced at the F A /F B center in PSI
complex of thylakoid membranes. The mid-point potentials of FA and F B are — 530 and — 580 mV, respectively (Ke
and Beinert 1973, Evans et al. 1974), low enough to reduce
FADs of MDAR, GR, and FNR, because the FADs of
MDAR and GR can be reduced by NADPH (NADPH/
NADP + ; Eo'; —320 mV) and the mid-point potential of the
FAD of FNR is - 3 6 0 mV (Knaff and Hirasawa 1991). In
fact, under anaerobic conditions, the photoreduction of
MDAR-FAD in thylakoids could be directly demonstrated
as a decrease in the absorption of FAD (Fig. 6). Although
the FAD of GOX can be reduced by glucose (glucose/gluconate, Eo'; —450 mV), GOX does not catalyze the photoreduction of dioxygen by thylakoid membranes. This is due
to structural disturbance for electron transfer to GOX
from F A /F B in PSI rather than to its redox potential.
MDAR in chloroplasts accounts for the photoreduction of dioxygen to superoxide radicals in chloroplasts—
Recently, Osmond and Grace (1995) estimated the photoreduction rate of dioxygen in intact leaves of Hirschfeldia incana. The production rate of the superoxide radical increases with increasing light intensities, as judged from
18
O2-uptake rates under conditions where little photorespiration occurs. A photoreduction rate of dioxygen of 40
fimol electrons m" 2 s"1 (Osmond and Grace 1995) at a
light intensity over 1,000/imol (mleaf area)~ 2 s~' gives an
electron flow to dioxygen of 240 ^mol electron (mg Chi)" 1
h" 1 if we assume 0.6 mmol Chi (m leaf area)" 2 (Badger et
al. 1984). This rate cannot be accounted for only by the photoproduction rates of superoxide in thylakoid membranes
reported so far (Asada et al. 1974, Takahashi and Asada
1982) and in the present work (Fig. 5), which were 30//mol
O2" (mg Chi)" 1 h" 1 at most. Although it has been reported
that the Fd reduced by PSI is autooxidized, leading to the
production of superoxide (Telfer et al. 1970, Misra and
Fridovich 1971, Furbank and Badger 1983), the reaction
rate constant between reduced Fd and dioxygen is very low
(Miyake and Asada, unpublished, Hosein and Palmer
1983). In fact, Fd does not stimulate the superoxide
radical-dependent photooxidation of epinephrine in thylakoid membranes (Asada et al. 1974).
The present findings indicate that the flavoenzymes
MDAR, GR, FNR and GlyOx are photoreduced at PSI of
thylakoid membranes. The reduced flavoenzymes then
donate electrons to dioxygen, with superoxide radicals as
the primary product, at the maximum rate of 300/imol
(mg Chl)~~' h"'. Thus, the flavoenzymes are likely mediators and may account for the high rates of in viyo photoreduction of dioxygen in chloroplasts.
MDAR occurs in the chloroplast stroma at 14 ^M
(Asada 1996) and is attached to thylakoid membranes, as
observed by immunogold electronmicroscopy (Hossain et
al., unpublished), suggesting that the local concentration
of MDAR on the thylakoid surface would be much higher
than 14 fiM. Thus, chloroplastic MDAR could well account for the suggested high rates of photoreduction of dioxygen to superoxide radicals, (300 fimol (mg Chi)" 1 h" 1 ).
MDAR in chloroplasts catalyzes, no doubt, the reduc-
Photoproduction of superoxide by MDA reductase
tion of MDA to ascorbate using NAD(P)H as an electron
donor. However, since MDA produced by ascorbate peroxidase localized in the vicinity of PSI complex of thylakoid
membranes is rapidly reduced to ascorbate by the Fd photoreduced at PSI (Miyake and Asada 1992b, 1994, Asada
1996), the steady-state concentration of MDA in illuminated chloroplasts would be low. Thus, under illuminated conditions, MDAR on thylakoid membranes would not have
the substrate, MDA, and the MDAR photoreduced by PSI
would mainly function in its second physiological role reducing dioxygen to superoxide radical.
GR also has been found to be localized in the stroma
of chloroplasts. Its concentration, however, is only about
1 fiM, at least one order of magnitude lower than that of
MDAR, as estimated from its specific activity, molecular
weight, and activity in spinach chloroplasts (Foyer and
Halliwell 1976, Halliwell and Foyer 1978). Thus, the contribution of GR to the photoreduction of dioxygen at PSI
should be much lower than that of MDAR, even though
the concentration of GR in the stroma would in principle
support the superoxide production if GR is microcompartmentalized on the stromal surface of the thylakoids.
FNR also is localized in chloroplasts but binds to thylakoid membranes (Palatnik et al. 1997). The Fd-dependent
photoreduction of NADP + is observed without the addition of FNR to thylakoid membranes and is not stimulated
by the addition of FNR (Ben-Hayyim et al. 1969). FNR
added to the thylakoid membranes would be directly reduced by PSI, producing superoxide radical via autooxidation of redued FNR (Fig. 4), but the thylakoid-bound FNR
is supposed to be unable to accept electrons from the PSI
complex without the mediation of Fd. GlyOx also stimulated the photoproduction of superoxide (Fig. 4), but it cannot contribute to the photoreduction of oxygen in chloroplasts because of its location in peroxisomes. Thus, among
theflavoenzymeswhich enhance the photoproduction of superoxide, MDAR is the most likely candidate for enhancement of the photoreduction of dioxygen in chloroplast to
the observed in vivo level.
Water-water cycle—We have described the MehlerAscorbate Peroxidase-Cycle as an effective thylakoid-scavenging system for superoxide and hydrogen peroxide generated in PSI, which is composed of SOD, APX, and Fd
(Miyake and Asada 1994, Schreiber et al. 1995). In chloroplasts, this scavenging system is equivalent to a "waterwater cycle" (Fig. 7), where dioxygen is photoreduced to
superoxide radical by an FAD-enzymes; the most likely
candidate is MDAR.
The superoxide radicals such produced in PSI are
disproportionated to hydrogen peroxide and dioxygen by
thylakoid-bound or -attached superoxide dismutase (SOD)
in the vicinity of the PSI complex (Hayakawa et al. 1985,
Ogawa et al. 1995). The hydrogen peroxide is rapidly reduced to water by ascorbate catalyzed with ascorbate perox-
2H 2 O
AIPZ
827
WEW
IPSH-MIDAE
H2O2
Fig. 7 Water-water cycle. The cycle consists of: (a) photooxidation of water in PSII, (b) Flavoenzyme, most likely MDAR,
catalyzed photoreduction of dioxygen in PSI, (c) superoxide
dismutase (SOD) catalyzed disproportionation of superoxide radical (Of), (d) ascorbate peroxidase (APX) catalyzed reduction of
hydrogen peroxide (H2O2) by ascorbate, (e) photoreduction of
monodehydroascorbate radical (MDA) to ascorbate (AsA) by Fd
in PSI. For every turnover of the cycle (transient formation of one
molecule of H2O2), at least 8 quanta are consumed, leading to the
transport of at least 16 H + from the stroma into the thylakoid
lumen (see also Schreiber et al. 1995).
idase (APX) bound to the PSI complex (Miyake and Asada
1992a). In the APX reaction, ascorbate is univalently oxidized to MDA, and the thus produced MDA is photoreduced to ascorbate by Fd localized at the acceptor side of
the PSI complex in thylakoid membranes (Miyake and
Asada 1992b, 1994, Asada 1996). Based on the molecular
activities and local concentrations of these components
on the stromal surface of thylakoid membranes in the
millimolar range, it has been simulated that superoxide radicals and hydrogen peroxide can be scavenged only within a
5- to 10-nm layer at the surface of thylakoid membranes
prior to its diffusion to the stroma (Asada 1996).
We have preliminarily estimated the apparent rate constant of the reduction of dioxygen at PSI catalyzed by
MDAR to be 100 s~', which is 2 to 4 orders of magnitude
lower than those of the scavenging reactions (Asada 1996).
Thus, the rate-limiting step of the water-water cycle is the
photoreduction of dioxygen, even if it is stimulated by
MDAR. The photoreduction of dioxygen to superoxide radical at PSI would be stimulated under conditions in which
the Calvin-Benson cycle cannot turn over and the ratio of
NADPH to NADP + would be high. In fact, the electron
flux associated with the water-water cycle increases under
high light and/or CO2 stress in intact chloroplasts (Hormann et al. 1994, Schreiber et al. 1995), H.incana (Os-
Photoproduction of superoxide by MDA reductase
828
mond and Grace 1995), Ficus insipida (Lovelock and Winter 1996) and Pisum sativum (Park et al. 1996). Further,
dissipation of excess photon energy through the waterwater cycle has been shown in drought-stressed wheat
(Biehler and Fock 1996), chilled-stressed maize (Fryer et al.
1998) and salt-stressed mangrove (Cheeseman et al. 1997).
Since the contribution of the water-water cycle is around
30% total electron flux and cannot be accounted for by the
photoreduction of dioxygen in thylakoids, the MDAR-mediated photoreduction of dioxygen in chloroplasts would
be stimulated under photon energy-excess environments.
The water-water cycle would have the following physiological functions. First, rapid scavenging of the potentially dangerous superoxide radicals and hydrogen peroxide
prior to their interaction with target molecules in the stroma (Asada 1996). Second, the control of the photoproduction ratio of ATP/NADPH, depending on the stromal reactions. Under anaerobic conditions, chloroplasts cannot
start photosynthesis because of initial shortage of ATP
required for the operation of the Calvin-Benson cycle
(Egneus et al. 1975, Radmer and Kok 1976). Third, the
down-regulation of PSII associated with the zlpH across
thylakoid membranes (Ruban et al. 1992, Gilmore et al.
1994, Krieger et al. 1992), a high value for which relies on
an effective water-water cycle (Schreiber and Neubauer
1990, Schreiber et al. 1995). The last mentioned, but probably most important, function of the water-water cycle leads
to the safe dissipation of excess photon energy. Without
protection by the water-water cycle, chloroplasts would
suffer damage from photoinhibition; the enhanced photoinhibition under anaerobic conditions has been repeatedly
reported after its first discovery by Trebst (1962).
The present work was supported by Grant-in-Aids for the International Cooperation Research and for Scientific Research on
Priority Areas (No. 04273101) from the Ministry of Education,
Science and Culture, Japan, and also by grants from the Human
Frontier Science Program and from the Alexander von Humboldt
Award to KA. U. Sch. acknowledges support by the Deutsche
Forschungsgemeinschaft (SFB 176).
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(Received February 24, 1998; Accepted May 19, 1998)