NADPH and ferredoxin:NADP+ oxidoreductase-dependent reduction of quinones and their reoxidation

Article abstract of DOI:10.1016/S0031-9422(98)00540-8

Molecular oxygen uptake was initiated by adding NADPH (1 mM) to the buffered medium containing 0.6 μM spinach ferredoxin:NADP⁺ oxidoreductase and 20 μM quinone (plastoquinone-2, decyl-plastoquinone, decyl-ubiquinone, or duroquinone). At pH 7.7 the rate of oxygen uptake was 2- to 12-fold higher during an initial phase (V₁) than in a subsequent phase (V₂). Except for duroquinone, the initial rate of oxygen consumption was ca. 2.7-fold higher in alkaline than in acidic medium. Ferredoxin was not essential, although it stimulated the reaction investigated. Oxygen uptake was not detectable with plastoquinone-9 or α-tocoquinone. The possible mechanisms of the NADPH and ferredoxin:NADP oxidoreductase dependent reduction of some quinones and their reoxidation are discussed.

Full text of DOI:10.1016/S0031-9422(98)00540-8

PERGAMON
Phytochemistry 50 (1999) 203±208
NADPH and ferredoxin:NADP + oxidoreductase-dependent
reduction of quinones and their reoxidation
Monika Bojko, Stanislaw Wieckowski *
Department of Physiology and Biochemistry of Plants, The J. Zurzycki Institute of Molecular Biology, Jagiellonian University, al. Mickiewicza 3,
31-120 Krakow, Poland
Revised 22 July 1998
Abstract
Molecular oxygen uptake was initiated by adding NADPH (1 mM) to the bu€ered medium containing 0.6 mM spinach
ferredoxin:NADP + oxidoreductase and 20 mM quinone (plastoquinone-2, decyl-plastoquinone, decyl-ubiquinone, or
duroquinone). At pH 7.7 the rate of oxygen uptake was 2- to 12-fold higher during an initial phase (V1) than in a subsequent
phase (V2). Except for duroquinone, the initial rate of oxygen consumption was ca. 2.7-fold higher in alkaline than in acidic
medium. Ferredoxin was not essential, although it stimulated the reaction investigated. Oxygen uptake was not detectable with
plastoquinone-9 or a-tocoquinone. The possible mechanisms of the NADPH and ferredoxin:NADP oxidoreductase dependent
reduction of some quinones and their reoxidation are discussed. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Decyl-plastoquinone; Decyl-ubiquinone; Diaphorase activity; Dibromothymoquinone; Duroquinone; Ferredoxin:NADP+ oxidoreduc-
tase; Oxygen uptake; Plastoquinone-2; Plastoquinone-9; a-Tocoquinone
1. Introduction
whether diaphorase activity of FNR plays any role in
vivo in the oxidation of NADPH. Some evidence has
also been published for participation of this enzyme in
cyclic electron transport around photosystem 1 (Moss
& Bendall, 1984; Hosler & Yocum, 1985). However,
the mechanism of FNR-catalysed step(s) of cyclic elec-
tron transport in chloroplasts of higher plants is still
under debate (Bendall & Monasse, 1995; Arakaki et
al., 1997). One could assume that FNR mediates elec-
tron ¯ow from reduced Fd to other electron acceptors,
like cytochrome b6/f complex and/or plastoquinone
pool (Bendall & Monasse, 1995). We have previously
found (Bojko & Wieckowski, 1995) that dibromothy-
moquinone (2,5-dibromo-3-methyl-6-isopropyl-p-ben-
zoquinone, DBMIB) is a good electron acceptor in the
diaphorase activity of FNR. This suggests that re-
duction of plastoquinone may be mediated by FNR.
In this study we attempted to answer the question
whether FNR can catalyse the NADPH-dependent re-
duction of plastoquinone and some other quinones
which di€er from one another in the side chains and
to a lesser extent in the reducible heads. We have also
Chloroplastic ferredoxin:NADP + oxidoreductase
(EC 1.18.1.2, FNR) is involved in the last step of the
electron transport chain catalysing NADP + reduction
by electrons derived from reduced ferredoxin (for
recent review see Arakaki, Ceccarelli, & Carrillo,
1997). It has also been well established that in vitro
this enzyme exhibits diaphorase activity that catalyses
NADPH-dependent reduction of ferricyanide, dichlor-
ophenol indophenol, tetrazolium salts (Zanetti
&
Forti, 1966; Forti & Sturani, 1968; Zanetti, 1981),
cytochrome c (Forti & Sturani, 1968), methyl viologen
(Bowyer, O'Neill, Camilleri, & Todd, 1988), non-
physiological quinones (Anusevicius, Martinez-Julvez,
Genzor, Nivinskas, Gomez-Moreno & Cenas, 1997)
and some other arti®cial electron acceptors (for review
see Carrillo & Vallejos, 1987). It can also be involved
in the NADPH-dependent reduction of NAD + or its
analogues (Boger, 1971). It is still an open question
Preliminary results were presented at the 10th FESPP Congress,
Florence, Italy (Plant Physiol. Biochem., Special issue, 1996, p. 99).
* Corresponding author. E-mail: wieckowski@mol.uj.edu.pl.
demonstrated (Bojko & Wieckowski, 1995) that
reduced DBMIB is reoxidized by molecular oxygen
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00540-8

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M. Bojko, S. Wieckowski / Phytochemistry 50 (1999) 203±208
Fig. 1. Typical record of NADPH (1 mM)-initiated oxygen uptake by the bu€ered (pH 7.7, 40 mM Tris±HCl) reaction mixture (1 cm3) contain-
ing 1 mM Na2EDTA, 1 mM MgCl2, 0.6 mM FNR and 20 mM duroquinone (or other quinones). V1, rate of oxygen uptake during the 1st minute
of measurement; V2, rate of oxygen uptake between 2nd and 3rd minutes of measurement.
and this process can be conveniently followed by
monitoring oxygen uptake.
the highest V2 value was with PQ-2 (0.013 mmol O2
per min). No relationship between V1 and V2 could be
established. The two phases of oxygen uptake may be
interpreted in the terms of sequences of quinone re-
ductions, e.g. ubiquinone-10 in protic medium may
occur in six states of oxidation and protonation
2. Results and discussion
(Morrison, Schelhorn, Cotton, Bering,
& Loach,
Adding NADPH to the bu€ered reaction mixture of
FNR and DBMIB caused oxygen uptake which pro-
ceeded in one or two phase kinetics (Bojko &
Wieckowski, 1995). Oxygen consumption was associ-
ated with the reoxidation of reduced quinone. Under
anaerobic conditions the pseudo-®rst-order rate con-
stant (k) and the Michaelis constant (Km) for
1982). Quinols interact rather slowly with molecular
oxygen (Kruk, Jemiola-Rzeminska, & Strzalka, 1997)
whereas mono- and di-valent anions or semiquinones
are very strong reductants (Sugioka et al., 1988). This
suggests that V1 is associated with the oxygen re-
duction by quinone anions and/or semiquinones,
whereas V2 may be connected with the oxygen re-
duction by quinols.
NADPH- and FNR-dependent DBMIB reduction
1
were 0.34 s
and 16 mM, respectively. The data pre-
The e€ects of pH on the NADPH dependent re-
duction of PQ-2 (Fig. 3) and decyl-PQ (Fig. 4) and
their reoxidation by molecular oxygen are comparable
with those observed for DBMIB (see also (Bojko &
Wieckowski, 1995)). In both cases the V1 values were
ca. 2.7-fold higher in pH 8.7 than in pH 6.7.
Ferredoxin (Fd) was not an essential component for
the reaction investigated, however, at pH 7.7 it stimu-
lated oxygen uptake about 1.6-fold with PQ-2 and 2.2-
fold with decyl-PQ. No clear dependence of V2 values
on the pH was found. On the other hand the pH's
6.7±8.7 had no e€ect on the rate of NADPH stimu-
lated oxygen uptake in the presence of duroquinone
sented in this paper clearly show that the uptake of
molecular oxygen was also induced by adding
NADPH to the reaction medium containing FNR and
PQ-2, decyl-PQ, decyl-UQ or duroquinone, but not to
that with PQ-9 or a-TQ. In most experiments these
rates were higher in an initial (V1) than in a sub-
sequent step (V2) (Fig. 1). Fig. 2 shows that under con-
ditions applied the highest V1 was supported by
duroquinone (0.058 mmol O2 per min) with decreasing
rates in the order PQ-2 (0.031 mmol O2 per min),
DBMIB (0.026 mmol O2 per min), decyl-UQ (0.013
mmol O2 per min) and decyl-PQ (0.007 mmol O2 per
min). V1 was 2- to 12-fold higher than V2 values and

M. Bojko, S. Wieckowski / Phytochemistry 50 (1999) 203±208
205
Fig. 2. NADPH (1 mM) initiated oxygen uptake (V1, V2 values) by the reaction mixture containing also 0.6 mM FNR and 20 mM quinone as
indicated. Other details as in Fig. 1. DBMIB, dibromothymoqinone; PQ-9, plastoquinone-9; decyl-PQ, decyl-plastoquinone; PQ-2, plastoqui-
none-2; a-TQ, a-tocoquinone; decyl-UQ, decyl-ubiquinone.
(Fig. 5). In this case the V1 values were high (about
0.055 mmol O2 per min) at all three pHs investigated
and V2 values were highest in alkaline pH. Ferredoxin
had no stimulatory e€ect on this process. The depen-
dence of oxygen uptake upon pH con®rms the assump-
tion that semiquinones or semiquinone anions are
involved eciently in O2 reduction because higher pH
(with a domination of unprotonated phenolic groups)
favored a greater speed of oxygen uptake as compared
with that in lower pHs (see also Sugioka et al., 1988).
Semiquinones should be rather stable in pH 7.7 and
8.7 because their pKa values are in the range 3.5±5.0
Fig. 3. E€ects of pH (6.7, 7.7, 8.7) or ferredoxin, Fd (0.6 mM, as indicated) on the NADPH (1 mM) initiated oxygen uptake in the reaction mix-
ture containing 0.6 mM FNR and 20 mM PQ-2. For the composition of the basic reaction mixture and other details see Fig. 1.

206
M. Bojko, S. Wieckowski / Phytochemistry 50 (1999) 203±208
Fig. 4. The e€ects of pH (6.7, 7.7, 8.7) or Fd (0.6 mM, as indicated) on the NADPH (1 mM) initiated oxygen uptake by the reaction mixture con-
taining 0.6 mM FNR and 20 mM decyl-PQ. For composition of the basic reaction mixture and other details see Figs. 1 and 2.
(Swallow, 1982) whereas those for many hydro-
quinones range from 8.0 to 13.3 (Morrison et al.,
1982). Higher activity of FNR in alkaline medium
(Batie & Kamin, 1981) should be also taken into
account. Independence of oxygen uptake in the pre-
sence of duroquinone on the pH suggests that this qui-
none is unprotonated in the pHs 6.7 and 8.7.
The FNR- and NADPH-dependent reduction of
PQ-9 and a-TQ were also not detectable under anaero-
bic conditions (data not shown). Since PQ-9 and a-TQ
di€er mainly from the other investigated quinones in
the structure of their non-reducible side chains, the
results obtained could be explained by assuming that
any binding site(s) on FNR molecules is not accessible
Fig. 5. E€ect of pH (6.7, 7.7, 8.7) or Fd (0.6 mM, as indicated) on the NADPH (1 mM) initiated oxygen uptake by the reaction mixture contain-
ing 0.6 mM FNR and 20 mM duroquinone. For composition of the basic reaction mixture and other details see Figs. 1 and 2.

M. Bojko, S. Wieckowski / Phytochemistry 50 (1999) 203±208
207
to the reducible groups of these two molecules. It is
3. Experimental
likely that the long side chains and high hydrophobi-
city prevent the interaction of PQ-9 and a-TQ mol-
ecules with FNR. It is known that PQ-9 exhibits
extremely low solubility in polar solvents compares
with the other quinones. In con®rmation of this we
did not observe for PQ-9 suspension a 2-nm shift of
the absorption peak towards the shorter wavelength,
but this shift was observed for the other quinones
investigated (data not shown). Probably PQ-9, like
Reagents: plastoquinone-2 and plastoquinone-9,
were gifts from Ho€man-La Roche, decyl-plastoqui-
none and decyl-ubiquinone were purchased from
Sigma and a-tocoquinone was from Merck.
Dibromothymoquinone was obtained from Aldrich.
The reagents for chromatography were purchased as
indicated previously (Bojko & Wieckowski, 1995).
Other reagents of the analytical grade came from the
Polskie Odczynniki Chemiczne, Poland.
Ferredoxin:NADP oxidoreductase and ferredoxin
were isolated from spinach purchased on the local
market by the modi®ed procedure described in Boger,
Black, and San Pietro (1966) and Apley, Wagner, and
Engelbrecht (1985) and their purities were con®rmed
by the Na-dodecyl sulphate polyacrylamide gel electro-
ubiquinone-10 (Lenaz, Espost,
& Castelli, 1982),
remained in our reaction medium in the form of
micelles resemble those of Triton x-100. The quinones,
other than PQ-9 and a-TQ, are partially soluble in
polar medium and it is likely that this enables them to
interact with active site(s) on FNR. The results
obtained con®rmed the suggestion of some authors
(Shahak, Crowther, & Hind, 1981; Ravenel, Peltier, &
Havaux, 1995) that under in vivo conditions FNR
may be involved directly in the plastoquinone pool re-
duction. It is likely that localization of PQ-9 molecules
in thylakoid membranes prevents its micellization and
favors its head interaction with FNR. The accessibility
of some quinones, including PQ-9, in unbroken thyla-
koid membranes to speci®c antibodies (Radunz &
Schmid, 1973; Schmid et al., 1996) con®rms this hy-
pothesis. Additional proteins, e.g. PSI-E subunit
(Nielsen, Andersen, & Scheller, 1995) and/or ndhB and
ndhJ gene products (Guedeney, Corneille, Cuine, &
Peltier, 1995) are thought to be associated with FNR
molecules and may also favor PQ-9 reduction in thyla-
koid membranes. The life times for mono- and/or
divalent PQ-9 anions, if they occur in vivo, should be
very short because ecient oxygen uptake associated
with PQ-9 autooxidation has not been revealed. One
can consider that the quinone binding site on FNR
corresponds to that for 2,6-dichlorophenol indophenol
(DCPIP) (Lenaz et al., 1982) and it di€ers from high
anities Fd or NADP+ (NADPH) binding sites since
eleven monoclonal antibodies raised against spinach
FNR blocked speci®cally only the DCPIP reduction
(Chang, Morrow, Hirasawa, & Kna€, 1991). The
NADP+ and Fd binding sites have been well de®ned
in the 1.7 A resolution structure (Bruns & Karplus,
1995) whereas that for DCPIP has not yet been recog-
nized in crystallographic studies. If we assume that the
DCPIP (plastoquinone?) binding site is also accessible
to electrons from Fdred no hypothetical ferredoxin:-
plastoquinone reductase (Bendall & Monasse, 1995) is
needed for explanation of the mechanism of plastoqui-
none pool reduction in cyclic electron ¯ow around
photosystem 1 (for review see also Wieckowski &
Bojko, 1997).
phoresis. The A495/A276 ratio for FNR and the A420
A276 ratio for Fd were 0.18 and 0.28, respectively.
/
FNR and Fd concns were calculated using extinction
1
1
coecients 9.8 mM
1
cm
at 420 nm and 11.3
at 459 nm, respectively. For other details
1
mM
see Bojko and Wieckowski (1995).
cm
The quinones were added to the reaction mixture as
MeOH soln and their concn determined spectrophoto-
metrically using molar absorption coecients given in
Kruk, Strzalka, and Leblanc (1992). The ®nal conc. of
MeOH in the reaction mixture did not exceed 2%.
The O2 uptake was monitored at 258C by a Clark-
type O2 electrode (Hansatech., UK) connected with
the TZ 4100 Line Recorder (Praha, The Czech
Republic).
The absorbance measurements were carried out by
the LSM/Aminco DW 2000 Spectrophotometer
(USA).
Acknowledgements
This study was ®nancially supported by the
Committee for Scienti®c Researches (KBN), grant No.
6P04A 049 08. The authors are grateful to Dr.
Grazyna Majewska for assistance in the isolation of
FNR and Fd.
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