Vol.2, No.1, 1-9 (2011)
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
Journal of Biophysical Chemistry
Advances in understanding of the primary reactions of
protochlorophyll(ide) photoreduction in cells and model
Olga B. Belyaeva, Felix F. Litvin
Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia; belyaeva0104@gmail.com
Received 6 November 2010; revised 15 November; accepted 19 November 2010.
The key step in chlorophyll biosynthesis is
photoreduction of its immediate precursor, pro-
tochlorophyllide. This reaction is catalyzed by a
photoenzyme, protochlorophyllide oxidoreduc-
tase (POR) and consists in the attachment of
two hydrogen atoms in positions C17 and C18
of the tetrapyrrole molecule of protochlorophyl-
lide; the double bond is replaced with the single
bond. Two hydrogen donors involved in pro-
tochlorophyllide photoreduction are NADPH [1,2]
and a conserved tyrosine residue Tyr193 of the
photoenzyme POR [3]. The structure of active
pigment-enzyme complex (Pchlide-POR-NADPH)
ensures a favorable steric conditions for the
transfer of hydride ion and proton. This review
does not examine the ternary complex structure,
but concentrates upon the mechanisms of pri-
mary photophysical and photochemical reac-
tions during formation of chlorophyllide from
protochlorophyllide in living objects (etiolated
leaves and leaf homogenates) and model sys-
Keywords: Protochlorophyllide; Chlorophyllide;
Photoreduction; Fluorescence; Spectroscopy
1.1. Labile Intermediates Stabilized at
Low Temperatures
Analysis of protochlorophyllide photoreduction in
vivo at very low temperatures, at which biochemical
temperature-dependent steps are inhibited, helped to
clarify the mechanism of this reaction. Rubin et al. [4]
reported that the fluorescence lifetime in etiolated leaves
was found to decreased upon elevation of sample tem-
perature from 77 K to 193 K. The further temperature
increase in darkness led to the appearance of fluores-
cence bands characteristic of chlorophyll. The authors
supposed that the absorption of fluorescence-exciting
light at low temperatures converted the protochlorophyl-
lide molecule into the intermediary state, which was
transformed to chlorophyllide during subsequent dark
reaction allowed to proceed at higher temperature. This
assumption was confirmed in later studies [5,6]. By il-
luminating leaves at 153 K, Raskin [7] successfully sta-
bilized the intermediate product of photochemical reac-
tion and could determine the spectral position of its ab-
sorption band (near 690 nm). Later, a similar position of
absorption maximum for the primary intermediate was
detected at room temperature [8,9]. The nonfluorescent
intermediate stabilized at low temperatures (intermediate
X690) was investigated in many research groups [10-19].
It was found that the primary photoreaction could pro-
ceed even at the temperature of liquid helium (4.2 K)
[20]. This finding revealed the elementary photophysical
nature of the process accompanied by quenching of pro-
tochlorophyllide fluorescence.
In the dark the nonfluorescent intermediate X690
transforms into chlorophyllide upon the increase in tem-
Pchlide655650 Chlide
1.2. Occurrence of SeveralShort-Lived
NonFluorescent Intermediates
Studies of primary events in protochlorophyllide
photoreduction at very low temperatures led to the con-
clusion that formation of intermediate X690 is preceded
by even earlier photochemical reactions. Evidence to
support this notion came from comparison of spectral
changes induced by illumination of etiolated leaves at
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
various temperatures [15,20-22].
Fluorescence and absorption spectroscopy studies
have shown the reversibility of the primary reaction that
converts protochlorophyllide into the nonfluorescent
intermediate [16]. Calculations of the rate constants and
quantum yields of the forward and backward photoreac-
tions under photostationary equilibrium showed that the
low quantum efficiency of the whole process could not
be due to photoreversibility of X690-producing reaction
because the quantum yield of the backward process is
significantly lower (by a factor of 20). Since the inter-
mediate X690 is stable in darkness, the authors assumed
that X690 formation is preceded by one additional in-
termediary step that comprises a fast backward reaction
responsible for the lowered yield of the whole process.
The putative intermediate was designated by the symbol
R (reversible):
Pchlide655650 Chlide
⎯⎯→→ →R X690
The above experimental data gave clue to explanation
why the quantum yield of the reaction at low tempera-
ture is lower than the quantum yield of the process at
room temperature when it is close to 0.5. The rate con-
stants of forward processes increase faster upon raising
the temperature than those of backward reactions, which
elevates the total yield of chlorophyllide formation.
Analysis of spectral changes led to the suggestion that
the absorption spectrum of the primary intermediate R,
whose production dominates at 77 K, is close or even
identical to the absorption spectrum of the protochloro-
phyllide active form: the primary action of light on etio-
lated leaves, evident in quenching of protochlorophyllide
fluorescence, occurs under conditions when X690 is
hardly discernible in absorption spectra [21].
Further evidence that the chain of protochlorophyllide
transformations includes the short-lived intermediate
with the absorption band similar to that of protochloro-
phyllide active form emerged from the time-resolved
spectroscopy studies performed at room temperature
We examined the early stages of protochlorophyllide
photoreduction by comparing low-temperature (77 K)
optical spectra (absorption and fluorescence) and ESR
spectra of whole etiolated leaves [21]. After illumination
of leaves at 77 K, when the production of nonfluorescent
intermediate R is predominant, a structureless singlet
ESR signal was observed with a bandwidth of 1.1 mT
and a g-factor of 2.0021 characteristic of the free elec-
tron. As the sample temperature was raised gradually to
200 K, the ESR signal amplitude increased in parallel
with narrowing of the signal width. Such a pattern of
ESR signal changes apparently corresponds to the trans-
formation of the primary intermediate R into the inter-
mediate X690. Upon further increase in temperature
above 200 K, the ESR signal amplitude dropped abruptly,
reaching its initial dark level at the temperature of about
250 K. These changes occurred synchronously with the
appearance of fluorescence spectral bands assigned to
primary forms of chlorophyllide.
Studies of the primary steps of protochlorophyllide
photoreduction in etiolated leaves by means of differen-
tial and derivative absorption spectroscopy [24] provided
evidence for even higher complexity of the photoprocess.
The intermediate X690 was found to exist in two forms
distinguished by absorption maxima at 697 and 688 nm
and by different rates of their production. These inter-
mediates were designated as R697 and R688. After
short-term illumination of leaves at 77 K, the subtracted
“light minus dark” spectrum contained a weak band with
the maximum at 697 nm (intermediate R697) while the
amplitude of the protochlorophyllide band (650 nm)
remained unchanged. During prolonged illumination, a
band at 688 nm appeared in the subtracted spectrum in
addition to the former band at 697 nm. At the same time,
the decrease in the absorption band of active pro-
tochlorophyllide form at 650 nm was observed, indicat-
ing that the protochlorophyllide molecule underwent at
this stage more profound changes than at the stage of
intermediate R697 formation, when the absorption at
650 nm remained almost unchanged. According to ESR
spectra, both the intermediates R697 and R688 exhibit
paramagnetic properties. The dark stage of chlorophyl-
lide formation during temperature increase was only
observed after accumulation of intermediate R697 in
combination with R688. A proposal was put forward that
these two intermediates are the products of two parallel
photoreactions proceeding at different rates; the tem-
perature increase promotes interaction of their chromo-
phores, giving rise to chlorophyllide molecule through
disproportionation of free radicals. The mechanism of
chlorophyllide formation by means of free radical dis-
proportionation was earlier proposed by Losev and
Lyalkova [14].
Summing up this section, we conclude that photore-
duction of protochlorophyllide in vivo comprises at least
two elementary reactions which can be detected by
spectral methods at very low temperatures required for
stabilization of nonfluorescent intermediates arising as
the products of these photoreactions:
Pchlide655650R650 X690R697 R688
⎯⎯→− →+
Since the intensity of the absorption band for the inter-
mediate R697 is very low (it is only observed in differen-
tial spectra), a possibility cannot be ruled out that this
primary product is identical to the intermediate R-/650.
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
otochlorophyllide fluores-
s of pro-
n alternative approach to detection of labile sho
ed intermediates involved in protochlorophyllide photo-
reduction was based on time-resolved spectroscopy em-
ployed in studies of chlorophyll photobiosynthesis in
etiolated leaves. The application of these methods al-
lowed researchers to detect short-lived intermediates at
physiological temperatures.
The kinetic studies of pr
nce and fluorescence changes of chlorophyllide pro-
duced in etiolated leaves after illumination by short (0.3
ms) flashes at room temperature revealed that the
chlorophyllide fluorescence intensity increased toward
maximum in 400 ms, whereas the protochlorophyllide
fluorescence dropped to the minimum within less than 8
ms [25]. The discrepancy between stabilization times of
chlorophyllide and protochlorophyllide fluorescence
intensities indicated the production of a nonfluorescent
intermediate, which was converted during the dark step
to the fluorescent form of chlorophyllide. The applica-
tion of even shorter light pulses (20 ns) and a sensitive
technique for scanning the absorption spectra of etio-
lated leaf homogenates at room temperature [8] revealed
the appearance within 0.5 µs after the flash of an unsta-
ble product with the absorption maximum around 695
nm, which transformed within few microseconds into
chlorophyllide with the absorption maximum near 675
nm. Similar results were also obtained in studies of rapid
changes in absorption spectra of etiolated leaves and
isolated pigment-protein complexes [9]. Inoue et al.
discovered the primary short-lived intermediate (0.2 μs)
with the absorption maximum at 690 nm.
Iwai et al. [23] investigated the primary step
chlorophyllide photoreduction using active pigment-
protein complexes isolated from etiolated leaves and the
nanosecond- and picosecond absorption spectroscopy. At
physiological temperature on the nanosecond and mi-
crosecond time scale, they observed the appearance of
three intermediates (X1,X2,X3) with characteristic time
constants of 2 ns, 35-250 ns, and 1-2 μs, which was fol-
lowed by chlorophyllide formation (12 μs). The absorp-
tion peak of X1 is close to the absorption maximum of
the initial protochlorophyllide ( 640 nm). The interme-
diates X2 and X3 are characterized by the absorption
maxima at 688 and 684 nm, respectively. Using flash
photolysis (a 30-ps flash), these authors showed the ex-
istence of even earlier intermediate, termed X0, which
appeared at room temperature within the period of 50
ps from the protochlorophyllide excitation and had the
lifetime of about 1-2 ns (compatible with the results
from other group [26]). The position of absorption
maximum of the primary product X0 was almost coinci-
dent with the band position of the initial protochloro-
phyllide. The time constant of X0 formation corre-
sponded to the relaxation time of the protochlorophyll
molecule from the Franck-Condon state (S1*) to the
equilibrium state (S1); hence the X0 entity is not an in-
termediate in a chemical sense. The following scheme
was proposed for the primary events of protochlorophyl-
lide photoreduction in active pigment-protein complexes:
h 50ps
Pchlde SPchlde S*Pchlde S
⎯⎯→ ⎯⎯⎯
12 ns35250 ns12s12s
xxx Chlde
−− −
⎯⎯⎯→ ⎯⎯⎯⎯→⎯⎯⎯→⎯⎯⎯
The authors supposed that the intermediate X3 corr
e comparative analysis of the research results
TX690, stabilized ef-
onds to the nonfluorescent intermediate X690 ob-
served in etiolated leaves after illumination at low tem-
peratures. However, according to other authors [27,28],
the intermediate X690 is more likely to correspond with
the intermediate X2 observed by Iwai et al. This view is
based on the absorption spectrum of this intermediate
(maximum at 688 nm) and on its rise time (35-250 ns),
which is comparable to the formation time of intermedi-
ate X690 determined by other researchers [8,29]. The
intermediate X1 [23] is likely identical to the intermedi-
ate R discovered in our work [16, 24], as evidenced from
similarity of its absorption spectrum to the absorption
spectrum of the initial protochlorophyllide. The interme-
diate X3 can be compared with one of the primary forms
of chlorophyllide arising in etiolated leaves after preil-
lumination at 77 K and subsequent return to higher tem-
Thus, th
ncerning intermediate stages of protochlorophyllide
photoreduction in vivo at physiological and low tem-
peratures leads to the conclusion that this process com-
prises several intermediary products, including two or
three short-lived intermediates characterized by strong
quenching of protochlorophyllide fluorescence.
he nonfluorescent intermediate
ctively at low temperatures, transforms into chloro-
phyllide in dark reactions after raising the temperature of
preilluminated sample. When white light was used for
illumination of etiolated leaves at 77 K, the return to
higher temperature was followed by almost simultaneous
formation of two primary forms of chlorophyllide with
fluorescence maxima at 684 and 695 nm and the respec-
tive absorption maxima at 676 and 684 nm [15]. There-
after, the long-wavelength form Chlide695/684 was
converted in the dark reaction to a short-wavelength
form Chlide684/676. This pigment is presumably the
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
chlorophyllide form, which is a starting structure in the
biosynthetic pathway giving rise to the reaction center
pigments of photosystem II and to the pigments of
light-harvesting complex (reviewed in [30]). The pro-
portion of 695 and 684 nm fluorescence bands arising
after illumination at low temperature depended on spec-
tral quality of actinic light. When blue light (with the
peak intensity at 470 nm) was used for illumination, the
increase in temperature resulted in formation of the only
band at 695 nm, which shifted gradually to 684 nm.
When the leaves were preilluminated with red light (with
wavelengths above 600 nm), the temperature rise was
followed by the appearance in the spectrum of only one
short-wavelength maximum at 684 nm. In etioplast
preparations only one short-wavelength chlorophyllide
form was produced. These data implied that etiolated
leaves contain two kinds of active protochlorophyl-
lide–protein complexes exhibiting nearly identical ab-
sorption and fluorescence bands in the red spectral re-
gion [15]. Both forms are converted to chlorophyllide
through the stage of formation of nonfluorescent inter-
mediate. One of these precursors is able to transform
immediately into a short-wavelength chlorophyllide
form Chlide684/676. The reaction scheme was presented
as follows:
Pchld655650 R 690 Chld695684
Pchld655650 R 690 Chld695684
⎯⎯→→ →
→→ →→
Later studies demonstrated the existence in young
insight into the earliest stages
d pro-
es of concentrated protochlorophyll so-
iolated leaves of an additional active protochlorophyl-
lide form Pchlide653/648 [30,31] possessing a blue-
shifted absorption band in the Soret region (440 nm), as
compared to the band position for the main active form
Pchlide655/650 (450 nm). Pchlide653/648 is produced
under the action of red light from the minor
long-wavelength form of the precursor Pchlide686/676
[31]. Furthermore, we cannot exclude the possibility that
one of the two reactions consists in the conversion of a
short-wavelength active precursor Pchlide 643/637,
whose fluorescence is quenched owing to highly effec-
tive energy transfer toward the main active form.
Belyaeva and Sundqvist [32] revealed the for
four primary fluorescent pigment forms. When etio-
lated leaves preilluminated at 77 K were heated to 190 K,
a broad structured band appeared in the red region of
their fluorescence spectrum. The decomposition of the
spectra to the Gaussian components proved that this
band consists of four components with maxima at 683,
690, 696, and 706 nm. Apparently, the formation of sev-
eral primary labile chlorophyllide forms points to the
early onset for differentiation pathways involved in for-
mation of functionally different native pigment forms.
tudies of protochlorophyll reduction in solutions
ovided another approach to elucidating the mechanism
of primary steps in chlorophyll biosynthesis from its
precursor and the role of biological structures in this
process. This line of research enables identification of
spectral changes related to the conversions of the chro-
mophore molecule per se.
In order to gain deeper
protochlorophyll(ide) reduction, based on the previous
studies with whole leaves, we examined this process in
solutions and pigment films in cooperation with Bystrova
and Timofeev [21,34]. The experimental conditions were
maximally similar to those proved successful for detect-
ing the primary stages of protochlorophyllide reduction
in plant leaves (high intensity short-term illumination at
77 K). We found out that the primary photophysical
stages of protochlorophyllide reduction could occur in
solutions and films in the absence of reducing agents. In
this case the role of hydrogen donor belongs apparently
to the solvent (ethanol, ethyl ether, and pyridine).
When diluted solutions of protochlorophyll an
chlorophyllide in ethyl ether (10–6 M) were illuminated
at 77 K, the fluorescence at 626 nm attributed to the
monomeric pigment form was quenched, but this
quenching was fully reversed upon the subsequent ele-
vation of temperature to 273 K. No changes in the ab-
sorption spectrum were detected [34]. The quenching of
protochlorophyll fluorescence occurred synchronously
with the appearance in the ESR spectrum of the singlet
signal with the g-factor of 2.0013 and the singlet band
width of 1.2 mT; this signal disappeared rapidly upon the
increase in temperature in parallel with the buildup of
protochlorophyllide fluorescence [21]. The addition of
reducing agents resulted in a severalfold acceleration of
fluorescence quenching. The comparison with the photo-
reduction of chlorophyll precursor in whole etiolated
leaves suggests that the model systems with monomeric
protochlorophyllide could mimick the earliest stage of
the pigment photoreduction producing the paramagnetic
intermediate R.
Spectral featur
tions in ethanol (10–4 M) imitated quite well the ab-
sorption and fluorescence spectra of three main forms of
chlorophyll precursor: the spectra of these solutions
contained absorption bands at 625, 635 and 645-7 nm
and fluorescence bands at 627, 637, and 651-2 nm.
Measurements of circular dichroism spectra for pro-
tochlorophyll in model systems [45,46] indicate that the
spectral band at 651-652 nm, in the farthest long-wave-
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
tary reactions of pigment photoreduction
Ia growing number of researchers
arly to observations with whole etiolated leaves,
protochlorophyllide photoreduction in reconsti-
Ition [15] we proposed that the cell
length position, is determined by the formation of pig-
ment dimers. Illumination of concentrated ethanolic so-
lution at 77 K led to quenching of all fluorescence bands
[21,34]. Fluorescence quenching developed synchro-
nously with the appearance in low-temperature absorp-
tion spectrum of a weak maximum around 690 nm,
which is close to the position of absorption peak for
nonfluorescent intermediate X690 discovered in plant
leaves. In the ESR spectrum, a singlet signal with the
g-factor of 2.0015 and bandwidth of 1.1 mT appeared
upon illumination (the signal parameters were almost
identical to those induced by illumination in etiolated
leaves). Hence, the aggregated protochlorophyll in model
systems, unlike the monomeric pigment, undergoes more
profound transformation including the formation of non-
fluorescent intermediate X690. Apparently, this stage of
the process involves the aggregated long-wavelength
pigment form.
Thus, elemen
curring in the plant cell can be reproduced in pro-
tochlorophyll solutions as a model system
n the last few years
rned to studying protochlorophyllide photoreduction
with the use of artificial ternary complexes (consisting of
protochlorophyllide, NADPH, and photoenzyme pro-
tochlorophyllide oxidoreductase), because the properties
of such complexes are most close to those of living sys-
tems. The main active protochlorophyllide form in such
systems is commonly represented by the form whose
spectral properties are similar to those of protochloro-
phyllide active form in vivo, Pchlide 643/637 [17,18,38].
However, the reconstituted ternary complexes obtained
in our joint studies with Griffiths were characterized by
spectra with fluorescence bands at 651 and 708 nm. The
position of the first band coincides with the maximum of
protochlorophyllide fluorescence in whole cells, while
the second band was ascribed to a small number of large
aggregates serving as a sink for effective energy transfer
e protochlorophyllide photoreduction in artificial ter-
nary complexes was found to produce unstable nonfluo-
rescent intermediate as a primary product arising after
illumination of samples at low temperatures [17,18,37,
38]. The absorption band of this product had a peak at
696 nm [18,38]. The quenching of fluorescence was par-
alleled by the appearance in the ESR spectrum of a
singlet signal with the g-factor characteristic of the free
electron [37]. Upon the increase in temperature, the non-
fluorescent intermediate was converted into chlorophyl-
lide. The production of chlorophyllide after the tem-
perature rise was evident from the appearance of char-
acteristic bands in spectra of methanolic extracts [37].
Heyes et al. [19] examined the protochlorophyllide
otoreduction in reconstituted ternary complexes at
room temperature using femtosecond spectroscopy
(50-fs laser flash with a wavelength of 475 nm). Under
these conditions, a small increase in absorption of the
initial protochlorophyllide (maximum at 642 nm) was
observed in the time range between 3 and 400 ps after
the flash, which was accompanied by a slight shift of the
peak position to the short-wavelength spectral region. At
the same time, a weak maximum around 677 nm emerged.
These results are consistent with investigations of the
living systems: in both cases the primary reaction was
characterized by the lack of protochlorophyllide bleach-
ing (in parallel with effective fluorescence quenching)
ted ternary complexes is implemented in at least two
stages: the light-induced formation of a short-lived non-
fluorescent intermediate characterized by weak ESR
signal with the free electron g-factor, followed by the
dark transformation of this intermediate into chlorophyl-
lide. The pattern of spectral changes is close to that ob-
served in etiolated leaves.
n our early pubica
imary reactions of protochlorophyllide photoreduction,
characterized by the production of paramagnetic inter-
mediates, proceed via consecutive transfer of two elec-
trons and two protons toward the semi-isolated double
bond C17 = C18 in the protochlorophyllide molecule,
resulting in the appearance of intermediary semireduced
form, in essential similarity to a stepwise reduction of
the double bond described for the photoreaction of por-
phyrin reduction in solutions. When the role of NADPH
as a hydrogen donor was established [1,2], an assump-
tion was put forward that the sequential transfer of two
electrons is unlikely because of the high energy of
nicotinamide radicals. Since the photoenzyme POR is
structurally analogous to short-chain alcohol dehydro-
genases, it seemed more probable that the protochloro-
phyllide photoreduction involves the transfer of hydride
ion from NADPH in essential similarity to the mecha-
nism ensuring substrate reduction by alcohol dehydro-
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
s, mani-
Raskin and Schwartz considering the investigation [23]
genases. When NMR spectroscopy was applied for
analysis of protochlorophyllide photoreduction in reac-
tion mixtures containing protochlorophyllide, etioplast
membranes, and NADPH isomers labeled with radioac-
tive hydrogen (3H), the results revealed that the hydride
ion delivered from the 4S position of nicotinamide moi-
ety of NADPH is attached to the C17 position of pro-
tochlorophyllide, thus performing trans-reduction of the
pigment molecule [39,40]. The notion that hydride ion
and proton transfer are time-separated events during
photoreduction of protochlorophyllide molecule is sup-
ported by the existence of several short-lived intermedi-
ates. The second proton is apparently donated by POR
from the conserved amino acid residue Tyr-193 located
in the vicinity of C17 = C18 bond [3,41,42]. According
to the homology model of POR enzyme [43], the D-ring
of protochlorophyllide molecule is fixed against
NADPH and the tyrosine residue, which facilitates the
transfer of hydride ion and proton. The conserved resi-
dues Lys197 and Cys226 play an important role in con-
figuring optimal structure of the ternary complex, thus
enabling adequate steric requirements for the reaction of
protochlorophyllide photoreduction [3,41-44]. The pro-
ton of tyrosine phenol group arrives at C18 atom of pro-
tochlorophyllide molecule. In reconstituted ternary com-
plexes comprising mutant POR where Tyr-193 was re-
placed with Phe, the reduction of protochlorophyllide
ceased at the stage of intermediary photoproduct, while
the second (light-independent) stage of the reaction was
inactivated [41]. The results indicate that the transfer of
hydride ion occurs prior to the proton transfer.
Paramagnetism of electron donors or acceptor
sted in the light-induced ESR signal is one of the most
convincing evidence that the charge-transfer complex
(CT complex) is formed in the donor-acceptor system.
The formation of charge-transfer complex is accompa-
nied by the appearance of new absorption band on the
condition that the oxidation-reduction potentials of in-
teracting partners are sufficient for completing the elec-
tron transfer from one to the other molecule within the
complex. When the CT complex formation is due to par-
tial charge transfer, the fluorescence quenching is ob-
served without the appearance of new bands in the ab-
sorption spectrum. The possibility that photoreduction of
protochlorophyllide in vivo proceeds through the stage
of charge-transfer complex was considered in a series of
studies [7,14,22,27,28]. Judging from spectral character-
istics of two primary intermediates in vivo (termed as R
and X690 in our publications [16,21,22] or X1 and X2
according to [23]), one may suppose that photoinduced
formation of these intermediates corresponds to the for-
mation of complexes with partial charge transfer [Dδ+
Aδ] and with complete charge transfer [D+ A] [22,25,28]:
h H
++ +
() ()
oposed that a comparatively long lifetime of the in-
termediate X1 (35-250 ns) indicates the existence at this
stage of two intermediates: the singlet exciton complex
(exiplex) and the triplet exiplex of protochlorophyllide
and the hydrogen donor. Examination of low-
temperature phosphorescence spectra of whole etiolated
leaves also supports the viewpoint that illumination of
the leaves at 77 K (the stage of formation of nonfluores-
cent intermediate) produces not only singlet exiplexes
but also triplet exiplexes of protochlorophyllide and the
hydrogen donor, because fluorescence quenching of ac-
tive protochlorophyllide form under these conditions
was concomitant with quenching of its phosphorescence
[45]. In our opinion, further experiments are needed to
answer the question about the role of triplet state of the
protochlorophyllide molecule in its photoreduction.
Heyes et al. [38] attempted to elucidate the photo-
ysical mechanism of protochlorophyllide reduction in
the ternary complexes by comparative study of this reac-
tion at 180 K using absorption spectroscopy, ESR spec-
troscopy, ENDOR spectroscopy, and Stark spectroscopy.
In illuminated samples the authors detected ESR spectra
suggesting the emergence of two paramagnetic products.
However, quantitative estimates based on ESR spectra
indicated that essentially complete transformation of the
active protochlorophyllide form (as estimated from
changes in absorption spectra) resulted in only 5% out-
put of the pigment free radicals. The appearance of non-
fluorescent intermediate with the absorption band at 696
nm corresponded to the broadband Stark effect charac-
teristic of charge transfer steps. The results obtained with
Stark spectroscopy provided evidence for the existence
of two constituents of the nonfluorescent intermediate.
This enabled the authors to suggest that the primary
stage of protochlorophyllide photoreduction is associ-
ated with the formation of charge-transfer complex. The
temperature dependences of the intermediate formation
and NADPH oxidation were identical. Therefore, the
authors supposed that the formation of nonfluorescent
intermediate involves the transfer of hydride ion for the
creation of charge-transfer complex. The authors as-
sumed that the photon absorption by the protochloro-
phyllide molecule leads to the temporary charge separa-
tion along the double C17 = C18 bond, which facilitates
the ultrafast transfer of hydride ion from NADPH to the
C17 atom of protochlorophyllide [46, 38]. The resulting
CT complex promotes the proton transfer toward the
C18 atom in the subsequent dark reaction.
Heyes et al. [47] studied temperature dependences and
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
ular pathways for pro-
flavins in the active pigment–protein
74) Source of reducing equivalent for
otopic effects of rate constants for two sequential reac-
tions - the hydride anion and proton transfer steps - and
analyzed data in the form of Eyring plots to obtain ther-
modynamic parameters for each step. The experimental
results and calculations based on the density functional
theory were consistent with a proton-tunneling, which
requires fast (sub-picosecond) promoting motions cou-
pled to the reaction coordinate.
Comparative studies of molec
chlorophyllide photoreduction in vivo in various model
systems allows the conclusion that the primary stages
characterized by fluorescence quenching and formation
of paramagnetic products are identical for whole etio-
lated leaves and all model systems examined, including
the most simple ones such as low-concentrated pigment
solutions. Nevertheless, some factors suggest that the
mechanism of protochlorophyllide photoreduction in
vivo is somewhat different from the mechanism of the
reaction in model systems. The configuration of active
complex in vivo seems to be more sophisticated than the
ternary complex reconstituted in vitro, comprising three
main components, i.e., Pchlide, NADPH, and POR. Spe-
cifically, this is indicated by different spectral character-
istics of artificial and natural active complexes. Etiolated
leaves contain several spectrally different active forms of
protochlorophyllide. The major forms include Pchlide
643/639, Pchlide655/650, and the form Pchlide653/648,
which is accumulated and metabolically converted in
juvenile leaves. There are also several minor long-
wavelength forms participating in the process of pro-
tochlorophyllide photoreduction [48]. The reconstituted
ternary active complexes usually comprise one or two
protochlorophyllide forms: Pchlide633/630 (the form
active only at room temperature) and Pchlide644/641,
which remains photoactive also at low temperatures. The
photoreduction of various protochlorophyllide forms in
vivo and in vitro may involve different mechanisms.
Evidence that protochlorophyllide photoreduction in
vivo is accomplished via more intricate mechanism is
evidenced by studies of this process in whole etiolated
leaves at low temperature [15,20], as well as by the re-
sults of time-resolved spectroscopy at physiological
temperatures [21]; all these studies demonstrated the
multistep nature of the reaction of protochlorophyllide
The presence of
mplex and their possible involvement in primary reac-
tions of protochlorophyllide photoreduction was sup-
posed in some studies [49-51]. Our investigations sup-
port this proposal, as we observed the drop of flavin
fluorescence at 520 nm after illumination of leaves at 77
K, which was reversed upon the return to higher tem-
perature [51]. The oxidation-reduction reactions between
nicotinamide and flavins are implemented by means of
hydride ion transfer [52], while many flavin-containing
enzymes participate in one-electron redox reactions. In
plant leaves flavins can act at the intermediate stage of
the reaction by acquiring hydride ion from NADPH and
donating electron to the pigment molecule. In this case
the reaction can include a stepwise transfer of two elec-
trons and two protons and the formation at the first step
of a semireduced pigment molecule with one attached
electron (intermediary semireduced form). This mecha-
nism is largely similar to that known for the reactions of
porphyrin photoreduction in solution. We cannot rule out
the existence of two mechanisms by which hydrogen
atoms are transferred to protochlorophyllide from
NADPH in whole etiolated leaves; i.e. , the direct transfer
of hydride ion from NADPH to C17 position in the pro-
tochlorophyllide molecule with the consequent attach-
ment of proton and (or) flavin-mediated electron transfer
from NADPH to the pigment. It is also possible that dif-
ferent mechanisms exist for the primary reactions of
spectrally distinct active forms of protochlorophyllide.
Thus, we arrive at the conclusion that the mechanism
the primary elementary photophysical reactions dur-
ing protochlorophyllide photoreduction is not yet com-
pletely elucidated. However, based on the results of nu-
merous investigations, it can be stated that the reduction
of active forms of the chlorophyll precursor is a multi-
step process comprising two or three short-lived inter-
mediates characterized by the ESR singlet signal; the
sequential conversions of these intermediates are ensured
by structurally sophisticated native enzyme-pigment com-
plex comprising protochlorophyllide, POR, and NADPH
as its main constituents. Owing to specific structure of
active ternary complex, favorable steric conditions are
enabled for the reaction of protochlorophyllide photore-
[1] Griffiths, W.T. (19
the in vitro synthesis of chlorophyll from protochloro-
phyll. FEBS Letters, 46, 301-304.
of the terminal
[2] Griffiths, W.T. (1975) Сharacterization
stages of chlorophyll(ide) synthesis in etioplast mem-
brane preparations. Biochemistry Journal, 152, 623-635.
[3] Wilks, H.M. and Timko M.P. (1995) A light-dependent
complexation system for analysis of NADPH: pro-
tochlorophyllide oxidoreductase identification and muta-
genesis of two conserved residues that are essential for
activity. Proceedings of the National Academy of the
Sciences of the USA, 92, 724-728.
[4] Rubin, A.B., Minchenkova, L.E., Krasnovsky, A.A. and
Tumerman, L.A. (1962) Investigation of protochloro-
phyllide fluorescence lifetime during greening of etio-
lated leaves. Biofizika, 7, 571-577.
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/JBPC/
.A.H. (1970) Fluores-[5] Goedheer, J.C. and Verhulsdonk, C
cence and phototransformation of protochlorophyll with
etiolated bean leaves from –196˚C to +20˚C. Biochemical
and Biophysical Research Communications, 39, 260-266.
[6] Sironval, C. and Kuyper, P. (1972) The reduction of pro-
A short-lived intermedi-
tochlorophyllide into chlorophyllide: IV. The nature of the
intermediate P688-676 species. Photosynthetica, 6, 254-275.
[7] Raskin, V.I. (1976) Mechanism of photoredution of pro-
tochlorophyllide in the intact etiolated leaves. Ve st i
Akademii Nauk BSSR, 5, 43-46.
[8] Franck, F. and Mathis, P. (1980)
ate in the photoenzimatic reduction of protochlorophyll
(ide) into chlorophyll(ide) at a physiological temperature.
Photochemistry and Photobiology, 32, 799-803.
[9] Inoue, Y., Kobayashi, T., Ogawa, T. and Shibata, K. (1981)
1977) The primary r
A short lived intermediate in the photoconversion of proto-
chlorophyllide to chlorophyllide a. Plant Cell Physiology,
22(2), 197-204.
[10] Dujardin, E. and Sironval, C. (eac-
tions in the protochlorophyll(ide) photoreduction. Plant
Science Letters, 10(1), 347- 353.
) Long-wavelength
bsorbing quen-
., Wilks, H.M. and Hunter, C.
[11] Dujardin, E. and Correia, M. (1979
absorbing pigment protein complexes as fluorescence
quenchers in etiolated leaves illuminated in liquid nitro-
gen. Photobiochemistry and Photobiophysics, 1, 25-32.
[12] Dujardin, E., Correia, M. and Sironval, C. (1981) Fluo
rescence quenching of protochlorophyllide, chlorophyl-
lide and chlorophyll in etiolated, greening and green
leaves. Proceedings 5th Internatioal Congress On Photo-
synthesis, Phyladelfia, 5, 21-29.
[13] Dujardin, E. (1984) The long-wavelength-a
chers formed during illumination of protochlorophyl-
lide-proteins. In: Sironval, C. and Brouers, M. Eds., Pro-
tochlorophyllide Reduction and Greening, Martinus Ni-
jhoff/Dr. W Junk Publisher, The Hague, 87-98.
[14] Losev, A.P. and Lyal´kova, N.D. (1979) Invest of
the primaries stages of protochlorophyllide photoreduc-
tion in the etiolated plants. Molecular Biology, 13, 837-844.
[15] Belyaeva, O.B. and Litvin, F.F. (1981) Primary reactions
of protochlorophyllide into chlorophyllide phototrans-
formation at 77 K. Photosynthetica, 15, 210-215.
[16] Litvin, F.F., Ignatov, N.V. and Belyaeva, O.B.
Photoreversibility of transformation of protochlorophyl-
lide into chlorophyllide. Photobiochemistry and Photo-
biophysics, 2, 233-237.
[17] Heyes, D.J., Ruban, A.VN.
(2002) Enzimology below 200 K: The kinetics and ther-
modinamics of the photochemistry catalyzed by pro-
tochlorophyllide oxidoreductase. Proceedings of the Na-
tional Academy of the Sciences of the USA, 99, 11145-
Hunter, C.N. (2003) Pro-[18] Heyes, D.J., Ruban, A.V. and
tochlorophyllide oxidoreductase: “Dark” reaction of a
light-driven enzyme. Biochemistry, 42(2), 523-528.
[19] Heyes, D.J., Hunter, C.N., van Stokkum, I.H.M., Gron-
delle, R. and Groot, M.L. (2003) Ultrafast enzymatic re-
action dynamics in protochlorophyllide oxidoreductase.
Nature Structural Biology, 10, 491-492.
[20] Belyaeva, O.B., Personova, E.R. and Litvin, F.F. (1983)
Photochemical reaction of chlorophyll biosynthesis at 4.2
K. Photosynthesis Research, 4(1), 81-85.
[21] Belyaeva, O.B., Timofeev K.N. and Litvin, F.F. (1988) The
primary reaction in the protochlorophyll(ide) photoreduc-
tion as investigated by optical and ESR-spectroscopy.
Photosynthesis Research, 15(3), 247-256.
doi:10.1007/BF00047 356
[22] Belyaeva, O.B. (2009) Light dependent chlorophyll bio-
. and Kobayashi, T. (1984)
. (1993)
synthesis. BINOM, Moscow.
[23] Iwai, J., Ikeuchi, M., Inoue, Y
Early processes of protochlorophyllide photoreduction as
measured by nanosecond and picosecond spectropho-
tometry. In: Sironval, C. and Brouers, M. Eds., Pro-
tochlorophyllide Reduction and Greening, Martinus Ni-
jhoff/Dr.W Junk Publisher, The Hague, 99-112.
[24] Ignatov, N.V., Belyaeva, O.B. and Litvin, F.F
Low temperature phototransformation of protochloro-
phyll(ide) in etiolated leaves. Photosynthesis Research,
38(2), 117-124.
d Sironval, C. (1980) Non-
[25] Frank, F., Dujardin E. an
fluorescent, short-lived intermediate in photoenzymatic
protochlorophyllide reduction at room temperature. Plant
Sciences Letters, 18, 375-380.
d van Grondelle, R.
1989) Photobiosynthesis
F., Sironval, C., Breton,
d Franck, F. (2000) Spectro-
[26] van Bochove, A.C., Griffiths, W.T. an
(1984) The primary reaction in the photoreduction of
protochlorophyllide. A nanosecond fluorescence study. In:
Sironval, C. and Brouers, M. Eds., Protochlorophyllide
Reduction and Greening, Martinus Nijhoff/Dr.W Junk
Publisher, The Hague, 113-125.
[27] Belyaeva, O.B. and Litvin, F.F. (
of chlorophyll. Moscow State University Press, Moscow.
[28] Raskin, V.I. and Schwartz, A. (2002) The charge-transfer
complex between protochlorophyllide and NADPH: An
intermediate in protochlorophyllide photoreduction. Phosin-
thesis Research, 74(2), 181-186.
[29] Dobek, A., Dujardin, E., Franck,
J. and Roux, E. (1981) The first events of protochloro-
phyll(ide) photoreduction investigated in etiolated leaves
by means of the fluorescence excited by short, 610 nm
laser flashes at room temperature. Photobiochemistry and
Photobiophysics, 2, 35-44.
[30] Schoefs, B., Bertrand, M. an
scopic properties of protochlorophyllide analyzed in situ
in the course of etiolation and in illuminated leaves.
Photochemistry and Photobiology, 72(1), 85-93.
[31] Ignatov, N.V. and Litvin, F.F. (2002) A new pathway of
chlorophyll biosynthesis from long-wavelength protochlor-
ophyllide Pchlide 686/676 in juvenile etiolated plants. Pho-
tosynthesis Research, 71(1), 195-207.
[32] Belyaeva, O.B. and Sundqvist, C. (1998) Comparative
investigation of the appearance of primary chlorophyllide
forms in etiolated leaves, prolamellar bodies and prothy-
lakoids. Photosynthesis Research, 55(1), 41-48.
O. B. Belyaeva et al. / Journal of Biophysical Chemistry 2 (2011) 1-9
Copyright © 2011 SciRes. http://www.scirp.org/journal/JBPC/Openly accessible at
[33] Belyaeva, O.B. and Litvin, F.F. (2009) Pathways of for-
.B., Bystrova, M.I., Safronova, I.A., Litvin,
vivo aggr
mation of pigment forms at the terminal photobiochemi-
cal stage of chlorophyll biosynthesis. Biochemistry, 74,
[34] Belyaeva, O
F.F. and Krasnovsky, A.A. (1985) Photoinduced reversi-
ble changes in protochlorophyll fluorescence in model
systems. Molecular Biology, 30, 933-938.
[35] Brouers, M. (1975) Optical properties of in e-
gates of protochlorophyllide in non-polar solvents. II.
Fluorescence polarization, delayed fluorescence and cir-
cular dichroism spectra. Photosynthetica, 9, 304-310.
[36] Böddi, B., Soos, J. and Lang, F. (1980) Protochloroph
forms with different molecular arrangements. Biochimica
et Biophysica Acta, 593(1), 158-165.
[37] Belyaeva, O.B., Griffiths, W.T., Kovalev, J.V., Timofeev,
, P., Rigby, S.E.J., Palacios, M.A.,
K.N. and Litvin, F.F. (2001) Participation of free radicals
in photoreduction of protochlorophyllide to chlorophyl-
lide in artificial pigment-protein complexes. Biochemis-
try, 66(2), 173-177.
[38] Heyes, D.J., Heathcote
Grondelle, R. and Hunter, C.N. (2006) The first catalytic
step of the light-driven enzyme protochlorophyllide oxi-
doreductase proceeds via a charge transfer complex. The
Journal of Biological Chemistry, 281(37), 26847-26853.
[39] Valera, V., Fung, M., Wessler, A. N. and Richards, W.R.
.1016/0006-291X(87) 9114 1-7
(1987) Synthesis of 4R- and 4S-tritium labeled NADPH
for the determination of the coenzyme sterespecificity of
NADPH: Protochlorophyllide oxidoreductase. Biochemical
and Biophysical Research Communications, 148(1), 515-
[40] Begley, J.R. and Young, M. (1989)
reductase. I. Determination of the regiochemistry and the
stereochemistry of the reduction of protochlorophyllide
to chlorophyllide. Journal of the American Chemical So-
ciety, 111, 3095-3096.
., McIvor, W. and Timko, [41] Lebedev, N., Karginova, OM.
(2001) Tyr275 and Lys279 stabilize NADPH within the
catalytic site of NADPH: Protochlorophyllide oxidore-
ductase and are involved in the formation of the enzyme
photoactive state. Biochemistry, 40(42), 12562-12574.
[42] Menon, B.R.K., Waltho, J.P., Scrutton, N.S. and Heyes,
D.J. (2009) Cryogenic and laser photoexitation studies
identify multiple roles for active site residues in the
light-driven enzyme protochlorophyllide oxidoreductase.
Journal of Biologycal Chemistry, 284(27), 18160-18166.
[43] Townley, H.E., Sessions, R.B., Clarke, A.R., Dafforn,
T.R. and Griffiths, W.T. (2001) Protochlorophyllide oxi-
doreductase: A homology model examined by sitedirected
mutagenesis. Proteins: Structure, Funktion, and Genetics,
Vo l . 44(3), 329-335.
, P.A., Hunter, C.N., Scrutton N.S. [44] Menon, B.R.K., Davison
and Heyes, D.J. (2010) Mutagenesis alters the catalytic
mechanism of the light-driven enzyme protochlorophyllide
oxidoreductase. Journal of Biologycal Chemistry, 285(3),
a, O.B., Kovalev, Yu.V.,
[45] Krasnovsky, A.A. Jr., Belyaev
Ignatov, N.V. and Litvin, F.F. (1999) Phosphorescence
of intermediates of the terminal photochemical stage of
chlorophyll biosynthesis. Biochemistry, 64(5), 703-708.
[46] Griffiths, W.T., McHugh, T. and Blankenship, R.E. (1996
The light intensity dependence of protochlorophyllide
photoreduction and its significance to the catalytic
mechanism of protochlorophyllide reductase. FEBS Let-
ters, 398, 235-238.
nd Scrutton, N.S.
hotoactive pig-
. (1988) Protochlorophyl-
[47] Heyes, D.J., Sakuma, M., Visser, S.P. a
(2009) Nuclear quantum tunneling in the light-activated
enzyme protochlorophyllide oxidoreductase. Journal of
biologibal chemistry, 284(6), 3762-3767.
[48] Belyaeva, O.B. and Litvin, F.F. (2007) P
ment-enzyme complexes of chlorophyll precursor. Bio-
chemistry, 72(13), 1458-1477.
[49] Walker, C.J. and Griffiths, W.T
lide reductase: A flavoprotein? FEBS Letters, 239(2),
Begley, T.P. (1992) [50] Nayar, P., Brun, A., Harriman, A.,
Mechanistic studies on protochlorophyllide reductase: A
model system for the enzymatic reaction. Journal of the
Chemical Society, Chemical Communications, 5, 395-397.
doi:10.1039/c399200003 95
[51] Ignatov, N.V., Belyaeva, O.B. and Litvin, F.F. (1993) The
ide-dependent one-electron
possible role of the flavin components of protochloro-
phylide-protein complexes in the primary processes of
protochlorophyll photoreduction in etiolated plant leaves.
Photosynthetica, 29, 235-241.
[52] Blenkenhorn, G. (1976) Nicotinam
and two-electron (Flavin) oxidoreductation: thermody-
namics, kinetics, and mechanism. European Journal of
Biochemistry, 67(1), 67-80.