Advances in understanding of the primary reactions of protochlorophyll(ide) photoreduction in cells and model systems

doi:10.4236/jbpc.2011.21001

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Vol.2, No.1, 1-9 (2011)

doi:10.4236/jbpc.2011.21001

Journal of Biophysical Chemistry

Advances in understanding of the primary reactions of

protochlorophyll(ide) photoreduction in cells and model

systems

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.

ABSTRACT

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-

tems.

Keywords: Protochlorophyllide; Chlorophyllide;

Photoreduction; Fluorescence; Spectroscopy

1. PRIMARY REACTIONS OF

PROTOCHLOROPHYLLIDE

PHOTORE DUCTION IN

PLANT LEAVES AND ISOLATED

PIGMENT-PROTEIN COMPLEXES

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-

perature:

Pchlide655650 Chlide

⎯⎯→→X690

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

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

[19,23].

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

Chlide

⎯⎯→− →+

→

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

LIDE T S

Art-

liv

otochlorophyllide fluores-

s of pro-

2. RAPID STAGES OF

PROTOCHLOROPHYL

PHOTOREDUCTION IN VIVO A

PHYSIOLOGICAL TEMPERATURE

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

123

xxx Chlde

μμ

−− −

⎯⎯⎯→ ⎯⎯⎯⎯→⎯⎯⎯→⎯⎯⎯→

The authors supposed that the intermediate X3 corr

e comparative analysis of the research results

3. PRIMARY FLUORESCENT FORMS OF

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-

perature.

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.

CHLOROPHYLLIDE

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

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

↓

⎯⎯→→ →

⎯

⎯→→ →→

Later studies demonstrated the existence in young

mation

HYLL(IDE) ONS

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.

4. OBSERVATION OF ELEMENTARY

REACTIONS OF

PROTOCHLOROP

PHOTORE DUCTION IN SOLUTI

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

tary reactions of pigment photoreduction

5. ELEMENTARY REACTIONS OF

Ia growing number of researchers

arly to observations with whole etiolated leaves,

protochlorophyllide photoreduction in reconsti-

6. POSSIBLE MECHANISMS IN

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

PROTOCHLOROPHYLLIDE

PHOTORE DUCTION IN

RECONSTITUTED TERNA

COMPLEXES

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

[37].

Simil

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)

[23,24].

Thus,

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.

PHOTORE DUCTION OF

PROTOCHLOROPHYLLID

MOLECULE

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

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

––

DADAD ADAH

νδδ

++ +

⎯⎯→→⎯⎯⎯→

() ()

()

X690

HADPHPChlideHADP Chlide

R+

−−

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

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

photoreduction.

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-

duction.

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