ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 830-839 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 903-914.
830
REVIEW
Study of Electron Transfer in PhotosystemI
Using High-Frequency EPR Spectroscopy.
In Memory of Professor Klaus Möbius (1936-2024)
Vasily V. Ptushenko
1
and Alexey Y. Semenov
1,a
*
1
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
119992 Moscow, Russia
a
e-mail: semenov@belozersky.msu.ru
Received April 11, 2025
Revised June 5, 2025
Accepted June 9, 2025
Abstract— Klaus Möbius, Professor at the Free University of Berlin, was an outstanding physical chemist
and biophysicist. He was a pioneer in the development of high-field/high-frequency EPR spectroscopy meth-
ods and their application in the study of photosynthesis. Among the most essential are the applications
in studying the charge transfer kinetics and properties of the ion-radical pairs in photosynthetic reaction
centers (RC). Under his leadership and with his direct participation a unique setup allowing registration of
the kinetics of the electron transfer between the (bacterio)chlorophyll dimer and quinone in the bacterial
photosynthetic RC and plant photosystem I (PSI) was created. This setup also allowed precise determining
of the distance between separated charges based on measuring the frequencies of the Electron Spin Echo
Envelope Modulation (ESEEM). This setup made it possible to prove that electron transfer in PSI occurs
mainly along the A branch of redox cofactors. The kinetics of backward electron transfer reaction (reoxida-
tion of the phyllosemiquinone anion A
1
−
and reduction of the photooxidized chlorophyll dimer P
700
+
) in PSI
were measured under the same conditions. The essential data on the bioprotective effect of the disaccha-
ride trehalose on the kinetics of forward and backward electron transfer in PSI complexes were obtained.
A significant slowdown in the kinetics of electron transfer due to the restriction of protein conformational
mobility, as well as long-term maintaining of functional activity of PSI dried in a vitreous trehalose ma-
trix at room temperature (i.e., subjected to a reversible anhydrobiosis) was demonstrated. These results ob-
tained in collaboration with Prof. Möbius and Prof. Venturoli (Bologna) allowed elucidating the role of hydro-
gen bond network and the conformational mobility of the protein subunits in facilitating electron transfer
in the photosynthetic RC.
DOI: 10.1134/S0006297925601091
Keywords: high-frequency EPR spectroscopy, ion radical pair, multiresonance methods, primary electron donor,
semiquinone, iron-sulfur clusters, trehalose
* To whom correspondence should be addressed.
INTRODUCTION
Professor Klaus Möbius (1936-2024) was one of
the pioneers in the development of high-frequency
EPR spectroscopy methods, including multiresonance
methods such as electron-nuclear double resonance
(ENDOR), electron-electron double resonance (ELDOR),
triple resonance (TRIPLE), and their applications in
photosynthesis studies. We were fortunate to interact
with this remarkable scientist. For one of the co-au-
thors of this article (V. V. P.), communications with
Klaus Möbius were (alas!) only episodic, occurring
during his several visits to Russia. However, during
these relatively short meetings and conversations, his
deep interest not only for scientific (including gener-
al scientific) issues, but also for the personality of his
interlocutor was always felt. Both his explanations of
some ideas on EPR spectroscopy and his benevolent
advice on life evoke grateful memories. Communica-
tion with Klaus Möbius for another co-author (A. Y. S.)
IN MEMORY OF KLAUS MÖBIUS (1936-2024) 831
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
was much closer and more regular (Fig.1); we would
like to devote a separate section to the memories of
this communication.
PERSONAL MEMORIES
OF ONE OF THE CO-AUTHORS (A.Y.S.)
ON MEETING KLAUS MÖBIUS
Klaus and I first met at the Gordon Conference
on the biophysics of photosynthesis in the small town
of Plymouth, New Hampshire (USA) in 1997. A year
later, he invited me to visit Berlin with my wife, and
we stayed in the hospitable home of Klaus and his
wife Uta in Dahlem. From that moment until 2024,
we often saw each other in Berlin, Moscow, Bologna,
Mülheim and Kazan. Based on common scientific in-
terests and mutual sympathy, we developed good sci-
entific and close personal relations.
Klaus was a friendly person with deep knowl-
edge in various fields, the breadth of his interests was
simply amazing. Klaus was exceptionally modest and
democratic; he never demonstrated his superiority to
any interlocutors. I was amazed that during one of
our first visits to Berlin, he considered it necessary to
show me many places connected with the Holocaust
of the Jews under the Nazi Regime, in particular, the
platform from which trains used to depart for the ex-
termination camps in 1943.
In 2003, together with the laboratories of S. Ciurli
(Bologna, Italy), R. Hasanov (Baku, Azerbaijan) and
A. N. Tikhonov (Moscow), we received a joint INTAS
grant, within the framework of which, during a visit
to Bologna, I introduced Klaus to my long-time col-
league Professor Giovanni Venturoli. This acquain-
tance developed into a long and successful collabo-
ration and friendship between Möbius and Venturoli.
As a result of the joint work of the laboratories of
Venturoli, Möbius, his student Anton Savitsky and
Professor Wolfgang Lubitz from the Max Planck In-
stitute in Mülheim (Germany), most important results
were obtained on the effect of the disaccharide treha-
lose (a bioprotector) on the kinetics of electron trans-
fer in bacterial photosynthetic reaction centers (RC).
Sometime later, similar work was performed with the
participation of our laboratory on photosystemI (PSI)
complexes from cyanobacteria. It was shown that dry-
ing in a glassy trehalose matrix at room temperature
leads to a slowdown in the kinetics of electron trans-
fer in both bacterial RC and PSI due to the limitation
of the conformational mobility of the protein and
long-term preservation of its functional activity in a
state of reversible anhydrobiosis.
Klaus was a great connoisseur of music, painting,
and literature; he was interested in the history of sci-
ence. In his house he organized a science museum,
where he collected unique examples of scientific in-
struments and working models of some technical de-
vices. Klaus was interested in the discoveries of his
famous ancestor, the mathematician August Möbius
(known to everyone for discovering the “Möbius
strip”). He knew a lot about the life of August Möbi-
us’s teacher, the great mathematician Carl Gauss.
Once Klaus surprised me by telling that Gauss had
specifically learned Russian in order to read Nikolai
Lobachevsky’s articles, which had been available only
in Russian at the time. His interest in the Möbius strip
made Klaus, together with his long-time colleague
Martin Plateau and AntonSavitsky, write and publish
a book about Möbius strip-like structures in mathe-
matics, astronomy, physics, chemistry, biology, music,
painting and architecture near the end of his life[1].
Klaus witnessed the important historical events
in Germany: the entry of Soviet troops into Berlin in
1945, the Soviet blockade of West Berlin in 1948-1949,
the construction of the Berlin Wall and the closing of
the border between East and West Berlin in 1961, the
youth movement of the 1960s and the destruction of
the Berlin Wall in 1989. His stories about these histor-
ical events, as well as the history of German science
at the Kaiser Wilhelm Institutes in Dahlem, were ex-
tremely interesting.
Fig. 1. K. Möbius and A. Semenov, Moscow, 2007. Photo by
W. Lubitz.
PTUSHENKO, SEMENOV832
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Klaus lived a long and eventful life, till the very
end he fully retained his interest in science, art, pol-
itics, and was a convinced supporter of the develop-
ment of environmentally friendly energy. His stories
and discussions with him are sorely missed by many
of his colleagues and friends in different countries of
the world.
A BRIEF SCIENTIFIC BIOGRAPHY
OF KLAUS MÖBIUS
In this brief introduction we will review the main
scientific achievements of K. Möbius, in which his fun-
damental research and methodological developments
are closely intertwined, forming an inherent unity in
his scientific journey.
Klaus Möbius’s scientific career was linked to EPR
spectroscopy from the very beginning. His supervisor
at the Free University of Berlin, where Klaus Möbius
studied (and later worked throughout his life), Richard
Honerjäger, was a specialist in microwave electromag-
netic radiation [2] – its generation, propagation, and
its various applications, from plasma physics to micro-
wave spectroscopy of molecules. In the second half of
the 1950s, the scientist’s attention was drawn to the
new microwave method of electron paramagnetic res-
onance and its prospects in chemistry[3]. K. Möbius’s
thesis, completed in 1960-1961, was devoted to anisot-
ropy of g-tensor and hyperfine interaction constants in
crystals of organic molecules. Although the era of seri-
al EPR spectrometers had already arrived, K. Möbius
had to assemble an EPR spectrometer from available
materials himself to complete his research. This first
experience became the basis for K. Möbius’s further
scientific studies always combining fundamental sci-
entific research with the development of new technol-
ogy for magnetic resonance studies, which ensured his
scientific achievements. The main focus of K. Möbius’s
instrument development was the high-frequency EPR
spectrometry. The spectrometer created by K. Möbius
and his colleagues at the Free University of Berlin is
one of the best in the world, exceeding commercial
instruments of well-known brand owners in its capa-
bilities. His work in this area brought Klaus together
with the famous Russian scientist Yakov Sergeevich
Lebedev, a pioneer in high-frequency EPR spectros-
copy. K. Möbius highly valued this researcher and
believed that his contribution to world EPR spectros-
copy was very significant, despite all the restrictions
on the international exchange of scientific knowledge
in the USSR in the 1970s and 1980s [4].
Nevertheless, the use of a non-self-made EPR
spectrometer (manufactured by the famous German
company AEG) which was the most modern at the
time ensured efficient start of scientific work of
K. Möbius [5]. Fritz Schneider, who was working at
the AEG Research Institute (AEG Forschungsinstitut),
provided to K. Möbius this fortunate opportunity [6].
It was a joint article with Schneider in the German
journal Zeitschrift für Naturforschung [7] – a review
introducing chemists to a new research method in-
cluding the introduction to the theory of hyperfine
structure (HFS) of EPR spectra and its application to
the analysis of organic radical structure– that appar-
ently became the first published work of K. Möbius.
His subsequent works in those years, including col-
laborations with colleagues, among whom was Martin
Plato, were also devoted mainly to EPR spectroscopy
of organic radicals (for example, Möbius and Plato
[8]). Later, K. Möbius turned to using ENDOR as a
promising method for studying organic radicals in
solutions [9]. This was facilitated by an internship
in the USA with August (Gus) Mackie, who had first
applied this method several years earlier together
with James Hyde. The method had been previously
proposed by J. Feher [10] for studying free radicals
in solutions[11, 12]. The internship resulted in a data
on changes in spin density in aromatic compounds
and the experience of creating ENDOR observation
equipment, which turned out to be very useful for
his subsequent scientific work [13].
Since the late 1970s, K. Möbius also began to use
the ENDOR, ELDOR, and TRIPLE methods [14-16], in-
cluding their applications with strong magnetic fields.
It should be noted that, despite the fact that high-
field EPR methods promised a breakthrough in free
radical research, by that time only a few research
groups around the world had attempted to master
these techniques and create the necessary equipment.
K. Möbius’s group was actually one of the first three
teams of researchers and developers in the field of
high-field EPR spectrometry, along with the groups of
Harold Box etal. [17] in the USA and Ya. S. Lebedev in
Moscow[18, 19]; only later J. Schmidt’s group in Leiden
[20] and the laboratories of Thomas Priesner [21]
and Jack Fried in the USA[22] joined these studies.
The “instrument-building” stage of K. Möbius’s ac-
tivity coincided remarkably with the emergence of his
interest in photosynthesis, a phenomenon that looks like
tailor-made for the application of magnetic resonance
methods. In his autobiographical notes, K. Möbius re-
called how this interest was sparked in him by Arnold
Hoff[6], with whom he published his first work on the
of bacteriochlorophyll cation radical (Hoff and Möbi-
us[23]). From this moment, K. Möbius began studying
electron transfer reactions in photosynthesis, first on
bacterial RCs[24], and then on photosystems I and II
(PSI and PSII) pigment–protein complexes from oxy-
genic organisms (see the reviews[4,25] and references
therein). The development of another resonance meth-
od, electron-spin-echo-envelope-modulation (ESEEM),
IN MEMORY OF KLAUS MÖBIUS (1936-2024) 833
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
specifically in the high-frequency W-band[26], proved
invaluable for these studies, with this approach re-
maining one of the most modern methods of EPR spec-
troscopy to this day. In subsequent years, the use of
this particular technique allowed K. Möbius to obtain
the most interesting results related to photosynthesis.
All the scientific activities of K. Möbius had also
another aspect: he never closed himself off within
his laboratory or even the university, and many of
his works were carried out within the framework of
international cooperation. Together with his outstand-
ing scientific achievements, this humanistic result of
his work was recognized by many international and
national prizes and honorary titles, of which Klaus
Möbius especially valued the Order of Merit of the
Federal Republic of Germany (2006), awarded for the
development of broad international cooperation; the
Max Planck Prize of the Alexander von Humboldt
Foundation (jointly with Chaim Levanon; 1992); the
E. K. Zavoisky Prize (jointly with James Norris and
Ya. S. Lebedev; 1994) and the V. V. Voevodsky Prize of
the Siberian Branch of the Russian Academy of Scienc-
es (2006); Silver (1996) and Gold (2001) Medals of the
International EPR Society; and the AMPERE (Atomes
et Molécules Par Études Radio-Électriques) Prize, 1998.
He was elected a member of a number of academies
and scientific societies, including the International
Society of Magnetic Resonance (ISMAR) (2009); Inter-
national EPR/ESR Society (IES) (2011) and the Istituto
Veneto di Scienze, Lettere ed Arti in Venice (2002).
COLLABORATIONS WITH KLAUS MÖBIUS
AND HIS COLLEAGUES
Our laboratory was fortunate to collaborate with
Klaus Möbius for the last 15 years of his life.
Asymmetry of electron transfer in PSI. Our first
joint work was the study of the asymmetry of elec-
tron transfer along the branches of redox cofactors in
PSI complexes[27]. All photosynthetic RCs are known
to have two practically symmetrical branches of re-
dox cofactors between the dimer of chlorophyll (Chl)
(or bacteriochlorophyll) and quinone molecules, but
electron transfer in PSII complexes and in bacterial
RCs occurs only along one of these branches. At the
same time, electron transfer in PSI occurs along both
branches (A and B), but with different efficiency and
predominantly along branchA.
Conservative Met amino acid residues (M688 in
the cofactor branch A and M668 in the branch B) are
axial ligands of the third Chl molecule pair (called
Chl
3A
and Chl
3B
in the literature), which correspond
to the primary electron acceptors A
0A
and A
0B
in the
symmetric A and B branches of redox cofactors in PSI
(Fig. 2). Substitution of Met molecules by Leu in any
of the redox cofactor branches can lead to a change
in the degree of asymmetry of electron transfer along
A and B branches from the primary acceptors A
0A
and
A
0B
to the secondary quinone acceptors A
1A
and A
1B
. In
PSI from cyanobacterium Synechocystis sp. PCC 6803
the degree of asymmetry of electron transfer along the
branches of redox cofactors was investigated on com-
plexes isolated from the wild type and from mutant
strains with symmetrical point substitutions of Met to
Leu in A (M688LPsaA) and B (M668LPsaB) branches
of cofactors.
The results obtained using femtosecond laser
spectroscopy [28], time-resolved EPR spectroscopy in
microwave X (~9 GHz) [29] and Q (~35 GHz) rang-
es [30] showed that in PSI from the M688LPsaA mu-
tant, the efficiency of electron transfer from the pri-
mary PSI donor, P
700
, to the terminal F
A
/F
B
acceptors
is reduced, while an increase in the formation of the
triplet state P
700
* was assumed. In our joint work,
it was shown that the decay kinetics of the laser-in-
duced EPR signals of the oxidized primary PSI donor
P
700
•+
from wild type Synechocystis sp. and from the
M668LPsaB mutant are very similar, while in the case
of the M688LPsaA mutant, the decay kinetics is signifi-
cantly slowed down [27]. For an illustration of this,
see Fig. 3, which demonstrates the kinetics of P
700
•+
decay (which is formed as a result of excitation of
an isolated PSI complex by a nanosecond laser pulse).
Fig. 2. Electron transfer between redox cofactors in PSI.
Thearrangement of redox cofactors within the protein com-
plex and the pathways of forward (blue arrows) and back-
ward (red arrows) electron transfer are shown. P
700
– the
primary electron donor, a dimer of Chl molecules; A
1A
,A
1B
–
phylloquinone electron acceptors in A and B branches
of cofactors; F
X
, F
A
, F
B
– 4Fe4S clusters. Electron transfer
occurs predominantly along branchA.
PTUSHENKO, SEMENOV834
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 3. Light-dependent changes in the magnitude of the P
700
•+
EPR signal in PSI from the wild type(1), M688NPsaA(2) and
M668NPsaB(3) mutants after illumination with a nanosecond laser pulse in linear and semi-logarithmic (insert) coordinate
systems (based on the data from Savitsky et al. [27]).
This result showed that at least under the exper-
imental conditions, electron transfer occurs asymmet-
rically in favor of branchA.
Registration of the decay kinetics of the P
700
•+
and A
1
•−
signals. Another important result that was
obtained by measuring the kinetics of the EPR sig-
nal in the W-band of microwave frequencies is the
demonstration of the possibility of recording the de-
cay of the signals of the cation radical P
700
•+
and the
anion radical A
1
•−
in one experiment. Following the
illumination of PSI photosynthetic complex by a laser
flash, P
700
transitions to the excited state P
700
* and
subsequent electron transfer from P
700
* to A
1
occurs.
As a result, an ion-radical pair P
700
•+
A
1
•−
is formed, the
lifetime of which is limited both by the recombination
of charges within the pair and by the further forward
electron transfer to the distal PSI acceptors, iron-sul-
fur clusters F
X
, F
A
, and F
B
. Since EPR signals of the
paramagnetic centers P
700
•+
and A
1
•−
are characterized
by different g-factor values[27,31], this makes it pos-
sible to observe them in different (non-overlapping)
ranges of magnetic field strength using high-frequency
EPR spectroscopy methods (Fig. 4) and, consequently,
to simultaneously record the kinetics of EPR signal
decay for both of these radicals after the excitation
by light pulse. As demonstrated on Fig.5, the kinetics
of EPR signal decay for P
700
•+
and A
1
•−
differ signifi-
cantly, since the disappearance of the P
700
•+
signal is
due only to the backward electron transfer (i.e., re-
combination, A
1
•−
→ P
700
•+
), while the A
1
•−
signal can
be associated with both the recombination of charges
in the ion radical pair P
700
•+
A
1
•−
and with the for-
ward electron transfer from A
1
•−
to the subsequent
acceptors (4Fe4S clusters). Thus, the obtained data
Fig. 4. Calculated EPR spectra of P
700
•+
and A
1
•−
radicals in the W-band (95GHz). The relative positions of these cofactors
in PSI structure are shown. (The spectra were calculated in the article by Savitsky et al. [27] based on the values of the
g-tensor components obtained in the work by Zechet al. [31]).
IN MEMORY OF KLAUS MÖBIUS (1936-2024) 835
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 5. Kinetics of signal decay in the W-band, obtained in the EPR absorption maxima of P
700
•+
and A
1
•−
radicals for PSI
complexes from M668NPsaB strain cells (data obtained in the work by Savitsky et al. [27] were used).
demonstrated that, under the experimental conditions,
at least in some PSI complexes, direct electron trans-
fer occurs from the reduced semiquinone anion A
1
•−
to the iron-sulfur clusters.
Measuring the distance between the spin den-
sity centers of P
700
•+
and A
1
•−
. To determine the
distance between the components of the ion-rad-
ical pair, the pulse EPR method (ESEEM) was used,
which allows to obtain signals sensitive to the inter-
action of magnetic moments of two unpaired elec-
trons (P
700
•+
and A
1
•−
). These interactions depend on
the distance between the interacting unpaired elec-
trons (or, more precisely, on the spin density distri-
bution in the radicals). The accuracy of the method
is such that it allows distinguishing distances that
differ by 0.3 Å. Experiments have shown that in PSI
from the M668NPsaB mutant, smaller distances are
observed between the spin density centers in P
700
•+
and A
1
•−
radicals than in PSI from M688NPsaA. This
difference reflects the different charge distribution
in the radical pairs formed in PSI of the two mutant
strains.
According to the structural data, the distance
between the centers of P
700
and A
1B
quinone ring
should be 25.2 Å, and the distance between P
700
and
A
1A
should be 25.0 Å [32]. Since the spin density in
P
700
•+
cation radical dimer is significantly shifted to-
wards Chl
1B
[33, 34], taking into consideration the
preferential electron transfer along the Abranch, the
observed distance between the spin density centers of
P
700
•+
and A
1
•−
should be 25.8 Å (Fig. 6). Experiments
using pulse ESEEM showed that the distance between
P
700
•+
and A
1
•−
, measured in PSI from the wild type
and the mutant strain M668LPsaB, is 26.0 ± 0.3 Å,
i.e. close to the distance corresponding to electron
transfer along the A branch. A similar distance was
measured for PSI complexes containing plastoqui-
none-9 and 2,3-dichloro-1,4-naphthoquinone in the A
1
binding site [35], as well as for PSI dried in a treha-
lose matrix at 150 and 220 K [36].
Fig. 6. The relative positions of the primary PSI electron
donor (P
700
, a dimer of Chl
1A
andChl
1B
molecules) and phyl-
loquinone molecules A
1A
and A
1B
based on the structure of
PSI [32]. The distances between the Chl P
700
dimer and A
1A
(25.8 Å) in branch A and P
700
and A
1B
(24.6 Å) in branch B
correspond to the distances between the spin density center
in the P
700
radical (see [33, 34]) and the geometric centers
of the quinone rings of phylloquinonesA
1A
and A
1B
.
PTUSHENKO, SEMENOV836
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 7. Kinetics of reduction of the laser-flash photooxidized P
700
•+
in PSI, recorded at a wavelength of 820nm, at different
degrees of relative humidity of the trehalose matrix (shown as a percentage near the kinetic curves) (data obtained in the
work by Malferrari et al. [37]).
Thus, the data obtained by using pulse ESEEM
demonstrate that the distances between the spin den-
sity centers P
700
•+
and A
1
•−
in various PSI complexes
from the Synechocystis sp. PCC 6803 cyanobacterium
correspond to preferential electron transfer along the
A branch of PSI redox cofactors. It should be empha-
sized that such structural and functional data can-
not be obtained by such methods as X-ray structural
analysis or even by cryoelectron microscopy, which is
the most popular approach today.
The effect of dehydration in trehalose matrix
on electron transfer in PSI. The kinetics of electron
transfer in pigment–protein complexes depends on
many factors, including the distance between redox
cofactors, the free energy of reactions and the reorga-
nization energy, which in turn depends on the confor-
mational mobility of proteins. It has been previously
shown that successive dehydration of bacterial RCs in
a trehalose matrix, as well as a decrease in tempera-
ture, leads to a slowdown in the forward reactions
between the primary and secondary ubiquinone ac-
ceptors Q
A
and Q
B
and an increase in the contribu-
tion of backward electron transfer reactions to the
observed kinetics of reduction of the laser flash-ox-
idized primary donor, the bacteriochlorophyll dimer
(see reviews[4,25] and references therein). In a joint
study with the laboratories of K. Möbius, G. Venturoli,
and W. Lubitz, we investigated the effect of succes-
sive decreases in the relative humidity of a glassy
trehalose matrix at room temperature on the kinet-
ics of reduction of the photooxidized Chl dimer P
700
in PSI complexes from cyanobacteria. W-band EPR
spectroscopy showed structural homogeneity of the
protein-trehalose matrix and the absence of changes
in the distances and mutual orientation of the redox
cofactors upon drying [37]. At high humidity, the ki-
netics of P
700
•+
reduction was mainly due to the back-
ward electron transfer from the terminal iron-sulfur
F
A
/F
B
clusters. With successive drying, the kinetics of
P
700
•+
reduction accelerated and became less monoex-
ponential and more heterogeneous (Fig. 7).
The kinetics analysis using the Maximum Entro-
py Method (MEM), demonstrated in Fig. 8, showed
that dehydration is accompanied by a decrease in
the contributions of the slow components (τ ≈ 300 ms
and ≈ 60 ms) due to charge recombination between
[F
A
/F
B
]
−
and P
700
•+
, in parallel with an increase in
the contribution of the fastest component (τ ≈ 150 μs)
due to the backward electron transfer from phyllo-
quinone A
1
−
and the intermediate components in the
1-10 ms time range (see the diagram in Fig. 8a). The
intermediate components can most likely be attributed
to the backward electron transfer from the F
X
−
cluster,
captured in one of several conformational states.
In our joint work with the laboratories of John
Golbeck and Klaus Möbius, it was shown that in PSI
dried in a trehalose matrix at room temperature, a
significant slowdown in direct electron transfer from
A
1
•−
to the F
X
cluster occurs. Meanwhile, the preferen-
tial electron transfer along the A branch of redox co-
factors slows down from 220ns in solution to 13-14 μs
in a trehalose matrix. A similar result was obtained
both using EPR spectroscopy in the W-band and by
recording the electrochromic carotenoid shift [38].
PSI dehydration in the trehalose matrix changes
the kinetics of forward and backward electron transfer
IN MEMORY OF KLAUS MÖBIUS (1936-2024) 837
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 8. Energy diagram of electron transfer in PSI(a) and recombination distribution functions of the P
700
•+
reduction over
various hydrations levels (11%, 33%, 43%, 53%, 63%, and in solution) for PSI in a trehalose glass (b). Italic numbers near
the peaks on the lifetime (τ) distribution graph indicate the corresponding charge recombination pathways in PS I (data
obtained in the work by Malferrari et al. [37] were used).
reactions, similar to what occurs when temperature
decreases in a water–glycerol mixture below the phase
transition temperature [4, 25, 39]. This is apparently
due to the fact that in both cases there is a decrease in
the conformational mobility of the PSI pigment–pro-
tein complex. It is assumed that the effect of drying
in a trehalose matrix is due to changes in the hydro-
gen bond network of the protein complex. Meanwhile,
a layer of water molecules surrounded by trehalose
molecules is preserved around the protein globule.
Under such conditions, the protein complex transits
into a state of reversible anhydrobiosis and can be
preserved for many months at room temperature.
CONCLUSION
As a result of joint works with Professor Klaus
Möbius, important results were obtained that clarify
the molecular mechanisms of PSI functioning.
1. Using high-frequency EPR spectroscopy in the
W-band for studying PSI from wild type cyanobacteria
and mutant strains with point substitutions of axial
ligands to the primary chlorophyll electron acceptor
A
0
in the symmetric A and B branches of redox cofac-
tors, it was shown that at low temperatures, electron
transfer occurs along the Abranch.
2. It was demonstrated that the decay of the A
1
•−
anion radical signal at liquid nitrogen temperature
is caused not only by backward electron transfer
to P
700
•+
, but also by forward transfer to the subse-
quent acceptors, 4Fe4S clusters. It was shown that the
distance between the spin density centers of the P
700
•+
cation radical and the A
1
•−
anion radical in native PSI
complexes containing a full set of redox cofactors and
in PSI complexes lacking the terminal 4Fe4S F
A
and
F
B
clusters corresponds to electron transfer along
branchA.
3. It was found that successive drying of PSI in a
glassy trehalose matrix at room temperature results in
slowing down the forward reactions kinetics and the
increase in contribution of backward electron trans-
fer reactions. Meanwhile, the distance and mutual ar-
rangement of the redox cofactors P
700
and A
1
remain
unchanged.
4. When dried in a trehalose matrix, PSI enters
a state of reversible anhydrobiosis and can be pre-
served at room temperature for many months. Sub-
sequent rehydration leads to a complete restoration
of electron transfer in PSI.
Abbreviations. A
1
, phylloquinone, the electron
acceptor in photosystem I; Chl, chlorophyll; ESEEM,
Electron Spin Echo Envelope Modulation; PSI (PSII),
photosystem I (II); P
700
, primary electron donor in
photosystem I; RC, photosynthetic reaction center.
Acknowledgments. The authors are grateful to
G. E. Milanovsky for assistance in writing this review.
Contributions. V. V. Ptushenko– writing sections
of the manuscript, discussion, editing; A. Y. Semenov–
concept of the work, writing sections of the manu-
script, discussion, editing.
PTUSHENKO, SEMENOV838
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Funding. This work was carried out within the
framework of the state assignment of Lomonosov
Moscow State University.
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
REFERENCES
1. Möbius, K., Plato, M., and Savitsky, A. (2022)
The Möbius Strip Topology: History, Science,
and Applications in Nanotechnology, Materials,
and the Arts, Jenny Stanford Publishing, https://
doi.org/10.1201/9781003256298.
2. Honerjäger, R. (1951) Microwave spectroscopy [in
German], Naturwissenschaften, 38, 34-39, https://
doi.org/10.1007/BF00716170.
3. Honerjäger, R. (1958) Paramagnetic electron reso-
nance [in German], Fortsch. Chem. Forsch., 3, 722-
737, https://doi.org/10.1007/BFb0051770.
4. Möbius, K., Savitsky, A., Malferrari, M., Francia, F.,
Mamedov, M. D., Semenov, A. Y., Lubitz, W., and
Venturoli,G. (2020) Soft dynamic confinement of mem-
brane proteins by dehydrated trehalose matrices: high-
field EPR and fast-laser studies, Appl. Magn. Reson.,
51, 773-850, https://doi.org/10.1007/s00723-020-01240-y.
5. Möbius, K. (1965) Investigation of simple π-electron
systems using electron spin resonance and polarog-
raphy: Part I. Experiment and theory [in German],
Zeitschr. Naturforsch.A, 20, 1093-1102.
6. Möbius, K. (2022) Autobiographical sketches (2021),
Appl. Magn. Reson., 53, 467-489, https://doi.org/10.1007/
s00723-021-01410-6.
7. Schneider, F., and Moebius, K. (1963) Report: the
evaluation of electron resonance spectra of organic
radicals [in German], Zeitschr. Naturforsch. B, 18,
1111-1119.
8. Möbius, K., and Plato, M. (1967) EPR-investigation
of dissolved free-radical ions of non-alternating hy-
drocarbons [in German], Zeitschr. Naturforsch. A,
22, 929-939, https://doi.org/10.1515/zna-1967-0612.
9. Möbius, K., and Dinse, K.-P. (1972) ENDOR of or-
ganic radicals in solution, Chimia, 26, 461, https://
doi.org/10.2533/chimia.1972.461.
10. Feher,G. (1956) Observation of nuclear magnetic res-
onances via the electron spin resonance line, Phys.
Rev., 103, 834, https://doi.org/10.1103/PhysRev.103.834.
11. Hyde, J. S., and Maki, A. H. (1964) ENDOR of a free
radical in solution, J.Chem. Phys., 40, 3117-3118,
https://doi.org/10.1063/1.1724957.
12. Hyde, J. S. (1965) ENDOR of free radicals in solu-
tion, J.Chem. Phys., 43, 1806-1818, https://doi.org/
10.1063/1.1697013.
13. Möbius, K., Van Willigen, H., and Maki, A. H. (1971)
ENDOR study of pentaphenylcyclopentadienyl rad-
icals in solution. Lifting of orbital degeneracy by
methyl substitution, Mol. Phys., 20, 289-304, https://
doi.org/10.1080/00268977100100271.
14. Möbius, K., and Biehl, R. (1979) Electron-nuclear-nu-
clear TRIPLE resonance of radicals in solutions, in
Multiple Electron Resonance Spectroscopy (Dorio,
M. M., and Freed, J. H., eds) Springer, pp. 475-507,
https://doi.org/10.1007/978-1-4684-3441-5_14.
15. Biehl, R., Moebius, K., O’Connor, S. E., Walter, R. I.,
and Zimmermann, H. (1979) Evaluation and assign-
ment of proton and nitrogen hyperfine coupling
constants in the free-radical 1-picryl-2, 2-diphenylhy-
drazyl. An NMR, electron-nuclear double resonance,
and electron-nuclear-nuclear triple resonance study,
J.Phys. Chem., 83, 3449-3456, https://doi.org/10.1021/
j100489a027.
16. Haindl,E., and Möbius,K. (1985) A94GHz EPR spec-
trometer with Fabry-Perot resonator, Zeitschr. Natur-
forsch. A, 40, 169-172, https://doi.org/10.1515/zna-
1985-0211.
17. Box, H. C., Budzinski, E. E., and Potter, W. (1971)
Effects of ionizing radiation on potassium hydro-
gen malonate, J.Chem. Phys., 55, 315-319, https://
doi.org/10.1063/1.1675523.
18. Grinberg, O. Ya., Dubinskii, A. A., Shuvalov, V. F.,
Oranskii, L. G., Kurochkin, V. I., and Lebedev, Ya. S.
(1976) Submillimeter EPR spectroscopy of free radi-
cals, Dokl. Akad. Nauk SSSR, 230, 884-887.
19. Galkin, A.A., Grinberg, O.Y., Dubinskii, A.A., Kabdin,
N. N., Krymov, V. N., Kurochkin, V. I., Lebedev, Y. S.,
Oranskii, L. G., and Shuvalov, V. F. (1977) EPR spec-
trometer in 2-mm range for chemical-research,
Instr. Exp. Tech., 20, 1229-1233.
20. Weber, R. T., Disselhorst, J., Prevo, L. J., Schmidt, J.,
and Wenckebach, W. T. H. (1989) Electron spin-echo
spectroscopy at 95GHz, J.Magn. Reson., 81, 129-144,
https://doi.org/10.1016/0022-2364(89)90272-2.
21. Prisner, T. F., Un, S., and Griffin, R. G. (1992) Pulsed
ESR at 140 GHz, Isr. J. Chem., 32, 357-363, https://
doi.org/10.1002/ijch.199200042.
22. Lynch, W. B., Earle, K. A., and Freed, J. H. (1988)
1-mm wave ESR spectrometer, Rev. Sci. Instrum., 59,
1345-1351, https://doi.org/10.1063/1.1139720.
23. Hoff, A. J., and Möbius, K. (1978) Nitrogen electron
nuclear double resonance and proton triple reso-
nance experiments on the bacteriochlorophyll cation
in solution, Proc. Natl. Acad. Sci. USA, 75, 2296-2300,
https://doi.org/10.1073/pnas.75.5.2296.
24. Lendzian,F., Lubitz,W., Scheer,H., Hoff, A.J., Plato,M.,
Tränkle, E., and Möbius, K. (1988) ESR, ENDOR and
TRIPLE resonance studies of the primary donor rad-
ical cation P960
+•
in the photosynthetic bacterium
Rhodopseudomonas viridis, Chem. Phys. Lett., 148,
377-385, https://doi.org/10.1016/0009-2614(88)87191-4.
IN MEMORY OF KLAUS MÖBIUS (1936-2024) 839
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
25. Gorka, M., Cherepanov, D. A., Semenov, A. Y., and
Golbeck, J. H. (2020) Control of electron transfer by
protein dynamics in photosynthetic reaction centers,
Crit. Rev. Biochem. Mol. Biol., 55, 425-468, https://
doi.org/10.1080/10409238.2020.1810623.
26. Prisner, T.F., Rohrer,M., and Möbius,K. (1994) Pulsed
95GHz high-field EPR heterodyne spectrometer with
high spectral and time resolution, Appl. Magn. Reson.,
7, 167-183, https://doi.org/10.1007/BF03162610.
27. Savitsky, A., Gopta, O., Mamedov, M., Golbeck, J. H.,
Tikhonov, A., Möbius, K., and Semenov, A. (2010)
Alteration of the axial met ligand to electron accep-
tor A
0
in photosystem I: effect on the generation of
P
700
•+
A
1
•−
radical pairs as studied by W-band tran-
sient EPR, Appl. Magn. Reson., 37, 85-102, https://
doi.org/10.1007/s00723-009-0052-0.
28. Dashdorj, N., Xu, W., Cohen, R. O., Golbeck, J. H.,
and Savikhin, S. (2005) Asymmetric electron trans-
fer in cyanobacterial photosystem I: charge separa-
tion and secondary electron transfer dynamics of
mutations near the primary electron acceptor A
0
,
Biophys. J., 88, 1238-1249, https://doi.org/10.1529/
biophysj.104.050963.
29. Cohen, R.O., Shen,G., Golbeck, J. H., Xu,W., Chitnis,
P. R., Valieva, A. I., van der Est, A., Pushkar, Y., and
Stehlik, D. (2004) Evidence for asymmetric electron
transfer in cyanobacterial photosystemI: analysis of
a methionine-to-leucine mutation of the ligand to the
primary electron acceptorA
0
, Biochemistry, 43, 4741-
4754, https://doi.org/10.1021/bi035633f.
30. Salikhov, K. M., Pushkar, Y. N., Golbeck, J. H., and
Stehlik, D. (2003) Interpretation of multifrequency
transient EPR spectra of the P
700
+
A
0
Q
K
−
state in pho-
tosystem I complexes with a sequential correlated
radical pair model: wild type versus A
0
mutants, Appl.
Magn. Reson., 24, 467-482, https://doi.org/10.1007/
BF03166949.
31. Zech, S.G., Hofbauer,W., Kamlowski, A., Fromme,P.,
Stehlik, D., Lubitz, W., and Bittl, R. (2000) A struc-
tural model for the charge separated state in pho-
tosystem I from the orientation of the magnetic in-
teraction tensors, J.Phys. Chem.B, 104, 9728-9739,
https://doi.org/10.1021/jp002125w.
32. Jordan, P., Fromme, P., Witt, H. T., Klukas, O.,
Saenger, W., and Krauß, N. (2001) Three-dimen-
sional structure of cyanobacterial photosystem I
at 2.5 Å resolution, Nature, 411, 909-917, https://
doi.org/10.1038/35082000.
33. Plato, M., Krauß, N., Fromme, P., and Lubitz, W.
(2003) Molecular orbital study of the primary elec-
tron donor P
700
of photosystem I based on a re-
cent X-ray single crystal structure analysis, Chem.
Phys., 294, 483-499, https://doi.org/10.1016/S0301-
0104(03)00378-1.
34. Lubitz, W. (2006) EPR studies of the primary elec-
tron donor P700 in photosystem, in Photosystem I.
Advances in Photosynthesis and Respiration (Gol-
beck, J. H., ed) Vol. 24, Springer, Dordrecht, https://
doi.org/10.1007/978-1-4020-4256-0_17.
35. Mula,S., Savitsky,A., Möbius,K., Lubitz,W., Golbeck,
J. H., Mamedov, M. D., Semenov, A. Y., and van der
Est, A. (2012) Incorporation of a high potential qui-
none reveals that electron transfer in photosys-
tem I becomes highly asymmetric at low tempera-
ture, Photochem. Photobiol. Sci., 11, 946-956, https://
doi.org/10.1039/c2pp05340c.
36. Sukhanov, A. A., Mamedov, M. D., Möbius, K.,
Semenov, A. Y., and Salikhov, K. M. (2018) The de-
crease of the ESEEM frequency of P
700
+
A
1
−
ion-rad-
ical pair in photosystem i embedded in trehalose
glassy matrix at room temperature can be explained
by acceleration of spin-lattice relaxation, Appl. Magn.
Reson., 49, 1011-1025, https://doi.org/10.1007/s00723-
018-1017-y.
37. Malferrari, M., Savitsky, A., Mamedov, M. D.,
Milanovsky, G. E., Lubitz, W., Möbius, K., Semenov,
A.Y., and Venturoli,G. (2016) Trehalose matrix effects
on charge-recombination kinetics in photosystemI of
oxygenic photosynthesis at different dehydration lev-
els, Biochim. Biophys. Acta, 1857, 1440-1454, https://
doi.org/10.1016/j.bbabio.2016.05.001.
38. Shelaev, I., Gorka, M., Savitsky, A., Kurashov, V.,
Mamedov,M., Gostev,F., Möbius,K., Nadtochenko,V.,
Golbeck, J., and Semenov, A. (2017) Effect of dehy-
drated trehalose matrix on the kinetics of forward
electron transfer reactions in photosystemI, Zeitschr.
Phys. Chem., 231, 325-345, https://doi.org/10.1515/
zpch-2016-0860.
39. Milanovsky, G., Gopta, O., Petrova, A., Mamedov, M.,
Gorka, M., Cherepanov, D., Golbeck, J. H., and
Semenov, A. (2019) Multiple pathways of charge re-
combination revealed by the temperature depen-
dence of electron transfer kinetics in cyanobacterial
photosystemI, Biochim Biophys Acta Bioenerg., 1860,
601-610, https://doi.org/10.1016/j.bbabio.2019.06.008.
Publisher’s Note. Pleiades Publishing remains
neutral with regard to jurisdictional claims in published
maps and institutional affiliations. AI tools may have
been used in the translation or editing of this article.
