Applied Magnetic Resonance (2024) 55:1481–1514 Applied
https://doi.org/10.1007/s00723-024-01713-4 Magnetic Resonance
REVIEW
Nitroxide Spin Labels for Exploring Relationships 
Between Molecular Structure, Microenvironment and EPR 
Parameters: A Mini‑review Dedicated to Carlo Corvaja
Klaus Möbius1 · Anton Savitsky2 · Martin Plato1 · Wolfgang Lubitz3
Received: 14 August 2024 / Revised: 30 August 2024 / Accepted: 31 August 2024 /  
Published online: 14 September 2024 
© The Author(s) 2024
Abstract
This mini-review is dedicated to Carlo Corvaja (University of Padova) in recogni-
tion of his important contributions to the study of biomimetic donor–acceptor model 
dyads and triads and to the understanding of spin exchange in excited fullerene–
nitroxide derivatives. We report on attractive examples of multi-frequency and 
multi-resonance EPR spectroscopy, highlighting recent work in Padova and Berlin/
Mülheim. The examples selected include TR-EPR, ENDOR, and EDNMR experi-
ments on photoexcited spin-labeled macromolecules, such as fullerene–nitroxide 
complexes or photosynthetic bacterial reaction centers, which were optionally NO 
spin-labeled. From the spin interaction parameters measured, detailed information 
about structure and dynamics of macromolecules embedded in liquid-solution or 
solid-state microenvironments could be extracted.
Prologue
We dedicate this mini-review to Prof. Carlo Corvaja (University of Padova) on 
the occasion of his 85th birthday (which actually had been celebrated already 
in 2022).
*  Klaus Möbius 
 moebius@physik.fu-berlin.de
*  Anton Savitsky 
 anton.savitsky@tu-dortmund.de
*  Wolfgang Lubitz 
 wolfgang.lubitz@cec.mpg.de
1 Department of Physics, Free University Berlin, Arnimallee 14, 14195 Berlin, Germany
2 Faculty of Physics, Technical University Dortmund, Otto-Hahn-Str. 4a, 44227 Dortmund, 
Germany
3 Max-Planck-Institute for Chemical Energy Conversion, Stiftstr. 34-36, 
45470 Mülheim an der Ruhr, Germany
Vol.:(0123456789)
 1482 K. Möbius et al.
Carlo Corvaja (born July 25, 1937) 
[Photo: Fabio Corvaja, 2024]. 
Q: When did you meet Carlo Corvaja for the first time and where was it?
A: It was almost 60 years ago, when the German Mark (DM) was equivalent to 
156 Italian Lira (ITL) and Martin Plato, Klaus Möbius and Carlo Corvaja were 
29 ± 1 years old. In addition, it was in England.
The first encounter of Klaus Möbius and Martin Plato with Carlo Corvaja was in 
March 1965 when they attended the international EUCHEM Conference on “Chem-
ical Aspects of Electron Spin Resonance” in Cirencester (England). It was the first 
international conference in our young EPR career. We enjoyed the opportunity to 
talk with high-profile EPR researchers, among them Jack Freed, Jim Hyde, Fabian 
Gerson, Joan van der Waals, Vladislav Voevodsky, Giovanni Giacometti—and his 
graduate student Carlo Corvaja.
Together with Pier Luigi Nordio and Marina Brustolon, Carlo Corvaja was 
among the first-generation members of the renowned Padova group of Giovanni 
Giacometti, who had pioneered EPR spectroscopy in Italy. It was no surprise then 
that the group members knew (almost) everything about EPR spectroscopy. But 
beyond that, they had an amazing knowledge of political, scientific and medical 
history. They were fond of old maps and historic books, and were fascinated by 
antique scientific instruments. They were familiar with the behavior of snakes and 
birds and watched them in the free nature. They were involved in the preservation 
of Cultural Heritage using up-to-date physico-chemical tools. In addition, they 
admired the masterful craftsmanship of finely chiseled contemporary gold jew-
elry made from thin gold filaments. What a stimulating environment for guest sci-
entists! In addition, all this against the background of the day-to-day excitement 
Nitroxide Spin Labels for Exploring Relationships Between… 1483
Galileo Galilei (From: THE EDWARD WORTH LIBRARY, Dublin) 
Fig. 1  Galileo Galilei (1564–1642). He was a teacher at the University of Padova from 1592 to 1611. 
Padova University, founded in the year 1222, is one of the oldest in the world. Galileo Galilei is con-
sidered the founder of modern science. He accomplished numerous seminal discoveries in Padova. For 
example, with his novel telescope, he observed the four largest satellites of Jupiter (1610). His priority 
of the invention of scientific instruments is not always undisputed [1]. (The etching is from the Edward 
Worth Library in Dublin, Ireland)
of an extended teaching and research stay at the University of Padova, one of the 
oldest universities in the world; the place where Galileo Galilei had worked as a 
teacher and researcher (see Fig. 1). In addition, the place which is also famous for 
its ancient Medical Faculty (see Fig. 2). Where you can see an important section 
of the history of medicine, that of anatomy including public dissection of human 
corpses. For teaching purposes, they were carried out there in early times of the 
 1484 K. Möbius et al.
Fig. 2  The University of Padova is famous also for its ancient Medical Faculty, which attracted scholars 
from all over Europe in the Middle Ages and the Renaissance. The university’s main building, Palazzo 
del Bo, houses the old anatomical theater built by Girolamo Fabricius D’Acquapendente in 1584. This is 
probably the first permanent anatomical theater known to history. The elliptical-shaped theater has six 
tiers carved from walnut and can accommodate up to 300 spectators. The seating is arranged so that each 
student would have an uncompromised view of the dissecting table. Public dissections of human corpses 
were often carried out under candlelight. (The picture was taken by an unknown photographer)
Middle Ages and the Renaissance, under suspicious surveillance by the Catholic 
Church.
1 Introduction
In the subsequent decades after their first encounter in 1965, Carlo Corvaja and 
K. M., together with their respective co-workers, were orbiting independently 
around their common center of gravity, which was EPR spectroscopy. They did 
so in various sub-worlds of EPR spectroscopy, predominantly “Spin Chemistry” 
and “ENDOR”, where they met again and again at intersections of biologically 
inspired photo-chemistry and photo-physics. Occasionally, they also met at visits 
of EPR laboratories and conferences all over the world. Unforgettable for us, in 
Padova, Venice, Berlin, Kazan—and on the island of Rhodes, where Carlo Cor-
vaja received the 2001 Silver Medal Award in Chemistry from the International 
EPR Society. Whenever they encountered, they enthusiastically discussed their 
ideas of science and society, democracy and tyranny. No less enthusiastically, 
their ideas of peace and justice, the arts and history. Then they would return to 
their various sub-worlds. But always looking forward to their next encounter to 
exchange new ideas and old experiences.
Nitroxide Spin Labels for Exploring Relationships Between… 1485
In Spin Chemistry as well as in ENDOR, the decisive quantum phenomena 
have their origin in weak magnetic interactions within and between reaction part-
ners. In photoreactions, the reaction partners are often transient and/or stable par-
amagnetic intermediates of radical and triplet chemistry. The resulting character-
istic electron- and nuclear-spin polarization phenomena of the intermediates can 
be observed in the EPR spectra. By analyzing the spectra, the quantum mechani-
cal control mechanisms of fundamental (bio)chemical reaction pathways can be 
revealed.
With Carlo Corvaja and his co-workers in Padova, we share common interests 
in EPR spectroscopy of transient states in (bio)chemistry, e.g., photosynthetic sys-
tems and their biomimetic model complexes. However, while our main interest was 
in photosynthetic Reaction Centers for light-induced charge separation of chloro-
phyll–quinone donor–acceptor complexes, Carlo Corvaja was primarily interested 
in photosynthetic Antenna complexes for light-induced energy transfer. For instance, 
the mechanisms of light-induced singlet–singlet and triplet–triplet energy trans-
fer between carotenoids and chlorophyll pigments. An exemplary publication by 
the Padova group with Donatella Carbonera, Marilena di Valentin, Carlo Corvaja, 
Giovanni Giacometti, Giancarlo Agostini, and their co-workers from the Arizona 
State University group with Paul A. Liddell, Ana L. Moore, Thomas A. Moore, and 
Devens Gust is Ref. [2]. There, carotenoid triplet detection by time-resolved tran-
sient EPR (TR-EPR) spectroscopy in carotenopyropheophorbide dyads (see Fig. 3) 
is described. The TR-EPR technique, when combined with laser photolysis, provides 
detailed information on reactive species in their excited states.
Carotenoid triplets play a photoprotective role in natural photosynthesis. The 
main process of carotenoid triplet formation is known to be triplet–triplet energy 
transfer from chlorophyll triplets. The structural requirements for high transfer 
yields are still an active field of research and the presence of competitive triplet 
formation pathways has not been excluded. Transient EPR measurements of triplet 
states formed by photoexcitation allow detection of the initial spin polarization. The 
characteristic polarization pattern derives from the mechanism of triplet formation. 
In the case of triplet–triplet energy transfer, if the condition of spin angular momen-
tum conservation is fulfilled, simulation of the EPR spectra gives information about 
the donor–acceptor mutual orientation. In their publication, Corvaja et al. describe 
transient EPR experiments on two artificial photosynthetic dyads, consisting of a 
carotenoid covalently linked to a free-base or zinc-substituted pyropheophorbide 
moiety. The results are discussed in terms of possible dyad conformations owing to 
the flexibility of the linker bridge.
Carotenoids act as light-harvesting pigments and photoprotectants in photosyn-
thetic organisms. Their role as antenna pigments is accomplished by absorption of 
photons in the blue-green spectral range, followed by rapid singlet–singlet energy 
transfer to chlorophyll pigments. The photoprotective function involves the direct 
quenching of singlet oxygen or the quenching of chlorophyll triplets that could sensi-
tize the formation of singlet oxygen. The process of chlorophyll triplet quenching is 
generally regarded as triplet–triplet energy transfer from chlorophyll to carotenoids.
Energy transfer has been widely studied in photosynthetic organisms, includ-
ing both bacteria and higher plant systems. In parallel, investigations of artificial 
 1486 K. Möbius et al.
Fig. 3  Molecular structure of a free-base pyropheophorbide and b free-base carotenopyropheophorbide. 
The zinc pyropheophorbide and the zinc caroteno-pyropheophoride have the same molecular structures 
as the corresponding free-base compounds except for the substitution of the central protons with zinc [2]
photosynthetic systems have been carried out on molecular dyads and triads consist-
ing of carotenoid polyenes covalently linked to porphyrin and chlorophyll deriva-
tives. These artificial systems can mimic both the antenna and photoprotective 
functions.
It has been shown that the spin angular momentum is conserved during the 
transfer process. The relative populations of the triplet acceptor sublevels are pro-
portional to the squares of the projections of the donor principal magnetic axes on 
the acceptor magnetic axes. It is therefore possible for the chemical synthesis of the 
dyads to determine the conformational requirements of the model compounds for an 
efficient triplet–triplet energy transfer.
The transient triplet-state EPR spectra of the free-base pyropheophorbide and 
of the zinc pyropheophorbide are shown in Fig. 4a and b, respectively. The spectra 
have been detected at 0.6 µs delay after the laser flash. They are spin-polarized in an 
EAEAEA and AAEAEE fashion, respectively.
The analysis of the experimental results has been performed in terms of spin 
angular momentum conservation during triplet–triplet energy transfer from the 
pheophorbide triplet to the carotenoid triplet in an external magnetic field.
Nitroxide Spin Labels for Exploring Relationships Between… 1487
Fig. 4  Time-resolved triplet-state EPR spectra recorded by direct detection of a free-base pyropheophor-
bide, b zinc pyropheophorbide in 2-methyltetrahydrofuran. T = 20 K, PMW = 14.5 mW, delay time after 
the laser pulse = 0.6 µs, integration gate = 0.3 µs. A and E stand for absorption and emission, respectively. 
Figure taken from Ref. [2]
Scheme 1  Carotene-porphyrin-fullerene triad 1 and functionalized fullerene moiety 2, see ref. [3]
The authors conclude that the widely different initial polarization of the carot-
enoid triplet formed in a light-excited carotenopyropheophorbide, observed in the 
presence and absence of a Zn atom in the center of the porphyrin ring, are certainly 
not only due to the well-known differences in the population kinetics of the por-
phyrin moiety following metalation. Different conformations have to be invoked in 
order to account for the experimental data. Apparently, the test of the conforma-
tional change explanation requires additional experiments regarding the possibility 
of different mechanism to be invoked in the birth of the triplet. For a detailed discus-
sion of this issue, see Ref. [2].
An important step forward towards rigid donor–acceptor model systems was 
achieved by the same Padova/Arizona groups with the synthesis of a carotene–por-
phyrin–fullerene triad (see Scheme 1), and the analysis of the TR-EPR spectra of its 
transient radical-pair intermediate after laser-flash excitation [3].
This publication is an exciting piece of science about the essence of the photo-
chemical experiments on molecular donor–acceptor triads consisting of a porphyrin 
(P) covalently linked to a carotenoid polyene (C) and a fullerene derivative ( C60). 
The triads have been studied at 20  K by time-resolved EPR spectroscopy follow-
ing light excitation by short laser-flashes. Excitation of the porphyrin moiety yields 
C─1P─C60, which decays by photoinduced electron transfer to yield C─P·+─C60·−. 
This state rapidly evolves into a final charge-separated radical pair (RP) state 
C·+─P─C ·−60  whose spin-polarized EPR signal was detected and simulated. 
 1488 K. Möbius et al.
Fig. 5  Time-resolved EPR spectrum of triad 1 in 2-methyltetrahydrofuran. The spectrum was obtained at 
20 K, 0.4 µs, integration gate = 0.3 µs after the laser excitation pulse with 14.5 mW of microwave power 
and an integration time of 0.2 µs per point. The direct-detection spectrum appears in absorption (A) or 
emission (E), as indicated. a Expansion of the spectrum in the carotenoid triplet region. b Expansion of 
the spectrum of the C⋅+─P─C −60·  radical pair (1 G =  10–4 T) (figure taken from Ref. [3])
The exchange interaction between the electrons in the radical pair is rather weak 
(J = 1.2 G). The RP C·+─P─C60·− decays to give the carotenoid triplet in high yield 
with a time constant of 1.2 µs. The spin polarization of 3C─P─C60 is characteristic 
of a triplet formed by charge recombination of a singlet-derived radical pair. The 
kinetics of the decay of 3C─P─C60 to the ground state were also determined. The 
photoinduced electron transfer from an excited singlet state at low temperature and 
the high yield of charge recombination to a spin-polarized triplet indeed mimic simi-
lar processes observed in photosynthetic reaction centers.
Most of the photosynthetic reaction center models have employed quinones, 
porphyrins, or aromatic imides as electron acceptor moieties. However, it has been 
found recently that also fullerenes can function in this way. For example, a variety 
of molecular dyads consisting of fullerenes linked to donor pigments demonstrate 
photoinduced electron transfer to the fullerene, yielding a charge-separated RP state, 
see Ref. [2].
In Fig. 5, it is shown that Triad 1 (see Scheme 1) has undergone photoinduced 
electron transfer at low temperatures, followed by charge recombination to a triplet 
state, similar to natural photosynthetic reaction centers. In the present study, TR-
EPR was used to observe transient triplet and radical-pair states, their spin polariza-
tion, and their kinetic properties in order to compare the behavior of 1 with that of 
reaction centers and other model compounds.
In their Conclusions the authors emphasize that their TR-EPR studies on Triad 
1 are in complete accord with the results of their earlier optical studies. Excitation 
of the porphyrin moiety is followed by photoinduced electron transfer from the first 
excited singlet state to yield an intermediate charge-separated state, which evolves 
into a final radical pair state that can be detected by TR-EPR. The RP state mainly 
Nitroxide Spin Labels for Exploring Relationships Between… 1489
decays to the carotenoid triplet, 3C─P─C60, which then decays to the ground state. 
These events occur not only in solution at ambient temperatures, but also in a glass 
matrix at temperatures as low as 10 K. The temperature dependence of the charge-
recombination reaction is extremely weak.
The EAA EEA spin polarization pattern of the carotenoid triplet EPR signal is 
characteristic of a triplet formed by recombination of a singlet-born radical pair, 
rather than by normal spin–orbit coupling induced intersystem crossing.
It should be noted that all previous reaction center models employing pigments 
related to natural chlorophylls and carotenoids have demonstrated charge recombi-
nation to the ground state, rather than radical-pair induced triplet formation. In addi-
tion, the vast majority of these model systems failed to undergo photoinduced elec-
tron transfer in glasses at low temperatures.
Comparison of the results for 1 with those for other triads suggests that the unu-
sual properties of 1 must derive from the fullerene moiety and associated reor-
ganization and stabilization energies, rather than from differences in oxidation and 
reduction potentials and energies of excited states as measured in polar solvents. For 
a detailed discussion of this important work, see Ref. [3].
Somehow, it is surprising that we do not have joint publications with Carlo Cor-
vaja in which model systems mimicking primary photosynthesis are discussed. 
Donor–acceptor dyads and triads as model systems for light-induced electron-trans-
fer processes have long been the subject of our research. This research was carried 
out in close collaboration with the group of Harry Kurreck at the Chemistry Depart-
ment of FU Berlin [4–6].
In recent years, Carlo Corvaja’s favorite research area in Padova had shifted to 
Fullerene derivatives, specifically, to electron spin polarization effects in triplet, 
quartet and quintet states of fullerenes linked to one or more free nitroxide radicals 
[7]. In Berlin/Mülheim, our favorite research areas were photosynthetic Reaction 
Centers (RCs) and their interactions with the microenvironment. In this mini-review, 
we will focus on nitroxide spin-labeled protein complexes for which the research 
areas of Padova and Berlin/Mülheim overlap to a certain extent. We have singled out 
a few examples from both groups that we find particularly interesting. The common 
link between the selected examples is time-resolved single- or multiple-resonance 
EPR spectroscopy of light-irradiated macromolecules with short-lived doublet- 
or excited-state intermediates. The experiments are designed for investigating the 
relationships between molecular structure, microenvironment of the paramagnetic 
cofactors and their EPR properties [8, 9].
In Padova, the X-band TR-EPR measurements employed a Nd:YAG pulsed 
laser (second harmonic, λ = 532 nm) to illuminate the sample for times as short as 
5 ns. Using fullerene molecules linked to one or more nitroxide radicals, the Cor-
vaja group was able to observe photoexcited species with one, two, three, and four 
unpaired electron spins. The aim of their studies was to understand the basic pro-
cesses for photogeneration of high-spin organic molecules. The ultimate goal was 
to develop viable methods for controlling and modulating molecular magnetism by 
means of light beams.
In Berlin/Mülheim, we have capitalized on the improved spectral resolution 
of high-field EPR spectroscopy, primarily at W-band (TR-EPR, ENDOR, and 
 1490 K. Möbius et al.
EDNMR). The experiments were carried out in combination with selective isotope 
labeling to explore fundamental light-driven primary processes in bacterial reaction 
centers (bRCs). In certain cases, to elucidate subtle protein–water interactions with 
the solvent matrix, site-directed nitroxide spin labeling (SDSL) of the bRCs was 
used. Thereby, the relationships between the microenvironment of the spin label at 
the protein surface and molecular magnetic parameters were explored in detail. Ulti-
mately, our aim was to gain new insights into the interconnections between structure 
and function of electron-transfer cofactors embedded in their protein matrix.
2  Examples
2.1  Examples from Padova: Time‑Resolved EPR of Single and Double 
Spin‑Labeled  C60 Fullerene Derivatives. Observation of Triplet, Quartet 
and Quintet Excited States in Liquid and Solid Solutions
In the Chemistry Department of Padova University, the Corvaja group has exten-
sively studied, by TR-EPR, the magnetic properties of  C60 fullerenes (Fig. 6) linked 
to stable nitroxide radicals. Molecular magnetism in organic complexes obvi-
ously occurs when there is an odd number of electrons in the complex, as is the 
case of free radicals. Alternatively, it occurs in certain molecular high-energy states 
Fig. 6  Structure of the all-carbon molecule C 60 fullerene (ball-and-stick model). It is comprised of 12 
regular pentagonal faces and 20 regular hexagonal faces. The 60 carbon atoms are placed at the vertexes. 
The carbons of fullerenes are bound together to form closed cage structures. The fullerenes were discov-
ered in the early 1990s years (Nobel Prize in Chemistry 1996 to Robert F. Curl, Jr., Harold W. Kroto, and 
Richard E. Smalley). Fullerenes are formed in a variety of ways when carbon is heated to high tempera-
tures in an inert atmosphere. The product obtained contains several different fullerenes, but the one with 
the highest yield consists of 60 C atoms bound together in a truncated icosahedron structure
Nitroxide Spin Labels for Exploring Relationships Between… 1491
that are populated by the absorption of light. It is required that the electron spins 
in the excited molecule and in the radical partner couple in a way that a magnetic 
moment is created (total spin S > 0). When photoexcited in the nanosecond range, 
fullerene–nitroxide derivatives combine both sources of molecular magnetism. The 
mechanism of this spin–spin coupling was explored by analyzing the line shapes and 
decay times of the TR-EPR spectra of the different fullerene derivatives.
The TR-EPR research on fullerene derivatives in Padua was partly carried out in 
close collaboration with Seigo Yamauchi and his group who worked on related sub-
jects at the Tohoku University in Sendai. Important references of this work are, for 
instance, [10–14].
Fullerene  C60 molecules have a series of interesting properties connected with 
their capability to accept one extra electron from nearby donors and to form para-
magnetic excited states by light absorption [15]. For example, when a mixture of 
fullerene and a donor polymer is illuminated by visible light, an electron is trans-
ferred from the donor to C 60 and two charged radicals are formed. Such a charge 
separation process is exploited in organic photovoltaic devices [16].
Since long, nitroxide radicals, for example TEMPO [17], have been inserted as 
spin labels in a variety of molecular systems to study structural and dynamic proper-
ties [8, 9]. The unpaired electron predominantly resides on the NO group, i.e., delo-
calization on other atoms including the nearest neighbors is practically zero. How-
ever, population of the high-energy levels as well as the decay pathways and rates of 
the excited states can be strongly affected by the presence of the nitroxide unpaired 
spin. This is due to interactions with other electron spins. Although the coupling is 
small, it can give rise to large effects on the line intensity distribution in the mag-
netic resonance spectra [10, 11, 13, 18, 19]. To illustrate this,  C60 chromophores 
labeled with one or two nitroxide spins were examined in their light-excited state 
[13, 20].
As a chromophore,  C60 has various favorable characteristics which are preserved 
in  C60-nitroxide derivatives: (i) a long-lived excited triplet state produced with high 
yield, (ii) chemical stability in the excited state, (iii) absence of magnetic nuclei 
(neglecting 13C in the low natural abundance of about 1%). These properties facili-
tate the observation of the excited state and simplify the EPR spectrum. This makes 
the analysis of the spectrum unambiguous because only the hyperfine structure due 
to the nitrogen nuclei of the spin label occurs.
The Corvaja group made the first observation of radical–triplet pairs in the quar-
tet and quintet states of spin-labeled  C60 derivatives both in liquid solution and fro-
zen matrix [14]. The g factor and the hyperfine structure of the radical pairs are 
indicative of the spin multiplicity and were used to assign the total spin state of the 
excited complex.
The Corvaja paper reviewed here presents the results of a thorough TR-EPR 
investigation of four  C60 derivatives Ia and Ib, IIa and IIb, spin-labeled with one or 
two TEMPO radicals, respectively. Their chemical structures are shown in Fig. 7. 
The fulleropyrrolidines [21] were studied in their (dark) ground state by cw EPR, 
and by TR-EPR in their photoexcited states after ns-pulsed laser excitation, see 
Figs. 8 and 9.
 1492 K. Möbius et al.
Fig. 7  Spin labeled fullerene derivatives (fulleropyrrolidines) studied by the Corvaja group [14, 22]. 
Shown are monoradical (Ia, Ib) and biradical (IIa, IIb) fullerene derivatives, linked together at different 
distance and relative orientation to each other
In the primary stages of numerous chemical and photochemical reactions, radi-
cal pairs (RPs) and radical–triplet pairs (RTPs) are formed which eventually evolve 
to reaction products. Time-resolved EPR spectroscopy of these species is feasible, 
even if they are present in solution at low stationary concentration. This is due to the 
spin polarization of the radical species in the early instants of their formation, which 
strongly enhances the EPR sensitivity.
The strongly spin-polarized TR-EPR signals are the superposition of the spectra 
of 4[RTP] and of RP. The spectrum of RP is characterized by three lines separated 
by about 15 G and centered at g = 2.006. The spectrum of 4[RTP] consists of three 
lines centered around g = 2.003 with a hyperfine splitting of about 5 G. The signals 
occur either in Emission or in enhanced absorption and eventually change polariza-
tion as they evolve on timescales of a few microseconds.
Let us summarize the results of the 2006 papers [14, 22] from the Corvaja group 
on time-resolved EPR spectra of two single spin-labeled and two double spin-
labeled C 60 derivatives dissolved in liquid and frozen solutions. The spectra were 
observed after pulsed laser excitation with ns time resolution. Quartet and quintet 
excited species were identified which arise from the electron spin–spin exchange 
coupling J of the triplet-excited fullerene moiety with the unpaired spin(s) of the 
nitroxide label(s). Despite rather similar molecular structures in both series of single 
and double labeled derivatives (see Fig. 7), a very different behavior of the TR-EPR 
Nitroxide Spin Labels for Exploring Relationships Between… 1493
Fig. 8  X-band TR-EPR (3D presentation) of a  C60 derivative bearing two stable nitroxide radicals (struc-
ture IIa in Fig. 7) after light excitation at 290 K in liquid toluene solution. The spectrum is recorded by 
direct detection (i.e., without field modulation and lock-in detection, but with broad-band preamplifica-
tion and data collection by a fast digital oscilloscope). The transient EPR spectrum is extracted 0.5 μs 
after the laser pulse (fired at time = 0). The spectrum appears in enhanced absorption (above the noise 
floor) immediately after the laser pulse, later in emission (below the noise floor). It is assigned to a quin-
tet electron spin state (S = 2, four unpaired electrons). Adapted from Refs. [14, 22]
spectra was found. This is due to a substantial difference of the exchange coupling 
energy in the monoradicals Ia and Ib compared to the biradicals IIa and IIb.
In liquid solution at room temperature, the EPR spectra of derivatives Ia and 
Ib in their electronic ground state consist of three lines due to the 14N hyperfine 
coupling. They are further split by the small hyperfine coupling of the nitroxide 
methyl protons. When these derivatives are photoexcited, strong TR-EPR signals 
of different lineshape are recorded. They were analyzed on the basis of the forma-
tion of quartet states generated by the coupling of the S = 1 spin on the fullerene 
moiety with the radical nitroxide S = 1/2 spin (systems Ia and Ib).
The room temperature EPR spectra of IIa and IIb in the electronic ground state 
present three additional lines between the three hyperfine components typical of 
the nitroxide monoradicals. The spectral simulation is consistent with the pres-
ence of two weakly exchange-coupled nitroxide groups. After pulsed laser photo-
excitation, the TR-EPR spectra show an absorption/emission (A/E) pattern which 
were analyzed in terms of spin-polarized quintet states (S = 2) [22]. The electron 
exchange interaction J in radical pairs is an important parameter. Its value and 
sign determine the polarization pattern, the kinetic behavior, and the magnetic 
field effects on reactivity [23–28].
When a laser light pulse with wavelength λ ≈ 500 nm is absorbed by the  C60, 
the fullerene is promoted to the first excited singlet state S 1. Because of the 
attached nitroxide groups, the spin multiplicity of the whole molecule in this state 
 1494 K. Möbius et al.
Fig. 9  X-band EPR spectra of biradicals IIa (left panel) and IIb (right panel) recorded in glassy matrix 
of toluene at 120 K. a Integrated EPR spectra recorded in the dark. b TR-EPR spectra recorded at 0.4 μs 
after the laser pulse, c TR-EPR spectra recorded at 70 μs after the laser pulse (from Ref. [22])
is a doublet for the monoradicals Ia and Ib, and a mixed singlet–triplet state for 
the biradicals IIa and IIb. Intersystem crossing (ISC) is caused by spin–orbit and 
spin–spin exchange interactions of the unpaired electrons of the nitroxide with 
those in the fullerene SOMOs. This generates a number of electronic states char-
acterized by triplet excitation on the  C60 part, which interacts only weakly with 
the nitroxide spin states. In a profound quantum mechanical analysis, the Corvaja 
group has shown that the TR-EPR spectra of nitroxide spin-labeled fullerenes can 
be understood on the basis of the total spin Hamiltonian [22]:
H = HZ +Hhyp +Hexch +Hdip
where the first term on the right is the Zeeman interaction of the triplet state and 
of the radical(s); the second term is the hyperfine interaction term, which involves 
only the spin of the nitroxide groups of the radicals, neglecting the small coupling 
with 13C nuclei of the fullerene cage. The third term of the Hamiltonian comprises 
the exchange interaction between the triplet and the radical, and in the case of IIa 
and IIb, it also includes the exchange term between the radicals. The last term of the 
Hamiltonian represents the dipolar interaction between the triplet electron spin and 
the nitroxide spins. The magnitude of the electron exchange interaction between the 
two unpaired electrons in the ground state is obtained from the analysis of the EPR 
spectra in the dark.
Nitroxide Spin Labels for Exploring Relationships Between… 1495
In case of the quintet excited state, the assignment was confirmed by measuring 
the frequency of the echo-detected transient nutation. Fourier transformation of the 
transient spectra along the microwave pulse length allowed to disentangle the quintet 
transitions and to obtain precise measurements of the zero-field splitting parameters.
Single and double nitroxide spin-labeled C 60 derivatives in frozen matrices give 
quite different TR-EPR spectra depending on the magnitude of the exchange inter-
action between the unpaired spins on the triplet fullerene moiety and the nitroxide 
radical spin(s). In the fullerene derivatives Ib and IIb, where the nitroxide radical is 
bound to a malonate ester, the exchange coupling is weak and the frozen-solution 
TR-EPR spectra are very similar to those of triplet-excited fullerene monoadducts. 
In the phosphorylated methanofullerenes Ia and IIa, a strong spin–spin coupling is 
found probably caused by super-exchange promoted by the phosphorus atom. The 
frozen-solution spectra show the typical features of a quartet state (for the single 
labeled derivative) and of a quintet state (for the double labeled derivative).
As far as we know, this is the first determination of the magnitude and sign of the 
radical-triplet exchange coupling J in a radical-triplet pair (RTP).
For Ia,  JRTP is negative and its absolute value is large compared with the nitroxide 
14N hyperfine coupling. For both IIa and IIb in solution, the Corvaja group observed 
a quintet state arising from the exchange coupling of the fullerene triplet spins with 
the spins of the two nitroxides. From computer simulation of the TR-EPR spectra, a 
negative value of the radical–radical exchange interaction  JRP was inferred.
Sign and magnitude of J are indicative of the number of σ-bonds separating the 
π-centers of a biradical. According to the McConnell theory of exchange coupling 
J [29–32], the sign of J alternates with the number of σ-bonds. A single bond gives 
antiferromagnetic coupling (J < 0), and two bonds give ferromagnetic coupling 
(J > 0). The dipolar through-space contribution to J depends on the distance between 
the radicals and their relative orientation.
In the biradical units IIa and IIb, the second nitroxide label is linked to the 
fullerene in exactly the same way as the monoradical units Ia and Ib. This symme-
try allows to assume that the same interaction (dipolar and exchange) between the 
nitroxide group and the photoexcited fullerene exists in the monoradical and biradi-
cal systems. Therefore, a strong coupling between the nitroxide spins and the fuller-
ene triplet is expected in IIa, and a weaker one in IIb. Finally, we want to emphasize 
that the understanding of sign and magnitude of the exchange interaction between 
triplet-excited fullerenes and nitroxide radicals has been greatly advanced by the 
Corvaja group.
2.2 E xamples from Berlin/Mülheim: High‑Field EPR, EDNMR and ENDOR 
on Nitroxide–Solvent Interactions and Spin‑Labeled Photosynthetic Reaction 
Centers
The inherent limitation of EPR—the need for paramagnetic samples—is not a seri-
ous restriction in biomolecular spectroscopy since during the last decades chemical 
techniques have been developed to site-specifically introduce nitroxide spin labels 
into diamagnetic protein molecules as “spy probes” to explore local structures and 
 1496 K. Möbius et al.
dynamics. They allow EPR studies of the target molecules without significantly dis-
turbing their structure and function.
Wayne Hubbel at UCLA (USA) deserves credit for being the first to have real-
ized the potential of site-directed spin labeling (SDSL) for the exploration of pro-
tein structure and dynamics [8, 9, 33–35]. A commonly used nitroxide spin label 
is MTSSL (1-Oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate. 
MTSSL (or MTSL) is clearly the current “work-horse” and best-characterized 
nitroxide spin label for protein labeling [36]. W.L. Hubbell’s laboratory and those 
of his early and later collaborators, e.g., H.-J. Steinhoff (University of Osnabrück), 
systematically developed the SDSL technology into a quantitative tool for structural 
biology that complements NMR and crystallographic methods, see for instance 
Refs. [37–43], and for a more recent literature review, Ref. [44].
The investigation of nitroxide spin-labeled membrane proteins using high-field 
EPR, ENDOR, PELDOR, EDNMR, in conjunction with quantum chemical calcu-
lations, has a long tradition in our laboratories, dating back to the early 2000s [43, 
45]. The membrane protein complexes studied include bacteriorhodopsin, DNA-
photolyase, colicin A toxin, bacterial photosynthetic reaction centers with particu-
lar emphasis on H-bond networks and weak solvent interactions with the cofactors. 
Because of the large number of protein complexes and their solvent matrices, it was 
not easy for us to select examples that would match both scope and spirit of our 
mini-review. We have decided to limit ourselves to a few interesting examples taken 
from Refs. [46–52].
2.2.1  Nitroxide Spin Labels Probing Hydrogen‑Bond Formation
Using high-field EPR techniques either in cw or in pulsed mode of operation, the 
nitroxide spin-label approach has been successfully applied to various membrane 
and water-soluble proteins [40, 42]. The variation of the EPR parameters from site 
to site of the attached spin label was generally interpreted in terms of local polarity 
and/or hydrogen-bonding effects of the matrix [53–55].
The precision measurement of nitroxide magnetic interaction parameters provides 
encoded local information on solvent properties such as polarity (defined by the 
dielectric constant ε) and proticity (defined as propensity for hydrogen-bond forma-
tion). The appropriate interaction parameters are the g-tensor (reflecting the Zeeman 
and spin–orbit interactions of the unpaired electron spin), the nitrogen hyperfine 
A-tensor (reflecting the interaction of the unpaired electron with a 14N (I = 1) or 15N 
(I = 1/2) nuclear-spin), and the nitrogen quadrupole P-tensor (reflecting the interac-
tion of the 14N nuclear quadrupole moment with the electric field gradient of the 
electrons at this site).
In liquid solution, rapid Brownian motion averages out all anisotropic interac-
tions and only isotropic interaction parameters are retained. Characteristic variations 
of Aiso and giso (given by 1/3 trace of the A- and g-tensors) for nitroxide radicals in 
solvents of different polarity and proticity were recognized already many years ago 
[56–59]. Shifts of  giso to smaller values and Aiso to larger values were observed with 
increasing polarity of the environment. A linear correlation between (ε − 1)/(ε + 1) 
and Aiso could be obtained for aprotic solvents [57]. Interestingly, this correlation did 
Nitroxide Spin Labels for Exploring Relationships Between… 1497
not apply to protic solvents. In aprotic and protic solvents of comparable dielectric 
constant, larger Aiso shifts were observed in the protic solvent. This suggested two 
main effects to be responsible for the shifts: (a) non-site-specific interactions due 
to the bulk electric field and (b) site-specific interactions, mostly due to hydrogen 
bonds (H bonds), which result in local electric field changes affecting the radical’s 
electronic structure.
Hence, the information obtained from liquid-solution magnetic resonance stud-
ies is rather limited, i.e., a detailed data interpretation requires both the value and 
orientation of the interaction tensors to be known. Hence, for frozen-solution matri-
ces, the characteristics of the microenvironment are best determined by high-field/
high-frequency EPR below the glass-transition temperature of the solvent. Cw EPR 
at high magnetic fields allows one to resolve the nitroxide g-tensor principal com-
ponents. The  gxx component is particularly sensitive to the microenvironment. In 
addition, high-field ENDOR or ELDOR-detected NMR (EDNMR) can favorably be 
used to measure the nitrogen hyperfine-tensor and quadrupole-tensor components 
with high precision, e.g., Azz and Pzz, which react most sensitively on interactions 
with the environment.
We noticed [46] that for nitroxide radicals in frozen (protic) 2-propanol, a pro-
nounced heterogeneity of both gxx and Azz is present that can be resolved by W-band 
cw EPR and EDNMR techniques. The distinct correlation of gxx versus Azz values 
senses different nitroxide populations that are characterized by their different local 
environments. They were ascribed to a different hydrogen-bond network of the 
nitroxides, i.e., whether they develop one or two H bonds to the solvent molecules—
or no H bonds at all. In a subsequent study utilizing 275 GHz cw EPR and W-band 
EDNMR [55], it was demonstrated that also in frozen water solution, similar to 
2-propanol, two nitroxide fractions are observed. They were ascribed to nitroxide 
populations carrying one or two H bonds to the matrix.
Apparently, for nitroxide radicals in frozen solution, the relationship between the 
magnetic parameters and the hydrogen-bonding situation of the spin probe depends 
on the solvent matrix and is often more complex than assumed using simple mod-
els. SDSL methodology with nitroxide radicals thus requires differentiation between 
polarity and hydrogen bonding of the microenvironment to obtain meaningful inter-
pretations of the experimental EPR results. Both contributions, however, are theo-
retically expected to affect the nitroxide magnetic parameters in a similar way. This 
aggravates distinction between hydrogen bonding and other non-site-specific sol-
ute–solvent interactions.
For nitroxide radicals in their solution shells, absolute values of the magnetic 
interaction tensors are still rather elaborate and problematic to calculate, e.g., by 
DFT methods. This is due to the solvation-dependent structure of nitroxide labels 
[60, 61]. The situation is different for planar quinone anion radicals in solution [62, 
63]. For them excellent agreement between DFT calculations of magnetic interac-
tion parameters and experimental values could be achieved because of good geom-
etry data and sufficiently large solvent shell models.
To shed further light on the problem, we have investigated protic organic solvents 
that represent the most abundant H-bond donor groups of amino-acid side chains 
in proteins. In addition, we chose an aprotic and apolar solvent, ortho-terphenyl 
 1498 K. Möbius et al.
Scheme 2  Structure of the two 
nitroxides R1 and R2 described 
in this work
Fig. 10  Experimental a W-band (95 GHz) and b 244 GHz cw EPR spectra of nitroxide radical R1-D16 
(Scheme 2) in frozen solutions of ortho-terphenyl (OTP), 2-propanol and phenol recorded at 90 K and 
70 K, respectively. The spectral positions that correspond to different principal  gxx-tensor values and 14N 
 Azz values are indicated by dotted and dashed lines, respectively (adapted from Ref. [50])
(OTP). To maximize the EPR spectral resolution, we used a perdeuterated nitrox-
ide radical and perdeuterated solvents. The aim was to obtain reliable correlations 
of nitroxide magnetic parameters with the prevailing H-bond situation and the bulk 
solvent polarity. Since the investigated pyrroline-type nitroxide radical (3-hydroxy-
methyl-2,2,5,5-tetramethylpyrrolin-1-oxyl) represents the head group of the widely 
used MTS spin label, (S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl-
methanesulfonothioate). This information is particularly relevant for SDSL studies 
of spin-labeled proteins.
In our first example, we present a detailed high-field EPR investigation of mag-
netic parameters of the deuterated nitroxide radical R1 (see Scheme 2) dissolved in 
deuterated frozen solvents of polar or apolar, protic or aprotic character. The sol-
vents include ortho-terphenyl, methanol, propanol, glycerol, aniline, phenol, and 
water (see Figs. 10 and 11). The analysis of the highly resolved 95 GHz EDNMR 
and 244 GHz cw EPR spectra is detailed below. This yielded precise electron Zee-
man (g ), 14xx N hyperfine (A 14zz) and N quadrupole (Pzz) tensor components. Remark-
able changes in the solvation networks were found in the different matrices. The 
experimental data were discussed on the basis of previous semi-empirical and DFT 
quantum chemical calculations in an attempt to adequately model the nitroxide-
matrix interactions [50]. The obtained results demonstrate that the principal val-
ues of all the magnetic interaction parameters primarily depend on the nitroxide 
Nitroxide Spin Labels for Exploring Relationships Between… 1499
Fig. 11  a Comparison of the  gxx region of the 244  GHz cw EPR spectra of nitroxide radical R1-D16 
in frozen solutions of (from top to bottom) OTP, aniline, phenol, 2-propanol and glycerol. The spectra 
were recorded at 70 K. Best-fit simulations of the line(s) are shown for zero (red), one (green) and two 
(blue) H bonds to the nitroxide. The spectral range presented is indicated by the dotted box in Fig. 10b. b 
Molecular structures of solvent molecules used as hosts for the diluted nitroxide solutions (adapted from 
Ref. [50])
hydrogen-bond situation that depends on the donor group of the solvent. The solvent 
bulk polarity, as described by the static dielectric constant, is of minor importance.
In Fig.  10b, the 244  GHz EPR spectra of R1-D16 in 3 different solvents are 
shown. The analysis of the region at low field is indicative of differences in the 
H-bonding situations between the nitroxide and the solvent that is strongly affect-
ing the  gxx value: (i) with no H bonds for OTP (0), (ii) an equilibrium of nitroxides 
with one (1) and zero (0) H bonds for 2-propanol and (iii) with one (1) and two (2) 
H bonds in case of phenol as solvent. This is expressed in the drastic shift of the 
 gxx value of the nitroxide; examples including further solvent matrices are shown 
in Fig. 11. In the 244 GHz EPR spectra (Fig. 10b), the different nitrogen  Azz com-
ponents (also A yy and A xx) of the nitroxide are not well resolved due to the large 
linewidths; these splittings can be better observed at W-band EPR (95  GHz), see 
Fig. 10a, and are fully resolved in the W-band EDNMR spectra shown in Fig. 12, 
also the 14N quadrupole components  Pzz are resolved. The employment of a 15N 
labeled nitroxide with I = 1/2 lacks the quadrupole splitting and helped to precisely 
determine the nitrogen quadrupole parameters. Figure 13 shows a set of EDNMR 
spectra in different selected solvents. The formation of zero, one or two hydrogen 
bonds to the nitroxide has been detected and analyzed. There is a linear correla-
tion between the solvent-induced changes of g  and 14xx N Azz and also between Azz 
and Pzz values [50]. Note that the molecular differences of the solvents can lead to 
(slightly) different H-bond situations which is manifested in the high-field EPR and 
EDNMR spectra. Hydrogen bonding is clearly the dominant interaction leading 
 1500 K. Möbius et al.
Fig. 12  W-band EDNMR spectra of R1-D16 in phenol-D6 at 50  K. a The echo-detected EPR inten-
sity around the gzz, MI = -1 spectral position with indicated magnetic field positions of the individual 
EDNMR recordings. b Individual 14N EDNMR spectra at magnetic field positions indicated in a. c 
Example of two best-fit Gaussian lines to the experimental EDNMR spectrum marked in red in b. The 
two individual Gaussian components are shown by blue and green lines. d Experimental first-derivative 
EDNMR spectrum overlaid with the best-fit simulation performed in the first-derivative mode (dashed 
trace) (adapted from Ref. [50])
Fig. 13  W-band 14N EDNMR spectra of nitroxide radical R1-D16 in frozen solutions of OTP, aniline, 
phenol, 2-propanol, methanol, glycerol and water. The spectra were recorded close to the gzz, MI = − 1 
spectral position at 50 K. The experimental spectra (black traces) are superimposed with best-fit Gauss-
ian lines that are assigned to nitroxide radicals forming zero (red trace), one (green trace) or two (blue 
trace) H bonds with the solvent molecules. The EDNMR lines appear around half the 14N  Azz hyperfine 
coupling (see Fig. 12c). The fits were performed on the first-derivative mode of the experimental traces; 
for details of the analysis, see Ref. [50] (from Ref. [50])
Nitroxide Spin Labels for Exploring Relationships Between… 1501
Fig. 14  Top and side views of 
the energy-optimized structure 
of the R1 nitroxide complexed 
with one 2-propanol molecule. 
The principal directions of the 
calculated nitroxide g- and 
bridged proton hyperfine tensors 
are indicated (from Ref. [52])
to the observed changes of the nitroxide magnetic resonance parameters; the bulk 
polarity of the medium plays only a minor role, as shown in Ref. [50].
Calculations show that the H bonds to R1 are preferentially formed in the molec-
ular plane targeting the lone pairs of the oxygen of the nitroxide group carrying 
the electron spin (σ-type H bond). In Fig. 14, the geometry of the H-bond situation 
between R1 and 2-propanol is shown as an example [52]. Good agreement between 
the experimental and DFT-calculated spectroscopic data could be obtained. Note, 
however, that the H-bond situation is more complicated in case of non-planar nitrox-
ides as demonstrated, e.g., for piperidine-type nitroxides with a six-membered ring 
(see R2 in Scheme 2), in which out-of-plane H bonds with (partial) π-bond character 
(to the oxygen and/or nitrogen) are formed [52].
An interesting aspect of EPR/ENDOR on H-bonded systems lies in the possi-
bility to determine the hyperfine-tensor parameters of the proton (or deuteron) in 
the hydrogen bond. They are strongly affected by the bond length and its detailed 
geometry and allow an interpretation of the hydrogen bond strength affecting the 
physical properties of the system. Using ENDOR, this situation has been studied 
for nitroxide R1-D16 hydrogen bonded to 2-propanol-D7(OH) glass [52]. Even more 
information is available from the quadrupole coupling of 2H, in case the sample is 
prepared with a deuteron in the hydrogen bond. For more experimental details and 
the performed analyses, see Ref. [52]. Similar conclusions have been presented ear-
lier for H-bonding to quinone radicals in vitro and in proteins [62–67].
We summarize: high-field EPR, ENDOR and EDNMR have been used to deter-
mine changes of the nitroxide magnetic resonance parameters caused by different 
solvents. Hydrogen bonding was found to play a major role in the observed changes 
of the NO electronic structure. NO-complexes with zero, one and two H bonds 
 1502 K. Möbius et al.
could be observed and characterized. The planar five-membered ring system of the 
pyrroline-type nitroxide radical R1 was found to form well-defined in-plane σ-type 
hydrogen-bonded complexes with H-bond donor solvents in frozen solution. The 
measured hyperfine parameters of the hydrogen-bridge proton and the internal mag-
netic parameters describing the electron Zeeman and the electron-nuclear hyperfine 
and nuclear quadrupole interactions are in good agreement with values predicted by 
state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-pla-
nar six-membered ring system of piperidine-type nitroxide radicals (e.g., TEMPOL, 
R2) reveals a more complex situation, i.e., a mixture of a σ-type and an out-of-plane 
π-type complex, both present in comparable fraction in frozen solution. For such 
systems, it is more difficult to predict magnetic interaction parameters using quan-
tum chemical calculations, probably due to the considerable flexibility of the nitrox-
ide and hydrogen-bonded complex. The detailed information about nitroxide/solvent 
complexes is of particular importance for dynamic nuclear polarization (DNP) and 
site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents 
or spin labels, respectively.
2.2.2  Local Water Sensing: Water Exchange in Bacterial Photosynthetic Reaction 
Centers Embedded in a Trehalose Glass Studied by High‑Field Multi‑resonance 
EPR
Water plays an essential role in the chemistry of life. It governs the internal dynam-
ics of biological macromolecules, such as proteins. Unrestricted dynamics at a spe-
cific time scale is a crucial requirement for the specific biological activity of pro-
teins. This includes enzyme activity, macromolecular recognition, ligand binding 
and participation in electron and proton transfer processes for biological function. 
Under physiological conditions, (bio)macromolecules fluctuate between a multi-
tude of conformational sub-states in a rugged energy landscape that is hierarchically 
organized in energy tiers [68–70]. Their dynamics span an enormous time range, 
from sub-picosecond to tens of microseconds, and include a multitude of stochas-
tic local and collective motions, from bond vibrations to domain motions [69, 71, 
72]. Water drives protein folding through hydrophobic interactions [73–76], but also 
contributes to the stabilization of the 3D protein structure and modulates the pro-
tein dynamics [77]. For most proteins cooled below the glass-transition temperature 
(typically around 200 K), their biological function is blocked due to restricted con-
formational motion.
In biological systems, the solvent water is generally divided into three distinctly 
different classes of functionality: (i) internal, strongly bound water that cannot be 
removed even upon lyophilization; (ii) surface water in the hydration shell of the 
protein at the solute–solvent interface, and (iii) bulk water randomly distributed in 
the protein matrix. Water molecules in the protein hydration layer have restricted 
dynamics compared to water molecules in the bulk. The thickness of the hydration 
layer at the solute–solvent interface is still a matter of debate.
The “freezing out” of conformational dynamics by lowering the temperature 
is a common strategy for studying function-dynamics relationships in proteins. 
To minimize freezing damage by ice crystals, proteins are usually frozen in the 
Nitroxide Spin Labels for Exploring Relationships Between… 1503
presence of cryoprotectants. This approach is, however, problematic because it 
aggravates disentanglement of the influences of solvent and temperature on the 
protein dynamics. An elegant alternative approach is to embed the protein into 
amorphous matrices formed by disaccharides like trehalose (α-d-glucopyranosil-
α-d-glucopyranoside) [78, 79], see Fig. 16b. This allows the native protein fold-
ing to be preserved during extensive protein dehydration, even at temperatures 
well above room temperature. In nature, the extraordinary bio-protective capa-
bilities of disaccharide glasses are exploited by specific organisms, which are 
able to survive extreme conditions of temperature and dehydration by entering a 
state of reversibly arrested metabolic activity, called anhydrobiosis (“Life without 
water”) [79–81].
At room temperature, the stepwise dehydration of the trehalose matrix results in 
increasingly inhibited dynamics of the embedded protein. This was observed in both 
small globular proteins like myoglobin [78, 82] and large membrane proteins like 
bacterial photosynthetic reaction centers [79, 83] as well as photosystem I [84] and 
photosystem II [85] of oxygenic photosynthesis.
Trehalose is the most efficient sugar for bio-protection against extreme dehy-
dration and osmotic stress. It is an active stabilizer of enzymes, proteins, vaccines, 
pharmaceutical preparations and even organs for transplantation. The mechanistic 
details of trehalose efficiency are, however, not yet clear, but probably involve a 
combination of several factors that include its extraordinarily high glass-transition 
temperature (385 K) [86], its distinct propensity for hydrogen-bonding and the pro-
nounced rigidity of its dehydrated glass matrix [87].
In the following, we wish to address the biologically important question of detec-
tion and quantification of local water, in contrast to bulk water, in membrane pro-
teins. For water sensing, we chose the bacterial reaction center (bRC) from Rhodo-
bacter (Rb.) sphaeroides R26, in which the paramagnetic  Fe2+ had been replaced by 
diamagnetic Z n2+. The Zn-bRC was embedded into a trehalose glass matrix [49]. 
Local water sensing was made possible by monitoring the electron–nuclear hyper-
fine interaction of isotope-enriched water  (D2O and  H 172 O) with the paramagnetic 
sites of the bRC.
The bRC of Rb. sphaeroides is an integral membrane protein (see Fig. 15a, b) that 
catalyzes the primary photochemical processes to convert light energy into chemical 
free energy [88–90]. The three protein subunits, L, M, and H host several cofactors 
[91] sequentially involved in light-driven electron transfer. Two bacteriochlorophyll 
a (BChl a) molecules (blue) near the periplasmic side of the membrane form the 
“special pair” P 865, the primary electron donor. After photoexcitation to its singlet 
state, the subsequent electron transfer proceeds predominantly via the protein branch 
A [88] to the Q A acceptor. In the present study, the secondary electron-transfer step, 
Q∙− → Q B, is blocked using the inhibitor stigmatellin [92]. Thus, in the charge sepa-A
ration process the radical pair state P∙+ Q∙− is created in very high yield with two 
865 A
radical species located in a different protein environment.
The native bRC contains five cysteine residues buried within the protein domains, 
with the exception of cysteine 156, which is moderately solvent-exposed in subu-
nit H. The cysteine 156 residue can be site-specifically spin labeled with an exter-
nal paramagnetic probe molecule. We used MTSSL [93, 94]. The nitroxide label in 
 1504 K. Möbius et al.
Fig. 15  a X-ray structural model of the bRC from Rb. sphaeroides R26 [112] with the protein subunits L, 
M, and H carrying the electron-transfer cofactors P 865 (a bacteriochlorophyll a (BChl) dimer that forms 
the “special pair” electron donor) near the periplasmic side of the membrane, two monomeric BChl’s, 
two BPhe’s (bacteriopheophytin a), two ubiquinone-10 molecules ( QA and  QB) and a non-heme iron 
F e2+. The paramagnetic  Fe2+ cofactor is replaced by diamagnetic  Zn2+. Q A and  QB form the primary and 
secondary electron acceptor, respectively. Light-induced electron transfer proceeds predominantly along 
the A branch of the cofactors (“unidirectionality” enigma). Here, the quinone  QB is replaced by stigma-
tellin to block the secondary electron-transfer step from Q∙− to  QB. b Molecular surface representation A
of the spin-labeled bRC (SL-bRC) from Rb. sphaeroides R26, indicated are the primary electron donor 
 P865 (in blue), the primary electron acceptor Q A (in red) and the nitroxide spin label MTSSL attached to 
Cys156 in the H protein subunit (in green). Strongly bound water molecules within 1 nm radius of each 
paramagnetic center are shown as greenish colored spheres, they were obtained from the  a high resolu-
tion X-ray structure  [113] (adopted from Ref. [49])
the bRC protein [95] provides a third paramagnetic probe within the bRC besides 
the “natural” radical ions P∙+  and Q∙− . The W-band EPR spectra of the 3 radicals 
865 A
are schematically depicted in Fig. 16a. According to the protein 3D structure, the 
three paramagnetic reporter groups are in distinctly different local environments (see 
Fig. 15b) and serve as local probes to identify water molecules via dipolar and quad-
rupolar hyperfine interactions with either D (2H) or 17O nuclei.
In bRCs, a coating with trehalose influences the lifetime of the charge-sepa-
rated radical pair state, P∙+ Q∙− . Upon progressive dehydration of the trehalose 
865 A
matrix, the kinetics of P∙+ Q∙− charge recombination becomes faster and exhibits 
865 A
widely distributed rate constants [79, 81, 96, 97]. Two relative hydration levels, 
r, were chosen in this work, which result in a different extent of slaving the pro-
tein dynamics to the embedding sugar matrix: (i) r = 11% for which the protein 
dynamics is arrested on the time scale of seconds [97], and (ii) r = 74% for which 
the dynamics is only mildly retarded as compared to solution [98]. Note that in 
the dehydrated (dry) case r = 11% the solution contains only ~ 0.5 waters per tre-
halose molecule.
Nitroxide Spin Labels for Exploring Relationships Between… 1505
Fig. 16  a Simulated rigid limit W-band EPR absorption spectra of P∙+  (blue trace), Q∙− (red trace), and 
865 A
of the nitroxide radical R1 (green trace). b Chemical structures of the pyrroline-type nitroxide radical R1 
and the disaccharide trehalose (from Ref. [49])
The relative hydration levels r were adjusted by applying the isopiestic method 
of controlled sample dehydration [99]. It has been employed successfully both 
in EPR [100] and IR spectroscopy [101–103]. The samples were transferred into 
a sealed box where further dehydration of the sample occurred via controlled 
exposition to an atmosphere of low relative humidity over a saturated aqueous 
salt solution at room temperature using  H2O with natural isotope abundance. For 
measurements on samples equilibrated to the desired relative humidity with either 
 D 172O or H 2 O vapor, the saturated solutions of an adequate salt were prepared 
using the respective isotope labeled water at room temperature.
In a first series of experiments, the trehalose glass was characterized after dehy-
dration at different humidity levels using the homogeneously dispersed spin label 
R1 (Fig.  17b) in absence of the protein (bRC). The D 2O content was determined 
by W-band EDNMR monitoring the amplitude of the D-NMR signal associated 
with the nitroxide signal (at position gzz, MI = 0), see Fig. 17. The spectra in panel 
a show the signal for the sample directly prepared in  D2O (top, red) and for a series 
of exchange times (6–170 h) of the sample after it had been equilibrated at r = 11% 
(LiCl/D2O). A control prepared with H 2O shows no D matrix signal. The D matrix 
line at the D Larmor frequency (21.9  MHz) shows a clear dependence on time 
depicted in panel c, reaching a plateau after approx. 3 days. It only reaches ~ 50% of 
the intensity of the sample prepared directly in  D2O. This indicates that the trehalose 
hydroxyl groups exchange slower and do not contribute significantly to the water 
exchange experiment [49]. In the range, 30–55  MHz signals from the 14N of the 
nitroxide appear with similar intensity over the whole time range.
 1506 K. Möbius et al.
Fig. 17  a W-band EDNMR spectra recorded for R1/trehalose glasses at 60 K. The glasses were equili-
brated with a saturated LiCl/D2O solution for the indicated time. The red trace corresponds to the R1/tre-
halose glass prepared in  D2O. The spectra were recorded at the field position indicated in b by the green 
arrow. b Echo-detected field-swept W-band EPR spectrum of the nitroxide radical R1 embedded in treha-
lose glass. c Intensity of the deuterium EDNMR line as a function of equilibration time. The red dashed 
line corresponds to the deuterium line intensity evaluated for R1/trehalose glass prepared in D 2O. The 
error margins are determined by data analysis from two independent series of measurements. d W-band 
Mims ENDOR spectra recorded for R1/trehalose glasses at 60  K. The glasses were equilibrated with 
a saturated LiCl/D2O solution for the indicated time. The red trace corresponds to R1/trehalose glass 
prepared directly in D 2O. The spectra were recorded at the field position indicated in b by the blue arrow 
(adopted from Ref. [49])
The “matrix line” detected at νD could be simulated with a Gaussian lineshape 
for all spectra, no sign of splitting or shoulders were observed. No D nuclei hyper-
fine coupled to the nitroxide could be detected, which are expected for H bonds to 
the NO group. To check on this interaction, a series of Mims ENDOR spectra were 
measured in the gxx–gyy range where the largest D ENDOR couplings were expected 
(see Fig. 17d). Here, after 6 h of exchange a D hyperfine splitting of 1.8 ± 0.3 MHz 
was observed, in addition to the matrix line. Thus, both distant and local (H bonded) 
deuterium signals could be detected. However, the linewidth is rather broad in the 
rigid dry trehalose glass preventing detection of accurate hyperfine and nuclear 
quadrupole interactions. A much better resolution was observed in case of rehydra-
tion of trehalose at r = 74% where the nitroxide molecules are expected to sit pre-
dominantly in water-rich domains that allow an optimum geometry of the nitrox-
ide–hydrogen bond complexes to be formed (see Fig. S3 and S5 in Ref. [49]). Here, 
an increasing fraction of nitroxides is forming not only one but two H bonds to the 
solvent.
This series of experiments performed on the nitroxide R1/trehalose system 
shows how water sensing can be performed using labeled water  (D2O) in combina-
tion with high-field EPR and EDNMR/ENDOR. It also demonstrates how directly 
coordinated (e.g., H bonded) and distant water can be distinguished. This approach 
has also been used in exactly the same way to study spin-labeled proteins, e.g., the 
Zn-substituted bRC introduced above (see Fig. 4a-b in Ref. [49]). In this work, we 
noticed that other protons at the protein could undergo H/D exchange and add inten-
sity to the D matrix line leading to complications in the analysis. In such cases it is 
advantageous to use  H 172 O, i.e., 17O as nuclear spin probe as is demonstrated below.
Nitroxide Spin Labels for Exploring Relationships Between… 1507
Fig. 18  a W-band EDNMR spectra recorded at the field position of maximum signal intensity of the Q∙− 
A
EPR spectrum (marked by the black arrow in b). b The sum of EPR absorption spectra of P∙+  and Q∙− 
865 A
ionic states of cofactors (black trace). The simulated spectra corresponding to Q∙− (red trace) and P∙+  
A 865
(blue trace) are also shown. c W-band ENDOR spectra for bRCs in solution dissolved in Tris/HCl buffer 
prepared in  D2O, and for bRC/trehalose glass equilibrated at r = 74% (NaCl/D2O) recorded at the field 
position indicated by the black arrow in (b) for Q∙− . d W-band EDNMR spectra for bRCs dissolved in 
A
Tris/HCl buffer prepared in D 2O recorded at field positions indicated by green and blue arrows in b for 
P∙+  . For details, see Ref. [49] (adopted from Ref. [49])
865
In the Zn-bRC, two additional paramagnetic centers P∙+  and Q∙− are present 
865 A
under illumination and can be used for water sensing in the surrounding of the rad-
icals in the protein (see Fig.  18). For Q∙−(Fig.  18a), W-band EDNMR detects an 
A
(unsplit) 14N resonance in all spectra that is assigned to nitrogen(s) from His(M219) 
and/or Ala(M260), i.e., from residues known to be involved in formation of tight 
hydrogen bonds to the oxygens of Q∙−[63–67]. At the D Larmor frequency νD, a A
deuterium matrix line is observed, its intensity is approximately the same in fro-
zen  D2O and Zn-bRC/trehalose rehydrated at r = 74% (NaCl/D2O). This shows that 
the diffusion of water within the protein in rehydrated trehalose is not hampered as 
compared to that of protein in solution. In case of the dry protein/trehalose glass 
(r = 11%, LiCl/D2O) the D matrix line is also present but is only 1/10 of that in fro-
zen solution. Clearly, trehalose coating does not prevent water exchange, which hap-
pens between trehalose and protein on the same time scale as between trehalose and 
the vapor atmosphere [49].
The large linewidth prevents resolution of the D hfcs in the EDNMR. In Fig. 18c, 
D ENDOR spectra are shown for Zn-bRCs/trehalose (r = 74% rehydration) and in 
buffer/D2O solution. The spectra are identical and could be analyzed, and they result 
from D in hydrogen bonds to Q∙− in well-defined geometry with dipolar and quad-
A
rupolar contributions. These have been observed and analyzed earlier [63–67]. It 
should be noted that embedding in a trehalose matrix is not changing the H-bonding 
geometry to the quinone that is important for the function of the ET in the bRC.
Water exchange in the surrounding of P∙+  could also be probed by selecting a 
865
position for EDNMR and ENDOR in the W-band EPR where only P∙+  absorbs (see 
865
Fig. 18b). In the EDNMR spectrum, strong 14N resonances from the BChl a mole-
cules in P∙+  are observed at the ν(14N) Larmor frequency. In addition, the respective 
865
 1508 K. Möbius et al.
Fig. 19  a W-band EDNMR spectra for SL-bRC/trehalose glass samples equilibrated for 70 h at r = 74% 
(NaCl/H 172 O) black trace; r = 11% (LiCl/H 172 O) magenta trace; and r = 11% (LiCl/H2O) red trace. The 
spectra were recorded at the same nitroxide spectral position (gzz, MI = 0) indicated in Fig. 14b by the 
green arrow. EDNMR spectra are shown for ν < 0 to obtain a better separation between 17O and 14N sig-
nal contributions (see Fig. S2b in SI of ref. [49]). b The 17O EDNMR spectra for r = 74% (NaCl/H 172 O) 
(black trace) and r = 11% (LiCl/H 172 O) (magenta trace) samples. The gray dashed lines show the best-
fit simulation of experimental recordings to a Gaussian line. c Microwave field amplitude dependence 
of 17O EDNMR spectra of SL-bRC/trehalose glasses equilibrated for 3  days at r = 11% (LiCl/H 172 O). 
The black spectra were recorded using an HTA pulse length of tHTA = 20  μs. The blue trace shows 
the EDNMR spectrum acquired with tHTA = 7  μs. The green line shows the Davies ENDOR spectrum 
recorded at the same spectral position (from Ref. [49])
double-quantum transitions are detected which overlap with the D matrix signal at 
21.8 MHz. The lack of a (detectable) D matrix line is possibly due to the absence 
of exchangeable water in the vicinity of P∙+  ; however, this conclusion needs fur-
865
ther confirmation. The delocalized spin density distribution over the large P∙+  dimer 
865
could be the reason for the absence of D hfcs large enough for detection [104]. This 
is, however, not the case for liquid-solution ENDOR of P∙+  [105, 106] or the related 
865
BChl a radical cation [107].
In the last example, 17O is used as nuclear-spin probe instead of D, i.e.,  H 172 O 
water is employed. This is necessary since with  D2O unwanted H/D exchange could 
take place in the water sensing experiments preventing quantitative conclusions. In 
Fig. 19, 17O EDNMR experiments are shown on spin-labeled bRC/trehalose equili-
brated at r = 11% (LiCl/H 172 O) and at r = 74% (NaCl/H 172 O) with the magnetic field 
positioned at the NO spin label (g , M  = 0). In both spectra, a resonance at the 17zz I O 
Larmor frequency is present at 19.4 MHz as well as 14N lines from the nitroxide 
nitrogen. At low hydration level (r = 11%), the 17O matrix line is ~ 6 times smaller 
than at the high hydration level (r = 74%). Here, the difference in water content is 
more pronounced as in the case of  D2O, where H/D exchange contributes to the D 
line intensity. Based on the lineshape, the water molecules are assumed to belong 
to distant waters in the “dry case”, whereas at higher hydration levels also, local 
water molecules contribute that are hyperfine-coupled to the nitroxide. This is seen 
from the spectra presented in Fig. 19b, showing clear wings for the hydrated sam-
ple. In Fig. 19c, we have tried to improve the resolution of the coupled 17O nuclei 
and distinguish them from distant nuclei by changing the mw field and HTA pulse 
Nitroxide Spin Labels for Exploring Relationships Between… 1509
amplitude [108] in the EDNMR spectra (blue trace). For comparison, a Davies 
ENDOR spectrum (green) is also shown in which the matrix line is suppressed. The 
latter 17O EDNMR and ENDOR experiments clearly show a direct bonding of the 
water to the nitroxide probably via hydrogen bonding. This forms a promising basis 
for further experiments using these techniques in combination with 17O labeled 
water.
Finally, we note that recently there appeared a publication by the group of M. 
Bennati (Göttingen, Germany) about 17O hyperfine spectroscopy of the hydration 
structure of nitroxide radicals in aqueous solutions with support of quantum chemi-
cal calculations and MD simulations [109].
Conclusions: In the presented work, water accessibility was measured at 3 differ-
ent sites within a membrane protein, the bRC of Rb. sphaeroides. The paramagnetic 
reporter groups have distinctly different surroundings in the protein and can probe 
different types of water, i.e., internal, bulk and surface water. The bRC was embed-
ded in a trehalose matrix and equilibrated at low (r = 11%) and high (r = 74%) rela-
tive humidity. At low humidity, the matrix is rigid (with only ~ 0.5 waters per sugar 
molecule), whereas at higher humidity, more water molecules can serve as solvation 
partners for the protein.
To obtain details of the related hydration/dehydration process, the bRC/trehalose 
system was studied by sensitive high-resolution techniques, namely high-field EPR, 
EDNMR and ENDOR. The results show that these methods are very well suited 
to detect water in the vicinity of radical sites in the protein via dipolar and quad-
rupolar hyperfine interactions of magnetic nuclei such  as D or 17O when, respec-
tively, labeled water  (D2O and  H 172 O) is used for water exchange. The use of (the 
rather expensive) H 172 O has the advantage to avoid H/D exchange of the protein and 
should thus be used for quantitative studies.
The detailed analysis of the data demonstrated the very high efficacy of the tre-
halose in bio-protection. Several of the various models existing in the literature try-
ing to explain the special impact of this sugar on proteins, could be excluded based 
on the obtained results (for a detailed discussion, see Ref. [49]. It could further be 
shown that the best approach to explain hindering of the protein dynamics is pro-
vided by the “anchorage model” [110], in which a tight anchoring of the protein 
surface to the rigid matrix by an extended H-bond network is proposed.
The obtained spectra show that water molecules, detectable by the NO spin label 
attached to the bRC surface, are retained in the first and second solvation shell, even 
under extensive drying conditions (r = 11%) that block the internal protein dynam-
ics. Experiments on an intrinsic spin probe (Q ∙− ) in the bRC showed that residual 
A
protein motion still allows for exchange of strongly bound water within the protein 
and H/D exchange near the quinone radical site. The hydrogen bonding to the accep-
tor quinones has been discussed as key factor for proton-coupled electron transfer 
(PCET) and for the functioning of the bRC [111].
Our preliminary experiments using W-band 17O EDNMR and ENDOR gave 
very promising results: the ratio of the matrix 17O line at low and high humidity 
levels mirrored the water/trehalose molar ratio of both hydration states. Further-
more, detection of the 17O hyperfine coupling at higher hydration levels could be 
 1510 K. Möbius et al.
accomplished. Future work along these lines promises to deliver exquisite details of 
the water–protein interaction.
The presented methodology, W-band 17O EDNMR and ENDOR in conjunction 
with 17O isotope labeled water is very well suited for local water sensing in bio-mac-
romolecules. Clearly, a key issue is the understanding of the different types of water 
in living matter. It has been recognized that water is far more than merely a simple 
solvent—it is also indispensable for biological function.
Epilogue
We return to the Prologue, which dealt with our first encounter with Carlo 
Corvaja. That was in 1965 during the International EPR Conference in Cirences-
ter. Despite the Iron Curtain in Europe, there were still possibilities to meet EPR 
colleagues from West and East, in rare cases even from the USSR. A quarter of 
a century later, the Iron Curtain got pretty big holes, and East–West get-togethers 
became more frequent. New promising opportunities for East–West scientific coop-
erations opened up. Like all of us, Carlo Corvaja was also enthusiastic about these 
new opportunities and determined to seize them for the benefit of both sides. An 
outstanding example was the close collaboration with Yakov S. Lebedev and his 
team at the Institute of Chemical Physics of the Academy of Sciences in Moscow. It 
started in the early 1990s and lasted until his much too early death in 1996. During 
these years, Carlo Corvaja met Yakov Lebedev and his coworker Alexander Dubin-
skii as well as Yuri Grishin from Novosibirsk while visiting our high-field EPR lab-
oratory in Berlin.
In these years of thaw between East and West, generous public research funding 
programs were launched, e.g., by the German Research Foundation (DFG) the Pri-
ority Area “High-Field EPR Spectroscopy”. The EPR groups in Padova, Moscow, 
Novosibirsk, Kazan and Rehovot (Israel) were also involved in this Priority Area, 
whose scientists benefited greatly from the new exchange options with the EPR lab-
oratories in Berlin and Mülheim. In addition, indeed, we have now been fortunate to 
meet with Carlo Corvaja at new conference venues, e.g., in Venice, Padova, Kazan 
and Novosibirsk.
One man was on everyone’s lips at the time who had made this possible on the 
part of the Soviet Union: Mikhail Gorbachev (Gorbatschow), General Secretary 
of the Communist Party of the USSR. “Glasnost” and “Perestroika” describe the 
direction of his reforms. This development also benefited the foundation of “Applied 
Magnetic Resonance” in Kazan, the first international scientific journal in the USSR 
that published articles exclusively in English. Interestingly, since 1990 advertising 
posters with the name Gorbatschow appeared all over Berlin, on buses, trains and 
buildings. In fact, they were advertising the Gorbatschow vodka brand, which had 
been founded in Berlin already in 1921. In a delightful ambiguity, sales of Gor-
batschow vodka increased rapidly.
At present, political, economic and cultural relations between East and West have 
drastically deteriorated again. We do hope that the political decision-makers will be 
guided in the near future by the spirit of peace.
The authors of this article can only add: “Dum spiro spero”, which translates to 
“As long as I breathe, I hope”.
Nitroxide Spin Labels for Exploring Relationships Between… 1511
Acknowledgements We thank all our collaborators of the work described in this mini-review for their 
valuable contributions. They are acknowledged in the original articles cited here. This work is supported 
by the Deutsche Forschungsgemeinschaft and the Max-Planck-Gesellschaft. K. M. and M. P. acknowl-
edge sustaining support by the Freie Universität Berlin.
Author Contributions K.M., A.S., M.P. and W.L. wrote the manuscript. All the authors reviewed the 
manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL.
Data Availability No datasets were generated or analyzed during the current study.
Declarations 
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, 
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long 
as you give appropriate credit to the original author(s) and the source, provide a link to the Creative 
Commons licence, and indicate if changes were made. The images or other third party material in this 
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line 
to the material. If material is not included in the article’s Creative Commons licence and your intended 
use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permis-
sion directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/
licenses/by/4.0/.
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