Electron Transfer Relay Research Articles

Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays amongst the [FeFe]-hydrogenases, however it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of F-cluster relays. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII), exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric EPR, we resolved two EPR signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV vs. NHE, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain.

The blue light using the flavin (BLUF) domain is one of the smallest photoreceptors in nature, which consists of a unique bidirectional electron-coupled proton relay process in its photoactivation reaction cycle. This perspective summarizes our recent efforts in dissecting the photocycle into three elementary processes, including proton-coupled electron transfer (PCET), proton rocking, and proton relay. Using ultrafast spectroscopy, we have determined the temporal sequence, rates, kinetic isotope effects (KIEs), and concertedness of these elementary steps. Our findings provide important implications for illuminating the photoactivation mechanism of the BLUF domain and suggest an engineering platform to characterize intricate reactions involving proton motions that are ubiquitous in nonphotosensitive protein machines.

H2-driven reduction of CO2 to formate using bacterial plasma membranes

Bacterial membranes shield the intracellular compartment by selectively allowing unwanted substances to enter in, which in turn reduces overall catalytic efficiency. This report presents a model system using the isolated plasma membranes of Citrobacter sp. S-77 that harbor oxygen-stable [NiFe]hydrogenase and [Mo]formate dehydrogenase, which are integrated into a natural catalytic nanodevice through an electron transfer relay. This naturally occurring nanodevice exhibited selectivity and efficiency in catalyzing the H2-driven conversion of CO2 to formate with the rate of 817 mmol·L–1·gprotein–1·h−1 under mild conditions of 30 °C, pH 7.0, and 0.1 MPa. When the isolated plasma membranes of Citrobacter sp. S-77 was immobilized with multi-walled carbon nanotubes and encapsulated in hydrogel beads of gellan-gum cross-linked with calcium ions, the catalyst for formate production remained stable over 10 repeated uses. This paper reports the first case of efficient and selective formate production from H2 and CO2 using bacterial plasma membranes.

A Photoresponsive Battery Based on a Redox‐Coupled Covalent‐Organic‐Framework Hybrid Photoelectrochemical Cathode

AbstractPhotoresponsive batteries promise flexible and low‐cost solar‐to‐electrochemical energy storage (SES), but suffer from a limited SES efficiency due to rapid charge recombination and sluggish redox. Here, we present a porous‐shell/core hybrid of covalent organic framework@carbon nanotube. This hybrid ensures long‐lived separated charges (τave=3.0 ns) by an electron transfer relay starting from the donor‐acceptor molecules to the nanoscale heterojunction. These charges are further allowed to drive high‐rate redox of −C=O/−C−O− and −C−N/−C=N+ with facile kinetics. Equipped with this photoelectrochemical cathode, a photoresponsive aqueous battery shows a 5‐fold enhancement in SES efficiency (1.1 % at 1 sun) over their counterparts. It is unveiled that the electron relay favors the formation of electron‐enriching −C−O− and hole‐enriching −C=N+ groups responsible for photoelectrochemical Zn2+ and OTf− storage cascade; and further, the general photo coupled ions transfer (PCIT) process is proposed. This work presents an inspiring photoelectrochemical cathode design and theoretical insight for photoresponsive batteries.

A Photoresponsive Battery Based on a Redox-Coupled Covalent-Organic-Framework Hybrid Photoelectrochemical Cathode.

Photoresponsive batteries promise flexible and low-cost solar-to-electrochemical energy storage (SES), but suffer from a limited SES efficiency due to rapid charge recombination and sluggish redox. Here, we present a porous-shell/core hybrid of covalent organic framework@carbon nanotube. This hybrid ensures long-lived separated charges (τave =3.0 ns) by an electron transfer relay starting from the donor-acceptor molecules to the nanoscale heterojunction. These charges are further allowed to drive high-rate redox of -C=O/-C-O- and -C-N/-C=N+ with facile kinetics. Equipped with this photoelectrochemical cathode, a photoresponsive aqueous battery shows a 5-fold enhancement in SES efficiency (1.1 % at 1 sun) over their counterparts. It is unveiled that the electron relay favors the formation of electron-enriching -C-O- and hole-enriching -C=N+ groups responsible for photoelectrochemical Zn2+ and OTf- storage cascade; and further, the general photo coupled ions transfer (PCIT) process is proposed. This work presents an inspiring photoelectrochemical cathode design and theoretical insight for photoresponsive batteries.

Harnessing Intermolecular Interactions to Promote Long-Lived Photoinduced Charge Separation from Copper Phenanthroline Chromophores

Facilitating photoinduced electron transfer (PET) while minimizing rapid charge-recombination processes to produce a long-lived charge-separated (CS) state represents a primary challenge associated with achieving efficient solar fuel production. Natural photosynthetic systems employ intermolecular interactions to arrange the electron-transfer relay in reaction centers and promote a directional flow of electrons. This work explores a similar tactic through the synthesis and ground- and excited-state characterization of two Cu(I)bis(phenanthroline) chromophores with homoleptic and heteroleptic coordination geometries and which are functionalized with negatively charged sulfonate groups. The addition of sulfonate groups enables solubility in pure water, and it also induces assembly with the dicationic electron acceptor methyl viologen (MV2+) via bimolecular, dynamic electrostatic interactions. The effect of the sulfonate groups on the ground- and excited-state properties was evaluated by comparison with the unsulfonated analogues in 1:1 acetonitrile/water. The excited-state lifetimes for all sulfonated complexes are similar to what we expect from previous literature, with the exception of the sulfonated heteroleptic complex whose metal-to-ligand charge-transfer (MLCT) lifetime in water has two components that are fit to 10 and 77 ns. For the sulfonated complexes, we detected reduced MV+• in both solvent environments following MLCT excitation, but control measurements in 1:1 acetonitrile/water with the unsulfonated analogues showed no PET to MV2+, indicating that electrostatically driven supramolecular assemblies of the sulfonated complexes with MV2+ facilitate the observed PET. Additionally, the strength of the intermolecular interactions driving the formation of these assemblies changes drastically with the solvent environment. In 1:1 acetonitrile/water, PET occurred from both sulfonated complexes with quantum yields (ΦET) of 2-3% but increased to a remarkable 98% for the sulfonated heteroleptic complex with a 3 μs CS-state lifetime in water.

Facilitating Molecular Activation and Proton Feeding by Dual Active Sites on Polymeric Carbon Nitride for Efficient CO2Photoreduction

AbstractPhotoreduction of CO2provides an appealing way to alleviate the energy crisis and manage the global carbon balance but is limited by the high activation energy and the rate‐limiting proton transfer. We now develop a dual‐site strategy for high‐efficiency CO2conversion through polarizing CO2molecules at pyridine N vacancies and accelerating the intermediate protonation by protonated pyridine N adjacent to nitrogen vacancies on polymeric carbon nitride. Our photocatalysts with atomic‐level engineered active sites manifest a high CO production rate of 1835 μmol g−1 h−1, 183 times higher than the pristine bulk carbon nitride. Theoretical prediction and experimental studies confirm that such excellent performance is attributed to the synergistic effect between vacant and protonated pyridine N in decreasing the formation energy of the key *COOH intermediates and the efficient electron transfer relay facilitated by the defect‐induced shallow trap state and homogeneous charge mediators.

Facilitating Molecular Activation and Proton Feeding by Dual Active Sites on Polymeric Carbon Nitride for Efficient CO2 Photoreduction.

Photoreduction of CO2 provides an appealing way to alleviate the energy crisis and manage the global carbon balance but is limited by the high activation energy and the rate-limiting proton transfer. We now develop a dual-site strategy for high-efficiency CO2 conversion through polarizing CO2 molecules at pyridine N vacancies and accelerating the intermediate protonation by protonated pyridine N adjacent to nitrogen vacancies on polymeric carbon nitride. Our photocatalysts with atomic-level engineered active sites manifest a high CO production rate of 1835 μmol g-1 h-1 , 183 times higher than the pristine bulk carbon nitride. Theoretical prediction and experimental studies confirm that such excellent performance is attributed to the synergistic effect between vacant and protonated pyridine N in decreasing the formation energy of the key *COOH intermediates and the efficient electron transfer relay facilitated by the defect-induced shallow trap state and homogeneous charge mediators.

An efficient photocatalytic CO2 reduction system improved by emodin as a redox mediator

Solar-driven photoreduction of CO2 to value-added fuels such as CO, CH4, and CH3OH is a promising approach for alleviating the greenhouse effect. Transition-metal-complex molecular catalysts exhibit high catalytic performance for CO2 reduction owing to their tunable structures. In this study, we developed an efficient, inexpensive, and noble-metal-free photocatalytic CO2 reduction system containing an organic photosensitizer proflavine, a cobalt molecular catalyst, and an organic electron transfer relay emodin in DMF/H2O (DMF: N,N-dimethylformamide) solvent. In the presence of water, a turnover number (TON) of 1333 was achieved for CO2-to-CO conversion under ambient conditions and visible light irradiation, which is a 1.7-fold improvement over the system without emodin. In addition, a high selectivity of 90% was obtained for CO production. Absorption spectroscopy and cyclic voltammetry confirmed the crucial role of emodin in facilitating electron transfer between the photosensitizer and catalyst. We expect that our system will promote the development of an effective homogenous photocatalytic solar-fuel production technology.

Immobilization of Iron Phthalocyanine on Pyridine-Functionalized Carbon Nanotubes for Efficient Nitrogen Reduction Reaction

An electrochemical nitrogen reduction reaction (NRR) under mild conditions offers a promising alternative to the traditional Haber–Bosch process in converting abundant nitrogen (N2) to high value-added ammonia (NH3). In this work, iron phthalocyanine (FePc) was homogeneously immobilized on pyridine-functionalized carbon nanotubes to form a well-tuned electrocatalyst with an FeN5 active center (FePc-Py-CNT). Synchrotron X-ray absorption and Fourier transform infrared spectroscopy proved the presence of an Fe–N coordination bond between FePc and surface-bound pyridine. The resulting hybrid exhibited notably enhanced electrocatalytic NRR performance compared to FePc immobilized on CNTs based on π–π stacking interactions (FePc-CNT), resulting in doubled NH3 yield (21.7 μg mgcat–1 h–1) and Faradaic efficiency (22.2%). Theoretical calculations revealed that the axial coordination on FePc resulted in partial electron transfer from iron to pyridine, which efficiently suppresses the adsorption of H+ and improves the chemisorption of N2 at Fe sites. Meanwhile, the interfacial electron transfer was facilitated by pyridine as an electron transfer relay between FePc and CNTs. This work provides a unique strategy for the design of highly efficient NRR electrocatalysts at the molecular level.

Photoelectrochemical water oxidation improved by pyridine N-oxide as a mimic of tyrosine-Z in photosystem II.

Artificial photosynthesis provides a way to store solar energy in chemical bonds with water oxidation as a major challenge for creating highly efficient and robust photoanodes that mimic photosystem II. We report here an easily available pyridine N-oxide (PNO) derivative as an efficient electron transfer relay between an organic light absorber and molecular water oxidation catalyst on a nanoparticle TiO2 photoanode. Spectroscopic and kinetic studies revealed that the PNO/PNO+˙ couple closely mimics the redox behavior of the tyrosine/tyrosyl radical pair in PSII in improving light-driven charge separation via multi-step electron transfer. The integrated photoanode exhibited a 1 sun current density of 3 mA cm−2 in the presence of Na2SO3 and a highly stable photocurrent density of >0.5 mA cm−2 at 0.4 V vs. NHE over a period of 1 h for water oxidation at pH 7. The performance shown here is superior to those of previously reported organic dye-based photoanodes in terms of photocurrent and stability.

Functional basis of electron transport within photosynthetic complex I

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme – the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

A semiconductor/molecular catalyst hybrid photoanode with FeOOH as an electron transfer relay

A Fe2 O3 /FeOOH/poly-Ru(bda)(vpy) (bda = 2,2'-bipyridine-6,6'-dicarboxylate,vpy = 4-vinylpyridine) photoanode has been fabricated by electropolymerization of molecular Ru(bda)(vpy) catalyst on FeOOH modified Fe2 O3 , in which a thin layer of FeOOH replicates the role of tyrosine residue in PSII as an efficient electron transfer mediator. The ternary hybrid photoanode produced a 2.4 times higher photocurrent density than that of previously reported Fe2 O3 /poly-Ru(bda)(vpy) under AM 1.5 G illumination and displayed a negative shift on the onset potential by 100 mV. In addition, the Fe2 O3 /FeOOH/poly-Ru(bda)(vpy) exhibited long-term stability for at least 10 h with a Faraday efficiency of ∼96%. The high performance shown here was attributed to the improved charge separation between excited semiconductor and the catalyst caused by FeOOH mediated electron transfer on the electrode surface.

Self-assembly of a Ni(I)-photocatalyst for plain water splitting without sacrificial agents

The present work describes a self-assembling photocatalytic system that is observed when illuminating grey titania nanoparticles in suspension with a plain aqueous Ni2+ solution. Such a suspension (in contrast to white titania), under UV illumination, increasingly produces H2 from neutral water. We show that the origin of this self-activation is not the light-induced deposition of Ni0 or another Ni-catalyst compound on the titania surface. Rather, in-situ electron paramagnetic resonance (EPR) clearly indicates that the activation is due to a light-induced formation of a suitable defect structure (Ti3+-OV) on grey titania, combined with the formation of an intermediate monovalent nickel (Ni(I)) electron transfer relay. Remarkably, the resulting Ni+/TiO2/Ti3+ photocatalyst operates in absence of any noble metal or sacrificial agent.

Theoretical investigation of perfect fullerene-like borospherene Ih-B20 protected by alkaline earth metal: multi-layered spherical electride molecules as electric field manipulated second-order nonlinear optical switches.

For the challenge of stabilizing a fullerene-like borospherene, a perfect fullerene-like borospherene B20 with Ih symmetry is stabilized theoretically for the first time by selected 12 η5 (pentahapto)-Mg atoms capped on the 12 B5 pentagons, forming an exohedral metalloborospherene Ih Mg12&B20 molecule. Owing to the pull-push electron transfer relay, the molecule is converted to a spherical electride molecule (Mg2+)12&B2018- + 6e- with multiexcess electrons and high-valent B2018- polyanion with a full-shell electronic configuration, performing the stabilization of the fullerene-like B20 cage. Furthermore, we embed the metal M atom in the B20 cage, forming new endohedral electride molecules (Mg2+)12&(Mn+@B2018-) + (6 + n)e- (M = Li, n = 1 and Mg, n = 2). The embedded atom M increases the excess electron number. Notably, these electride molecules possess two different types of superatomic characteristics, exhibiting the behavior of an electron reservoir. Moreover, a compound (Mg12&B20)2O is designed and obtained, so Mg12&B20 might serve as a nano-building block. In addition, the Mg12&B20 and Mg12&(Mg@B20) electride molecules are efficient external electric field-manipulated nonlinear optical (NLO) switches with high sensitivity and reversibility. The embedding metal atom in the B20 cage can also serve as a new strategy to manipulate NLO switching.

An artificial photosynthesis system comprising a covalent triazine framework as an electron relay facilitator for photochemical carbon dioxide reduction

A ternary electron transfer relay photocatalytic system for CO2 reduction was fabricated by decorating a porphyrin-based covalent triazine framework with α-Fe2O3 nanoparticles, and then further coupled with a Ru complex photosensitizer.

Accelerating photosensitized H2 evolution over in situ grown amorphous MoSx catalyst employing TiO2 as an efficient catalyst loading matrix and electron transfer relay

Although dye sensitized photocatalytic systems employing in situ grown amorphous MoSx as catalyst have shown promising performance for H2 evolution reaction (HER), their low activity and short durability need to be further improved. Herein, we report that employing TiO2 as an electron relay and catalyst loading matrix can greatly accelerate the H2 evolution activity of in situ grown MoSx in an Eosin Y (EY) sensitized system. The EY/TiO2/MoSx system exhibited 4.5 times higher H2 evolution activity than EY/MoSx and an apparent quantum yield (AQY) of 27.5% at 500 nm. The TiO2/MoSx catalyst also showed improved stability as compared to free MoSx. The enhanced H2 evolution activity of EY/TiO2/MoSx system was attributed to the presence of TiO2, which can not only serve as an effective loading matrix to grow MoSx with high dispersion and reduced size, affording an abundance of active sites toward HER, but also efficiently mediate the electron transfer from excited EY (EY∗) to MoSx, leading to the promoted oxidative quenching of EY∗, and thus suppressing the formation of unstable EY−·. This work provides an effective strategy to develop highly active noble-metal-free H2 evolution systems by simultaneously regulating electron transfer kinetics and existing state of catalyst with multifunctional mediators.

Biomolecular EPR Meets NMR at High Magnetic Fields

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

Quasi-homogenous dye-sensitized photocatalytic H2 evolution catalyzed by in-situ grown cobalt-promoted MoSx catalyst coupled with graphene quantum dots

The development of practical photocatalytic systems for large-scale and on-demand solar-to-hydrogen conversion requires that H2 evolution catalysts consist of earth-abundant materials and can be facilely obtained and modified. In this paper, we report a high-performance and quasi-homogenous dye-sensitized H2 evolution system by coupling graphene quantum dots (GQDs) with cobalt-promoted MoSx (Co-MoSx) catalyst. The Co-MoSx serving as H2 evolution catalyst can be facilely grown by a in-situ photochemical reduction method in the presence of an organic dye as the photosensitizer and simultaneously coupled with GQDs during photocatalytic reactions, forming GQDs/Co-MoSx catalyst. The resulted GQDs/Co-MoSx catalyst exhibits high activity for catalyzing H2 evolution under visible light due to the significant synergistic effects among GQDs, Co dopant, and MoSx. The doping of MoSx with Co ions results in an effective promotional effect on the H2 evolution activity of in-situ generated Co-MoSx catalyst, while the presence of GQDs can not only improve the dispersion of Co-MoSx catalyst by serving as a homogenous matrix but also lead to an efficient Förster resonance energy transfer (FRET) to sensitizing dye by acting as a quasi-homogenous energy donor for enhancing light utilization efficiency. In addition, the GQDs can also act as an electron transfer relay to facilitate the electron transfer from excited dye to Co-MoSx catalyst, thus retarding the back electron transfer and reducing energy loss. This work provides a new route to construct an efficient and quasi-homogenous dye-sensitized H2 evolution system by integrating inexpensive MoSx-based catalyst with transition metal modification and highly photoluminescent and soluble GQDs with outstanding optical properties.

Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I

EPR-based potentiometric titrations are a well-established method for determining the reduction potentials of cofactors in large and complex proteins with at least one EPR-active state. However, such titrations require large amounts of protein. Here, we report a new method that requires an order of magnitude less protein than previously described methods, and that provides EPR samples suitable for measurements at both X- and Q-band microwave frequencies. We demonstrate our method by determining the reduction potential of the terminal [4Fe-4S] cluster (N2) in the intramolecular electron-transfer relay in mammalian respiratory complex I. The value determined by our method, Em7=−158mV, is precise, reproducible, and consistent with previously reported values. Our small-volume potentiometric titration method will facilitate detailed investigations of EPR-active centres in non-abundant and refractory proteins that can only be prepared in small quantities.