Continuous Wave Electron Paramagnetic Resonance Research Articles

Magneto-Structural Correlations in a Mixed Porphyrin(Cu2+ )/Trityl Spin System: Magnitude, Sign, and Distribution of the Exchange Coupling Constant.

Tetrathiatriarylmethyl radicals (TAM or trityl) are receiving increasing attention in various fields of magnetic resonance such as imaging, dynamic nuclear polarization, spin labeling, and, more recently, molecular magnetism and quantum information technology. Here, a trityl radical attached via a phenyl bridge to a copper(II)tetraphenylporphyrin was synthesized, and its magnetic properties studied by multi-frequency continuous-wave electron paramagnetic resonance (EPR) spectroscopy and magnetic measurements. EPR revealed that the electron spin-spin coupling constant J between the trityl and Cu2+ spin centers is ferromagnetic with a magnitude of -2.3 GHz (-0.077 cm-1 , convention) and a distribution width of 1.2 GHz (0.040 cm-1 ). With the help of density functional theory (DFT) calculations, the obtained ferromagnetic exchange coupling, which is unusual for para-substituted phenyl-bridged biradicals, could be related to the almost perpendicular orientation of the phenyl linker with respect to the porphyrin and trityl ring planes in the energy minimum, while the J distribution was rationalized by the temperature weighted rotation of the phenyl bridge about the molecular axis connecting both spin centers. This study exemplifies the importance of molecular dynamics for the homogeneity (or heterogeneity) of the magnetic properties of trityl-based systems.

Electron spin echo spectra and characterization of electronic ground states for lanthanide ions in acidic water:ethanol glasses

X-band EPR absorption spectra were obtained by field-swept echo-detection for 10 mM Ce3+, Nd3+, Sm3+, Tb3+, Er3+, Tm3+, and Yb3+ in glassy acidic 1:1 water:ethanol at 4.2 to 6 K. The slowly-varying signals extend over thousands of gauss and are more readily detected by spin echo than by continuous wave EPR. For lanthanide ions spin–orbit coupling is strong and J is a good quantum number. The microwave power required to achieve a π/2 pulse depends on quantum numbers J and mJ and the gJ for the levels involved in the EPR transition, so it can be used to characterize the electronic ground state. For Nd3+, Sm3+, Er3+, and Tm3+ the power required for a π/2 pulse is consistent with values calculated for the expected value of gJ and transitions involving mJ = ±1/2 or mJ = ±1,0. However for Ce3+, Gd3+, Tb3+, and Yb3+ the microwave power required for a π/2 pulse is larger than predicted. For Ce3+, Tb3+, and Yb3+ it is proposed that mixing of levels results in contributions to the transitions from higher values of mJ. For Gd3+ the discrepancy is attributed to overlapping transitions with varying values of mJ.

Quantification of Age-Related Changes in the Lateral Organization of the Lipid Portion of the Intact Membranes Isolated from the Left and Right Eye Lenses of the Same Human Donor.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a "threshold" for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

Structural dynamics of the activation loop in the kinase domain of E.faecalis IreK.

IreK is a transmembrane Ser/Thr PASTA kinase found in Enterococcus faecalis, as well as most other Gram-positive bacteria, and plays a key role in resistance to cell wall-active antibiotics such as cephalosporins. IreK belongs to a family of bacterial transmembrane kinases that include multiple PASTA (Penicillin-and Ser/Thr-kinase Associated) domains in the extracellular segment and are ubiquitous in nearly all Gram-positive bacteria. PASTA kinases have conserved functions across Gram-positive bacteria involving monitoring and regulating processes associated with the cell wall. Enterococci are frequently found in the human gut and are a common cause of severe and difficult to treat hospital acquired infections, due to their intrinsic drug resistance. The underlying mechanisms of antibiotic resistance in enterococci are not well understood and are an excellent target for the creation of preventative treatments to eliminate the growth opportunity of resistant E. faecalis in the gut and transfer to other parts of the body. Based on the crystal structures of IreK kinase domain homologs, the activation loop structure is unknown and the phosphorylation sites within this loop are proposed to play a key regulatory role in IreK kinase activity. IreB has also been identified as one of the targets for phosphorylation by IreK. Here, continuous wave electron paramagnetic resonance (CW EPR) spectroscopy was applied to study the dynamics of the IreK activation loop, including the effects of phosphorylation and IreB binding on activation loop structure and dynamics. In addition, new functionally important non-phosphorylation sites in the activation loop that regulate IreK function in vivo were identified and characterized. This comprehensive investigation into the IreK activation loop structure and functional dynamics characterizes the role of this loop as a key regulatory domain in kinase function.

Probing structural topology and dynamics of human KCNE3 membrane protein in lipid bilayers using site-directed spin labeling and electron paramagnetic resonance spectroscopy.

KCNE3 is a single pass membrane spanning protein that modulates the function of the voltage gated potassium ion channel KCNQ1. Genetic mutations in the KCNE3 protein can result in multiple diseases such as long QT syndrome, atrial fibrillation, Mènière's disease, etc. Despite the biological significance, its structural and dynamic properties in a lipid bilayer are not yet fully understood. In this study, the structural topology and dynamic properties of KCNE3 were explored in lipid bilayers utilizing site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. Continuous wave EPR data indicated that the nitroxide spin labeled sidechains of KCNE3 in the transmembrane domain (TMD) are less mobile when compared to that of N and C-termini. Multiple nitroxide spin labeled amino acid sites have been studied using power saturation EPR to determine the membrane depth parameter (φ) of KCNE3 with respect to the POPC/POPG lipid bilayers. These results will be helpful for developing the topology model of KCNE3 in lipid bilayers.

Guidelines for the Simulations of Nitroxide X-Band cw EPR Spectra from Site-Directed Spin Labeling Experiments Using SimLabel.

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (gy, gz) and others are determined (Az, gx) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.

Vanadyl(IV) Porphyrin Dimers with Palladium(II) and Platinum(II) Linkages: Syntheses, Electronic Properties, and Magnetic Interactions between the Two Moieties

We report the synthesis of new vanadyl porphyrins bearing peripheral coordination sites. By using palladium(II) or platinum(II) as connecting ions, porphyrin dimers were isolated and characterized by X-ray crystallography. These paramagnetic species were studied by electronic spectroscopy and continuous wave electron paramagnetic resonance (CW EPR). Large bathochromic shifts were observed by comparing porphyrin monomers and dimers showing that electronic delocalization was present as demonstrated earlier for diamagnetic compounds. Despite the large distance (>14 Å) between the two vanadyl centers in these dimers, small dipolar interactions between the two paramagnetic ions could be determined.

Two-Electron Mixed Valency in a Heterotrimetallic Nickel–Vanadium–Nickel Complex

Mixed-valence complexes represent an enticing class of coordination compounds to interrogate electron transfer confined within a molecular framework. The diamagnetic heterotrimetallic anion, [V(SNS)2{Ni(dppe)}2]-, was prepared by reducing (dppe)NiCl2 in the presence of the chelating metalloligand [V(SNS)2]- [dppe = bis(diphenylphosphino)ethane; (SNS)3- = bis(2-thiolato-4-methylphenyl)amide]. Vanadium-nickel bonds span the heterotrimetallic core in the structure of [V(SNS)2{Ni(dppe)}2]-, with V-Ni bond lengths of 2.78 and 2.79 Å. One-electron oxidation of monoanionic [V(SNS)2{Ni(dppe)}2]- yielded neutral, paramagnetic V(SNS)2{Ni(dppe)}2. The solid-state structure of V(SNS)2{Ni(dppe)}2 revealed that the two nickel ions occupy unique coordination environments: one nickel is in a square-planar S2P2 coordination environment (τ4 = 0.19), with a long Ni···V distance of 3.45 Å; the other nickel is in a tetrahedral S2P2 coordination environment (τ4 = 0.84) with a short Ni-V distance of 2.60 Å, consistent with a formal metal-metal bond. Continuous-wave X-band electron paramagnetic resonance spectroscopy, electrochemical investigations, and density functional theory computations indicated that the unpaired electron in the neutral V(SNS)2{Ni(dppe)}2 cluster is localized on the bridging [V(SNS)2] metalloligand, and as a result, V(SNS)2{Ni(dppe)}2 is best described as a two-electron mixed-valence complex. These results demonstrate the important role that metal-metal interactions and flexible coordination geometries play in enabling multiple, reversible electron transfer processes in small cluster complexes.

Tuning Human Serum Albumin (HSA) Hydrogels through Albumin Glycation.

The changes of technological properties of albumin-based hydrogels induced by increasing degrees of post-translational modification of the protein are reported. Maillard-type modification of amino acids arginine and lysine of albumin is achieved through glyoxal as an α-dicarbonyl compound. The degrees of modification are fine-tuned using different molar ratios of glyoxal. Hydrogels are thermally induced by heating highly concentrated precursor solutions above the protein's denaturation temperature. While the post-translational modifications are determined and quantified with mass spectrometry, continuous-wave (CW) electron paramagnetic resonance(EPR) spectroscopy shed light on the protein fatty acid binding capacity and changes thereof in solution and in the gel state. The viscoelastic behavior is characterized as a measure of the physical strength of the hydrogels. On the nanoscopic level, the modified albumins in low concentration solution reveal lower binding capacities with increasing degrees of modification. On the contrary, in the gel state, the binding capacity remains constant at all degrees of modifications. This indicates that the loss of fatty acid binding capacity for individual albumin molecules is partially compensated by new binding sites in the gel state, potentially formed by modified amino acids. Such, albumin glycation offers a fine-tuning method of technological and nanoscopic properties of these gels.

Glass-like behavior of intercalated organic solvents in graphite oxide detected by spin-probe EPR.

Membranes based on graphite oxide (GO) are promising materials for the separation of polar liquids and gases. Understanding the properties of solvents immersed in GO is important for the development of various technological applications. Here, the molecular motions of the TEMPO nitroxide spin probe in acetonitrile intercalated into the GO inter-plane space were studied using electron paramagnetic resonance (EPR), including its pulsed version, and electron spin echo (ESE). For a sample containing 75% acetonitrile relative to equilibrium sorption at room temperature, ESE-detected stochastic librations were observed for TEMPO molecules above 135 K. Since these librations are an inherent property of molecular glasses, this fact indicates that intercalated acetonitrile forms a two-dimensional glass state. Above 225 K, an acceleration of stochastic librations was observed, indicating the manifestation of a glass-like dynamical cross-over. Continuous wave (CW) EPR spectra of TEMPO showed the absence of overall tumbling motions in the entire investigated temperature range of up to 340 K, indicating that the intercalated acetonitrile does not behave as a bulk liquid (the melting point of acetonitrile is 229 K). Dynamical librations of TEMPO molecules detected by CW EPR were found to accelerate above 240 K.

Selective adsorption of dihydrogen isotopes on DUT-8 (Ni,Co) monitored by in situ electron paramagnetic resonance.

In situ continuous wave electron paramagnetic resonance investigation has been proven as a powerful method by employing paramagnetic Ni2+-Co2+ pairs as spin probes to follow the isotope-selective gate opening phenomenon on the DUT-8(Ni0.98 Co0.02) framework. This method is very sensitive to detect the phase transition from the closed pore to the open pore phase in response to D2 adsorption in the framework, while no phase transformation has been observed during H2 gas adsorption. More interestingly, it is also able to sense local structural changes around the spin probe during the desorption of D2 gas. Based on these evidences, the in situ continuous wave electron paramagnetic resonance method can be implemented as an efficient and non-invasive technique for the detection of dihydrogen isotopes.

A new strategy for improving cytotoxicity of a copper complex toward metastatic melanoma cells unveiled by EPR spectroscopy.

A novel strategy of improving cytotoxicity against metastatic melanoma cells using an oxindolimine copper(ii) complex immobilized and dimerized on a modified Polyhedral Oligomeric Silsesquioxane (POSS) matrix was developed, as revealed by electron paramagnetic resonance (EPR) spectroscopy. An assured correlation between continuous-wave (CW) and pulsed EPR spectroscopies provided a complete characterization of the actual active species, its coordination environment, as well as the efficiency/selectivity of the bioconjugate materials.

Rapid Energy Exchange between InSitu Formed Bromine Vacancies and CO2 Molecules Enhances CO2 Photoreduction.

Photocatalytic reduction of CO2 into fuels provides a prospective tactic for regulating the global carbon balance utilizing renewable solar energy. However, CO2 molecules are difficult to activate and reduce due to the thermodynamic stability and chemical inertness. In this work, we develop a novel strategy to promote the adsorption and activation of CO2 molecules via the rapid energy exchange between the photoinduced Br vacancies and CO2 molecules. Combining insitu continuous wave-electron paramagnetic resonance (cw-EPR) and pulsed EPR technologies, we observe that the spin-spin relaxation time (T2) of BiOBr is decreased by 198 ns during the CO2 photoreduction reaction, which is further confirmed by the broadened EPR linewidth. This result reveals that there is an energy exchange interaction between insitu formed Br vacancies and CO2 molecules, which promotes the formation of high-energy CO2 molecules to facilitate the subsequent reduction reaction. In addition, theoretical calculations indicate that the bended CO2 adsorption configuration on the surface of BiOBr with Br vacancies caused the decrease of the lowest unoccupied molecular orbital of the CO2 molecule, which makes it easier for CO2 molecules to acquire electrons and get activated. Insitu diffuse reflectance infrared Fourier transform spectroscopy further shows that the activated CO2 molecules are favorably converted to key intermediates of COOH*, resulting in a CO generation rate of 9.1 μmol g-1 h-1 and a selectivity of 100%. This study elucidates the underlying mechanism of CO2 activation at active sites and deepens the understanding of CO2 photoreduction reaction.

An exchange coupled meso-meso linked vanadyl porphyrin dimer for quantum information processing.

We report here the synthesis of a new meso-meso (m-m) singly linked vanadyl-porphyrin dimer that crystallizes in two different pseudo-polymorphs. The single crystal continuous-wave electron paramagnetic resonance investigation evidences a small but crucial isotropic exchange interaction, J, between the two tilted, and thus distinguishable, spin centers of the order of 10-2 cm-1. The experimental and DFT studies evidence a correlation between J values and porphyrin plane tilting angle and distortion. Pulsed EPR analysis shows that the two vanadyl dimers maintain the coherence time of the monomer. With the obtained spin Hamiltonian parameters, we identify suitable transitions that could be used as computational basis states. Our results, coupled with the evaporability of porphyrin systems, establish this class of dimers as extremely promising for quantum information processing applications.

In-depth magnetometry and EPR analysis of the spin structure of human-liver ferritin: from DC to 9 GHz.

Ferritin, the major iron storage protein in organisms, stores iron in the form of iron oxyhydroxide most likely involving phosphorous as a constituent, the mineral form of which is not well understood. Therefore, the question of how the ca. 2000 iron atoms in the ferritin core are magnetically coupled is still largely open. The ferritin core, with a diameter of 5-8 nm, is encapsulated in a protein shell that also catalyzes the uptake of iron and protects the core from outside interactions. Neurodegenerative disease is associated with iron imbalance, generating specific interest in the magnetic properties of ferritin. Here we present 9 GHz continuous wave EPR and a comprehensive set of magnetometry techniques including isothermal remanent magnetization (IRM) and AC susceptibility to elucidate the magnetic properties of the core of human liver ferritin. For the analysis of the magnetometry data, a new microscopic model of the ferritin-core spin structure is derived, showing that magnetic moment is generated by surface-spin canting, rather than defects. The analysis explicitly includes the distribution of magnetic parameters, such as the distribution of the magnetic moment. This microscopic model explains some of the inconsistencies resulting from previous analysis approaches. The main findings are a mean magnetic moment of 337μB with a standard deviation of 0.947μB. In contrast to previous reports, only a relatively small contribution of paramagnetic and ferrimagnetic phases is found, in the order of maximally 3%. For EPR, the over 30 mT wide signal of the ferritin core is analyzed using the model of the giant spin system [Fittipaldi et al., Phys. Chem. Chem. Phys., 2016, 18, 3591-3597]. Two components are needed minimally, and the broadening of these components suggests a broad distribution of the magnetic resonance parameters, the zero-field splitting, D, and the spin quantum number, S. We compare parameters from EPR and magnetometry and find that EPR is particularly sensitive to the surface spins of the core, revealing the potential to use EPR as a diagnostic for surface-spin disorder.

Predictive Removal of Interfacial Defect-Induced Trap States between Titanium Dioxide Nanoparticles via Sub-Monolayer Zirconium Coating.

First principles modeling of anatase TiO2 surfaces and their interfacial contacts shows that defect-induced trap states within the band gap arise from intrinsic structural distortions, and these can be corrected by modification with Zr(IV) ions. Experimental testing of these predictions has been undertaken using anatase nanocrystals modified with a range of Zr precursors and characterized using structural and spectroscopic methods. Continuous-wave electron paramagnetic resonance (EPR) spectroscopy revealed that under illumination, nanoparticle-nanoparticle interfacial hole trap states dominate, which are significantly reduced after optimizing the Zr doping. Fabrication of nanoporous films of these materials and charge injection using electrochemical methods shows that Zr doping also leads to improved electron conductivity and mobility in these nanocrystalline systems. The simple methodology described here to reduce the concentration of interfacial defects may have wider application to improving the efficiency of systems incorporating metal oxide powders and films including photocatalysts, photovoltaics, fuel cells, and related energy applications.

Boronated Cyanometallates

Thirteen boronated cyanometallates [M(CN-BR3)6]3/4/5- [M = Cr, Mn, Fe, Ru, Os; BR3 = BPh3, B(2,4,6,-F3C6H2)3, B(C6F5)3] and one metalloboratonitrile [Cr(NC-BPh3)6]3- have been characterized by X-ray crystallography and spectroscopy [UV-vis-near-IR, NMR, IR, spectroelectrochemistry, and magnetic circular dichroism (MCD)]; CASSCF+NEVPT2 methods were employed in calculations of electronic structures. For (t2g)5 electronic configurations, the lowest-energy ligand-to-metal charge-transfer (LMCT) absorptions and MCD C-terms in the spectra of boronated species have been assigned to transitions from cyanide π + B-C borane σ orbitals. CASSCF+NEVPT2 calculations including t1u and t2u orbitals reproduced t1u/t2u → t2g excitation energies. Many [M(CN-BR3)6]3/4- complexes exhibited highly electrochemically reversible redox couples. Notably, the reduction formal potentials of all five [M(CN-B(C6F5)3)6]3- anions scale with the LMCT energies, and Mn(I) and Cr(II) compounds, [K(18-crown-6)]5[Mn(CN-B(C6F5)3)6] and [K(18-crown-6)]4[Cr(CN-B(C6F5)3)6], are surprisingly stable. Continuous-wave and pulsed electron paramagnetic resonance (EPR; hyperfine sublevel correlation) spectra were collected for all Cr(III) complexes; as expected, 14N hyperfine splittings are greater for (Ph4As)3[Cr(NC-BPh3)6] than for (Ph4As)3[Cr(CN-BPh3)6].

Electron Paramagnetic Resonance Measurements of Lifetime Doses in Teeth of Durham Region Residents, Ontario.

The Canadian Nuclear Safety Commission (CNSC) and Ontario Power Generation (OPG) determined the total dose contribution of nuclear power plants in Durham Region populations by analyzing environmental samples from the surrounding areas of both nuclear generating stations (Pickering and Darlington). However, the total doses from the various sources were unknown in Durham Region populations, Ontario. Electron paramagnetic resonance (EPR) dosimetry with tooth enamel has been successfully established as an effective tool for gamma dose assessment for chronic and acute exposures in individuals, groups, or populations to reconstruct the absorbed dose down to 30 mGy. This study collected the extracted teeth from people of different ages in Durham Region, Ontario, and analyzed them using the x-band continuous wave (CW) EPR spectrometer. The total dose rate from the natural and anthropogenic sources was 1.9721 mSv y-1. The anthropogenic dose rate from the various sources was 0.6341 mSv y-1, about 47.39% of the natural background dose (1.338 mSv y-1) in Durham Region, Ontario. The combined anthropogenic doses from these sources were lower than the local background dose in Durham Region and lower than the regulatory annual effective dose limit of 1 mSv y-1 in Canada. Based on these data, this study concluded that the anthropogenic dose contribution was lower than the regulatory limit to the local populations.

Improved Conductivity in Gellan Gum and Montmorillonite Nanocomposites Electrolytes.

Nanocomposite polymer electrolytes (NPEs) were obtained using gellan gum (GG) and 1 to 40 wt.% of montmorillonite (Na+SYN-1) clay. The NPEs were crosslinked with formaldehyde, plasticized with glycerol, and contained LiClO4. The samples were characterized by impedance spectroscopy, thermal analyses (TGA and DSC), UV-vis transmittance and reflectance, X-ray diffraction (XRD), and continuous-wave electron paramagnetic resonance (CW-EPR). The NPEs of GG and 40 wt.% LiClO4 showed the highest conductivity of 2.14 × 10-6 and 3.10 × 10-4 S/cm at 30 and 80 °C, respectively. The samples with 10 wt.% Na+SYN-1 had a conductivity of 1.86 × 10-5 and 3.74 × 10-4 S/cm at 30 and 80 °C, respectively. TGA analyses revealed that the samples are thermally stable up to 190 °C and this did not change with clay addition. The transparency of the samples decreased with the increase in the clay content and at the same time their reflectance increased. Finally, CW-EPR was performed to identify the coordination environment of Cu2+ ions in the GG NPEs. The samples doped with the lowest copper concentration exhibit the typical EPR spectra due to isolated Cu2+ ions in axially distorted sites. At high concentrations, the spectra become isotropic because of dipolar and exchange magnetic effects. In summary, GG/clay NPEs presented good ionic conductivity results, which qualifies them for electrochemical device applications.

Unexpected Heme Redox Potential Values Implicate an Uphill Step in Cytochrome b6f.

Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Qn and Qp). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b6f, the redox midpoint potentials (Ems) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b6f and revealed that Em of heme bn is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Qp and Qn sites and modulate the efficiency of cyclic and linear ET in photosynthesis.