EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling

The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands. Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands. Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein. Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II). The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II). Hence, three ligands from the protein are required to complete the sextet of ligands.     

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.     

Specific loss of the extrinsic 18 KDa protein from Photosystem II upon heating to 47 °C causes inactivation of oxygen evolution likely due to Ca release from the Mn-complex

Exposure of Photosystem II (PS II) membrane particles from spinach to a temperature of 47 °C caused the rapid release of the 18 kDa protein in parallel to inactivation of oxygen evolution. Previously, it has been suggested that the first heat-jump response involves rapid Ca release from the Mn complex of O₂-evolution, followed by the slower release of (2 + 2) Mn^II ions [Pospisil P et al. (2003) Biophys J 84: 1370–1386]. Here, the predicted biphasic Mn^II release to the bulk was verified by atomic absorption spectroscopy (AAS). Analysis of laser flash-induced delayed fluorescence transients suggests that the loss of the essential Ca ion from the Mn₄Ca complex in the dark is due to the loss of the 18 kDa protein. The S₂-state multiline EPR signal of the Mn complex was still generated in heat-treated PS II presumably lacking Ca, but retaining four Mn ions.Dedicated to Professor Norio Murata on the occasion of his retirement     

Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase

The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3-phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron paramagnetic resonance studies of MN(II) complexes with myosin subfragment 1 and oxygen 17-labeled ligands

Ligands in the first coordination sphere of Mn(II) in the complex of MnADP with myosin subfragment 1 from rabbit skeletal muscle have been investigated. EPR spectroscopy was used to detect superhyperfine coupling between unpaired electrons of the metal ion and the nuclei of oxygen atoms specifically labeled with oxygen 17. The results show that ADP is a beta-monodentate ligand for Mn(II) and that there are probably two water oxygens directly bound to Mn(II). The inhibitory complex of vanadate with subfragment 1 . MnADP was also investigated. Vanadate-dependent changes in the EPR spectra for enzyme-bound Mn(II) indicate that the coordination sphere of MN(II) changes upon binding of vanadate. ADP remains a beta-monodenate ligand in the complex and experiments with 17O-labeled water indicate that two oxygen atoms originally in water are ligands in the complex. However, the oxygens of vanadate equilibrate with those of water during sample preparation so that one of these ligands may be a vanadate oxygen. Three additional ligands, probably from the protein, are required to complete the sextet of ligands to Mn(II) in both complexes studied.     

The first spectroscopic model for the S1 state multiline signal of the OEC

The parallel-mode electron paramagnetic resonance (EPR) spectrum of the S(1) state of the oxygen-evolving complex (OEC) shows a multiline signal centered around g=12, indicating an integer spin system. The series of [Mn(2)(2-OHsalpn)(2)] complexes were structurally characterized in four oxidation levels (Mn(II)(2), Mn(II)Mn(III), Mn(III)(2), and Mn(III)Mn(IV)). By using bulk electrolysis, the [Mn(III)Mn(IV)(2-OHsalpn)(2)(OH)] is oxidized to a species that contains Mn(IV) oxidation state as detected by X-ray absorption near edge spectroscopy (XANES) and that can be formulated as Mn(IV)(4) tetramer. The parallel-mode EPR spectrum of this multinuclear Mn(IV)(4) complex shows 18 well-resolved hyperfine lines center around g=11 with an average hyperfine splitting of 36 G. This EPR spectrum is very similar to that found in the S(1) state of the OEC. This is the first synthetic manganese model complex that shows an S(1)-like multiline spectrum in parallel-mode EPR.     

Mn-Mn interaction in adenylylated and unadenylylated glutamine synthetase

The distance between the two catalytically important metal ions of glutamine synthetase was determined by electron paramagnetic resonance (EPR). Mn(II) binds more tightly to the n1 site of this enzyme in the presence of methionine sulfoximine and the influence of Mn(II) bound at the n2 site on the EPR spectrum of Mn(II) at n1 was studied. A monotonic increase in the EPR spectrum of Mn(II) was observed at Mn:E (subunit) ratios of 0 to 0.8. After this point as Mn(II) was added to about 1.8 Mn:E, a decrease in the EPR signal was observed. This phenomenon was found for both adenylylated and unadenylylated forms of glutamine synthetase. The data were analyzed using a theory for dipolar electron-electron relaxation and a distance of 10-12 A was computed for the Mn(II)-Mn(II) separation. These data demonstrate that both modified and unmodified forms of glutamine synthetase which have different catalytic activities have a similar spatial relationship between the two catalytic metal ion sites.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Characterization of the manganese O2-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramagnetic resonance and X-ray absorption spectroscopy

The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.     

Assignment of the g = 4.1 EPR signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: an X-ray absorption edge spectroscopy study

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O2-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S2 multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S2 state.     

Heterometallic manganese/zinc-phytate complex as a model compound for metal storage in wheat grains

Myo-inositol-1,2,3,4,5,6-hexakisphosphate, also known as phytate, is a natural metal chelate present in cereals, an important feedstock worldwide. This article reports the characterization of three metal storage model complexes: the homometallic Mn(II) myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6), the heterometallic Zn(II), Mn(II) analogue Na4MnZn4(C6H6O24P6) x (NO3)2 x 8H2O (MnZn4IP6) and the homometallic Zn(II) metal complex Na3Zn5(C6H6O24P6)OH x 9H2O (Zn5IP6). The techniques of high-resolution 23Na, 13C and 31P NMR, electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) were applied in this study. The complexation of Zn(II) and Mn(II) by phosphate groups of IP6 is demonstrated by NMR and XPS results. 13C NMR results show a conformation for IP6 consisting of five equatorial phosphate groups to one axial group showing only one chemical environment for Zn and two for Mn, when characterized by XPS and EPR, in both Mn complexes. These results support, for the first time, a probable supramacromolecular structure for phytate complexes of transition metals. Based on the similarity between the EPR spectra of wheat seeds and that of the MnZn4IP6 compound, the manganese storage centers in wheat grains can be assigned to similar heterometallic phytate complexes.     

Rice tolerance to excess Mn: Implications in the chloroplast lamellae and synthesis of a novel Mn protein

Rice (Oryza sativa L. cv. Safari) plants were grown over a 21-d period in nutrient solutions containing Mn concentrations varying between 0.125 and 32 mg·L^–1. The plant shoots and the thylakoid membranes showed similar overall Mn increases for this range of treatments, but different accumulation kinetics. It was found that the highest Mn treatment were associated to the synthesis of a new thylakoid protein with an average molecular mass of 36.5 kDa and a Mn:protein ratio of about 1. This protein exhibited superoxide dismutase activity, as well as a high content of Gln, Asp, Glu, Leu and Gly. Its EPR spectrum is characteristic of high-spin Mn(II), in a S=5/2 ground state. A comparative study of SDS polyacrylamide gel profiles of thylakoid polypeptides from the various treatments disclosed quantitative changes, as well as a new 37/36-kDa polypeptide band in the two highest Mn treatments. The photosynthetic electron transport rates coupled to PSII and PSI showed a significant increase until the 8-mg·L^–1 Mn treatment. The related superoxide production of thylakoids (monitored by EPR spectroscopy) showed minimum values from the 0.5-mg·L^–1 Mn treatment onwards, which, as shown by the thiobarbituric acid reaction was coupled to a non-significant variation of the acyl lipid peroxidation. It was concluded that Oryza sativa L. cv. safari has a high internal tolerance to Mn as it synthesises a new manganese protein that mimics superoxide dismutase functioning.     

Identification of the six ligands to manganese(II) in transition-state-analogue complexes of creatine kinase: oxygen-17 superhyperfine coupling from selectively labeled ligands

The complete coordination scheme for Mn(II) in transition-state-analogue complexes with creatine kinase has been determined by electron paramagnetic resonance (EPR) spectroscopy. Perturbations in the EPR spectra for Mn(II) due to superhyperfine coupling to 17O of selectively labeled ligands have been used to identify oxygen ligands in the first coordination sphere of the metal ion. The results show that in the complex of enzyme-MnADP-formate-creatine, Mn(II) is bound to oxygen ligands from both the alpha- and beta-phosphate groups of ADP, to an oxygen from the carboxylate group of formate, and to three water molecules. In the complex with thiocyanate replacing formate as the stabilizing anion, previous infrared experiments [Reed, G. H., Barlow, C. H., & Burns, R. A., Jr. (1978) J. Biol. Chem. 253, 4153-4158] indicated that the nitrogen from thiocyanate was bound to the Mn(II). The magnitudes of the 17O superphyperfine coupling constants from the O- ligands of the ADP phosphate groups and from the formate carboxylate are approximately equal and are larger than that for the water ligands. The symmetry of the zero-field-splitting tensor for Mn(II) indicates that the oxygens from the alpha- and beta-phosphate groups of ADP and the ligand donor atom from the anion occupy mutually cis positions in the octahedral coordination geometry. Water proton relaxation time measurements show that the three water molecules which are bound to Mn(II) are not in free exchange with the bulk solvent. Hence, an enclosed structure at the active site is indicated. The results suggest that for creatine kinase the activating metal ion is bound to all three phosphate groups in the transition state of the reaction.     

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease

19 F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with K _d values of  ≈ 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with K _d  ≈ 2 mM. Mn(II) paramagnetic broadening in ¹⁹F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease. Received: 30 March 1999 / Accepted: 28 September 1999     

Electron spin-lattice relaxation studies of different forms of the S2 state multiline EPR signal of the Photosystem II oxygen-evolving complex

The pulsed EPR inversion recovery sequence has been utilized to monitor the temperature dependence of the electron spin-lattice relaxation rate of the Mn cluster of the Photosystem II oxygen evolving complex poised in a variety of S ₂ state forms giving rise to g = 2 multiline EPR signals. A previous study (Lorigan and Britt (1994) Biochemistry 33: 12072–12076) showed that for PS II membranes treated with 5% ethanol, the S ₂ state Mn cluster relaxes via the Orbach spin-lattice relaxation mechanism, where the relaxation is enhanced via phonon scattering off an excited state spin manifold, in this case at an energy of Δ = 36.5 cm⁻¹ above the S = 1/2 ground state giving rise to the multiline EPR signal. Parallel experiments are reported for PS II membranes with 5% methanol, treated with ammonia, and following short and long term dark adaptation. In each case, the temperature dependence of the electron spin-lattice relaxation rate is consistent with Orbach relaxation, and the range of excited state energies is relatively narrow (33.8 cm⁻¹ ≤ Δ ≤ 39.7 cm⁻¹). In addition, short term dark adapted (6 min, ‘active state’) PS II membranes show biphasic recovery traces which indicate that a minority fraction of the oxygen evolving complexes are trapped in a form with greatly slowed spin-lattice relaxation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g2 EPR signal of mononuclear Mn(II) with A_av(⁵⁵Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramers doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions.     

Assignment of the g = 4.1 ERP signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: An X-ray absorption edge spectroscopy study

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O₂-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S₂ multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S₂ state.     

In Vitro,In Silico,ADME and Theoretical Analysis of Mn(II) and Co(II) Complexes Derived from Methyl-(Z)-N′-Carbamothioylcarbamohydrazonate Schiff Base Ligand

Mn(II) and Co(II) complexes of methyl-(Z)−N′-carbamothioylcarbamohydrazonate Schiff base ligand were synthesized. The ligand and metal salts were taken in 2 : 1 stoichiometric ratio. All the synthesized complexes were characterized using elemental analysis, molar conductance, magnetic moment and various spectroscopic techniques (FT-IR, UV/VIS, EPR) techniques. Elemental and spectroscopic results verified bidentate donor nature of the ligand and octahedral geometry of all the complexes. The non-electrolytic nature of Mn(II) and Co(II) complexes were suggested by conductivity data analysis. In vitro antibacterial (E. coli and S. aureus) and antifungal (C. albicans and C. tropicalis) screening were achieved by employing agar well diffusion method which revealed better antimicrobial activity of Co(II) complexes than Mn(II) complexes. In silico SwissADME study predicted the drug-likeness probability of ligand and complexes. The interaction of two bacterial proteins (E. coli and S. aureus) with compounds was also analyzed using molecular docking study, which corroborate the in vitro analysis.     

A multifrequency high-field EPR (9–285 GHz) investigation of a series of dichloride mononuclear penta-coordinated Mn(II) complexes

The electronic structure of a series of 11 penta-coordinated dichloride mononuclear Mn(II) complexes [Mn(L)Cl₂] (L = Cl-terpy, Br-terpy, OH-terpy, phenyl-terpy, tolyl-terpy, mesityl-terpy, EtO-terpy, Me₂N-terpy, tBu₃-terpy, py-phen, and dpya) has been investigated by a multifrequency EPR study (9–285 GHz). The X-ray structures of [Mn(Br-terpy)Cl₂], [Mn(EtO-terpy)Cl₂], [Mn(Me₂N-terpy)Cl₂] and [Mn(tolyl-terpy)Cl₂] are described. The spin Hamiltonian parameters have been determined for all complexes and show that the steric and electronic effects of the N-tridentate ligand L do not induce appreciable variations on the zero field splitting parameters. The magnitude of D, close to 0.3 cm⁻¹, is governed by the chloride anion. High-field EPR spectroscopy allows the determination of electronic parameters of mononuclear Mn(II) complexes characterized by relatively large magnitudes of D and the unambiguous interpretation of the X-band spectra of these kinds of complexes.