Water Exchange Kinetics Research Articles

On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation.

Water oxidation by photosystem II (PSII) sustains most life on Earth, but the molecular mechanism of this unique process remains controversial. The ongoing identification of the binding sites and modes of the two water-derived substrate oxygens ('substrate waters') in the various intermediates (Si states, i = 0, 1, 2, 3, 4) that the water-splitting tetra-manganese calcium penta-oxygen (Mn4CaO5) cluster attains during the reaction cycle provides central information towards resolving the unique chemistry of biological water oxidation. Mass spectrometric measurements of single- and double-labeled dioxygen species after various incubation times of PSII with H218O provide insight into the substrate binding modes and sites via determination of exchange rates. Such experiments have revealed that the two substrate waters exchange with different rates that vary independently with the Si state and are hence referred to as the fast (Wf) and the slow (WS) substrate waters. New insight for the molecular interpretation of these rates arises from our recent finding that in the S2 state, under special experimental conditions, two different rates of WS exchange are observed that appear to correlate with the high spin and low spin conformations of the Mn4CaO5 cluster. Here, we reexamine and unite various proposed methods for extracting and assigning rate constants from this recent data set. The analysis results in a molecular model for substrate-water binding and exchange that reconciles the expected non-exchangeability of the central oxo bridge O5 when located between two Mn(IV) ions with the experimental and theoretical assignment of O5 as WS in all S states. The analysis also excludes other published proposals for explaining the water exchange kinetics.

Computational insight into a mechanistic overview of water exchange kinetics and thermodynamic stabilities of bis and tris-aquated complexes of lanthanides.

A thorough investigation of Ln3+ complexes with more than one inner-sphere water molecule is crucial for designing high relaxivity contrast agents (CAs) used in magnetic resonance imaging (MRI). This study accomplished a comparative stability analysis of two hexadentate (H3cbda and H3dpaa) and two heptadentate (H4peada and H3tpaa) ligands with Ln3+ ions. The higher stability of the hexadentate H3cbda and heptadentate H4peada ligands has been confirmed by the binding affinity and Gibbs free energy analysis in aqueous solution. In addition, energy decomposition analysis (EDA) reveals the higher binding affinity of the peada4- ligand than the cbda3- ligand towards Ln3+ ions due to the higher charge density of the peada4- ligand. Moreover, a mechanistic overview of water exchange kinetics has been carried out based on the strength of the metal-water bond. The strength of the metal-water bond follows the trend Gd-O47 (w) > Gd-O39 (w) > Gd-O36 (w) in the case of the tris-aquated [Gd(cbda)(H2O)3] and Gd-O43 (w) > Gd-O40 (w) for the bis-aquated [Gd(peada)(H2O)2]- complex, which was confirmed by bond length, electron density (ρ), and electron localization function (ELF) at the corresponding bond critical points. Our analysis also predicts that the activation energy barrier decreases with the decrease in bond strength; hence k ex increases. The 17O and 1H hyperfine coupling constant values of all the coordinated water molecules were different, calculated by using the second-order Douglas-Kroll-Hess (DKH2) approach. Furthermore, the ionic nature of the bonding in the metal-ligand (M-L) bond was confirmed by the Quantum Theory of Atoms-In-Molecules (QTAIM) and ELF along with energy decomposition analysis (EDA). We hope that the results can be used as a basis for the design of highly efficient Gd(iii)-based high relaxivity MRI contrast agents for medical applications.

Stability, relaxometric and computational studies on Mn2+ complexes with ligands containing a cyclobutane scaffold.

The stability constants of Mn2+ complexes with ligands containing a trans-1,2-cyclobutanediamine spacer functionalized with picolinate and/or carboxylate functions were determined using potentiometric titrations (25 °C, 0.1 M KCl). The stability constant of the complex with a hexadentate ligand containing four acetate groups (L14-, log KMnL = 10.26) is improved upon replacing one (L24-, log KMnL = 14.71) or two (L34-, log KMnL = 15.81) carboxylate groups with picolinates. The [Mn(L1)]2- complex contains a water molecule coordinated to the metal ion in aqueous solutions, as evidenced by 1H NMRD studies and 17O chemical shifts and transverse relaxation rates. The 1H relaxivities determined at 60 MHz (3.3 and 2.4 mM-1 s-1 at 25 and 37 °C, respectively) are comparable to those of monohydrated complexes such as [Mn(edta)]2-. The exchange rate of the inner-sphere water molecule (k = 248 × 106 s-1) is slightly lower than that of the edta4- analogue. DFT calculations (M11/def2-TZVP) suggest that the water exchange reaction follows a dissociatively activated mechanism, providing activation parameters in reasonably good agreement with the experimental data. DFT calculations also show that the 17O hyperfine coupling constant A/ℏ is affected slightly by changes in the Mn-Owater distance and the orientation of the water molecule with respect to the Mn-O vector.

Hydration structure and water exchange kinetics at xenotime-water interfaces: implications for rare earth minerals separation.

Hydration of surface ions gives rise to structural heterogeneity and variable exchange kinetics of water at complex mineral-water interfaces. Here, we employ ab initio molecular dynamics (AIMD) simulations and water adsorption calorimetry to examine the aqueous interfaces of xenotime, a phosphate mineral that contains predominantly Y3+ and heavy rare earth elements. Consistent with natural crystal morphology, xenotime is predicted to have a tetragonal prismatic shape, dominated by the {100} surface. Hydration of this surface induces multilayer interfacial water structures with distinct OH orientations, which agrees with recent crystal truncation rod measurements. The exchange kinetics between two adjacent water layers exhibits a wide range of underlying timescales (5-180 picoseconds), dictated by ion-water electrostatics. Adsorption of a bidentate hydroxamate ligand reveals that {100} xenotime surface can only accommodate monodentate coordination with water exchange kinetics strongly depending on specific ligand orientation, prompting us to reconsider traditional strategies for selective separation of rare-earth minerals.

Reconsidering Calcium Dehydration as the Rate-Determining Step in Calcium Mineral Growth.

The dehydration of cations is generally accepted as the rate-limiting step in many processes. Molecular dynamics (MD) can be used to investigate the dynamics of water molecules around cations, and two different methods exist to obtain trajectory-based water dehydration frequencies. Here, these two different post-processing methods (direct method versus survival function) have been implemented to obtain calcium dehydration frequencies from a series of trajectories obtained using a range of accepted force fields. None of the method combinations reproduced the commonly accepted experimental water exchange frequency of 10–8.2 s–1. Instead, our results suggest much faster water dynamics, comparable with more accurate ab initio MD simulations and with experimental values obtained using neutron scattering techniques. We obtained the best agreement using the survival function method to characterize the water dynamics, and we show that different method combinations significantly affect the outcome. Our work strongly suggests that the fast water exchange kinetics around the calcium ions is not rate-limiting for reactions involving dissolved/solvated calcium. Our results further suggest that, for alkali and most of the earth alkali metals, mechanistic rate laws for growth, dissolution, and adsorption, which are based on the principle of rate-limiting cation dehydration, need careful reconsideration.

Density Functional Theory Studies on the Real and Apparent Water-Exchange Reaction Kinetics of Al3+ in Aqueous Solution

The dissociative (D) mechanistic water-exchange kinetics of aquated Al3+ in aqueous solution is systematically studied using the density functional theory-quantum chemical cluster model (DFT-CM) method. The modeled pathways include dehydration of the hexacoordinated Al(H2O)63+ and successive hydration of the pentacoordinated Al(H2O)53+. For the hydration of Al(H2O)53+, the attacking pathways corresponding to the second-shell solvent water molecules at different sites are investigated. The gas phase-supermolecule-polarizable continuum model (GP-SM-PCM) is used to simulate the explicit and bulk solvation effects. The reactant, transition state, and product geometries of the modeled reaction pathways are optimized at the B3LYP/6-311+G(d,p) level of theory. The real and apparent water-exchange reaction mechanisms of Al3+ are analyzed based on the Gibbs free energy changes for the dehydration and hydration pathways. The possible ligand competition with the solvent water molecules in the formation of aqueous Al...

Differences in the Relaxometric Properties of Regioisomeric Benzyl-DOTA Bifunctional Chelators: Implications for Molecular Imaging.

The bifunctional chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane- N, N', N″, N‴-1,4,7,10-tetraacetate (IB-DOTA) is on paper the most attractive of the commercially available bifunctional chelators for magnetic resonance imaging (MRI) applications. The preserved DOTA scaffold is known to produce extremely kinetically and thermodynamically robust chelates with the Gd3+ ion. Also, ligation through four acetate pendant arms should ensure that the rapid water exchange kinetics so, crucial to the function of an MRI contrast agent are retained. However, upon ligation of the Gd3+ ion, IB-DOTA differentiates into two distinct isomers defined by the positions of the benzylic substituent (corner or side). A relaxometric analysis of these two isomers revealed marked differences in the property and behavior of the two chelates. Most notably the side isomer is found to be substantially more likely to aggregate in aqueous solution than its corner counterpart. This aggregation results in higher relaxivity for the side isomer versus the corner isomer, an observation that potentially obscures the impact of differences in water exchange kinetics between the two isomers. The side isomer is composed of a significant fraction of a twisted square antiprismatic coordination geometry that exchanges water more rapidly than optimal (τM = 7 ns) for maximizing relaxivity. The impact of this excessively fast exchange is not observed in the relaxivity of the side isomer only because in isolation this chelate tumbles much more slowly than the corner isomer. However, this situation is not expected to persist when the chelate is employed in a typical bioconjugate. These results imply that the corner isomer of IB-DOTA may represent a better choice of bifunctional chelator for bioconjugation applications in which a large macromolecule is to be tagged for MRI applications.

NMR shutter-speed elucidates apparent population inversion of 1 H2 O signals due to active transmembrane water cycling.

PurposeThe desire to quantitatively discriminate the extra‐ and intracellular tissue 1H2O MR signals has gone hand‐in‐hand with the continual, historic increase in MRI instrument magnetic field strength [B 0]. However, recent studies have indicated extremely valuable, novel metabolic information can be readily accessible at ultra–low B 0. The two signals can be distinguished, and the homeostatic activity of the cell membrane sodium/potassium pump (Na+,K+,ATPase) detected. The mechanism allowing 1H2O MRI to do this is the newly discovered active transmembrane water cycling (AWC) phenomenon, which we found using paramagnetic extracellular contrast agents at clinical B 0 values. AWC is important because Na+,K+,ATPase can be considered biology’s most vital enzyme, and its in vivo steady‐state activity has not before been measurable, let alone amenable to mapping with high spatial resolution. Recent reports indicate AWC correlates with neuronal firing rate, with malignant tumor metastatic potential, and inversely with cellular reducing equivalent fraction. We wish to systematize the ways AWC can be precisely measured.MethodsWe present a theoretical longitudinal relaxation analysis of considerable scope: it spans the low‐ and high–field situations.ResultsWe show the NMR shutter‐speed organizing principle is pivotal in understanding how trans–membrane steady–state water exchange kinetics are manifest throughout the range. Our findings illuminate an aspect, apparent population inversion, which is crucial in understanding ultra‐low field results.ConclusionsWithout an appreciation of apparent population inversion, significant misinterpretations of future data are likely. These could have unfortunate diagnostic consequences.

Protonation and water exchange kinetics in sandwich polyoxometalates.

Density functional theory is used to explore the locus and consequences of protonation in [Zn4(HO)2(PW9O34)2]10-. The results are used to explain recent observations regarding the contrasting pH effects on the water-ligand exchange in [Mn4(H2O)2(P2W15O56)2]16- and [Co4(H2O)2(P2W15O56)2]16-, and the general effect of protonation on solvent exchange in metal oxides is discussed.

The First State in the Catalytic Cycle of the Water-Oxidizing Enzyme: Identification of a Water-Derived μ-Hydroxo Bridge.

Nature's water-splitting catalyst, an oxygen-bridged tetramanganese calcium (Mn4O5Ca) complex, sequentially activates two substrate water molecules generating molecular O2. Its reaction cycle is composed of five intermediate (Si) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S0. Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of 18O-labeled water incorporation into the product O2 is slow, on a millisecond to second time scale depending on the S state. Here, we demonstrate that the Mn4O5Ca complex poised in the S0 state contains an exchangeable hydroxo bridge. On the basis of a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15561]. This oxygen species is the most probable candidate for the slowly exchanging substrate water in the S0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.

Ultrasensitivity of Water Exchange Kinetics to the Size of Metal Ion.

Metal ions play a vital role in many biological processes. An important factor in these processes is the dynamics of exchange between ion bound-water molecules and the bulk. Although structural and dynamical properties of labile waters bound to metal ions, such as Na+ and Ca2+, can be elucidated using molecular dynamics simulations, direct evaluation of rates of exchange of waters rigidly bound to high charge density Mg2+, has been elusive. Here, we report a universal relationship, allowing us to determine the water exchange time on metal ions as a function of valence and hydration radius. The proposed relationship, which covers times spanning 14 orders of magnitude, highlights the ultrasensitivity of water lifetime to the ion size, as exemplified by divalent ions, Ca2+ (∼100 ps) and Mg2+ (∼1.5 μs). We show that even when structures, characterized by radial distributions are similar, a small difference in hydration radius leads to a qualitatively different (associative or dissociative) mechanism of water exchange. Our work provides a theoretical basis for determination of hydration radius, which is critical for accurately modeling the water dynamics around multivalent ions, and hence in describing all electrostatically driven events such as ribozyme folding and catalysis.

Theoretical reflections on the structural polymorphism of the oxygen-evolving complex in the S2 state and the correlations to substrate water exchange and water oxidation mechanism in photosynthesis

The structural polymorphism of the oxygen-evolving complex is of great significance to photosynthetic water oxidation. Employing density functional theory calculations, we have made further advisement on the interconversion mechanism of O5 transfer in the S2 state, mainly focusing on the potentiality of multi-state reactivity and spin transitions. Then, O5 protonation is proven impossible in S2 for irreversibility of the interconversion, which serves as an auxiliary judgment for the protonation state of O5 in S1. Besides, the structural polymorphism could also be archived by alternative mechanisms involving Mn3 ligand exchange, one of which with Mn3(III) makes sense to substrate water exchange in S2, although being irresponsible for the derivations of the observed EPR signals. During the water exchange, high-spin states would prevail to facilitate electron transfer between the ferromagnetically coupled Mn centers. In addition, water exchange in S1 could account for the closed-cubane structure as the initial form entering S2 at cryogenic temperatures. With regard to water oxidation, the structural flexibility and variability in both S2 and S3 guarantee smooth W2-O5 coupling in S4, according to the substrate assignments from water exchange kinetics. Within this theoretical framework, the new XFEL findings on S1-S3 can be readily rationalized. Finally, an alternative mechanistic scenario for OO bond formation with ·OH radical near O4 is presented, followed by water binding to the pivot Mn4(III) from O4 side during S4-S0. This may diversify the substrate sources combined with the Ca channel in water delivery for the forthcoming S-cycle.

Dependence of Water Exchange Kinetics on the Size and Charge of Metal Ion

While structural and dynamical properties of water on a labile metal ion such as Na+ and Ca2+ are straightforwardly evaluated using molecular dynamics (MD) simulations, direct evaluation of water exchange kinetics on a relatively more inert metal ion, Mg2+, is computationally demanding. Here, we propose a universal relationship which allows us to determine the water exchange time on a metal ion with varying charges and hydration radii. The proposed relationship highlights the ultra-sensitivity of water lifetime to the ion size, as exemplified between two divalent ions, Ca2+ (∼100 ps) and Mg2+ (∼1.5 μs). Importantly, the current MD force fields underestimate the hydration radius of Mg2+ by ∼2.5 %, which results in ∼ 400 fold overestimation of water exchange time. Accurate determination of ion size is critical for correctly modeling the water dynamics around multivalent ions.

The open-cubane oxo-oxyl coupling mechanism dominates photosynthetic oxygen evolution: a comprehensive DFT investigation on O-O bond formation in the S4 state.

The dioxygen formation mechanism of biological water oxidation in nature has long been the focus of argument; many diverse mechanistic hypotheses have been proposed. Based on a recent breakthrough in the resolution of the electronic and structural properties of the oxygen-evolving complex in the S3 state, our density functional theory (DFT) calculations reveal that the open-cubane oxo-oxyl coupling mechanism, whose substrates preferably originate from W2 and O5 in the S2 state, emerges as the best candidate for O-O bond formation in the S4 state. This is justified by the overwhelming energetic superiority of this mechanism over alternative mechanisms in both the isomeric open and closed-cubane forms of the Mn4CaO5 cluster; spin-dependent reactivity rooted in variable magnetic couplings was found to play an essential role. Importantly, this oxygen evolution mechanism is supported by the recent discovery of femtosecond X-ray free electron lasers (XFEL), and the origin of the observed structural changes from the S1 to S3 state has been analyzed. In this view, we corroborate the proposed water binding mechanism during S2-S3 transition and correlate the theoretical models with experimental findings from aspects of substrate selectivity according to water exchange kinetics. This theoretical consequence for native metalloenzymes may serve as a significant guide for improving the design and synthesis of biomimetic materials in the field of photocatalytic water splitting.

Evaluation of Water Exchange Kinetics on [Ln(AAZTAPh-NO2)(H2O)q](x) Complexes Using Proton Nuclear Magnetic Resonance.

Water exchange kinetics on [Ln(AAZTAPh-NO2)(H2O)q](-) (Ln = Gd(3+), Dy(3+), or Tm(3+)) were determined by (1)H nuclear magnetic resonance (NMR) measurements. The number of inner-sphere water molecules was found to change from two to one when going from Dy(3+) to Tm(3+). The calculated water exchange rate constants obtained by variable-temperature proton transverse relaxation rates are 3.9 × 10(6), 0.46 × 10(6), and 0.014 × 10(6) s(-1) at 298 K for Gd(3+), Dy(3+), and Tm(3+), respectively. Variable-pressure measurements were used to assess the water exchange mechanism. The results indicate an associative and dissociative interchange mechanism for Gd(3+) and Dy(3+) complexes with ΔV(⧧) values of -1.4 and 1.9 cm(3) mol(-1), respectively. An associative activation mode (Ia or A mechanism) was obtained for the Tm(3+) complex (ΔV(⧧) = -5.6 cm(3) mol(-1)). Moreover, [Dy(AAZTAPh-NO2)(H2O)2](-) with a very high transverse relaxivity value was found as a potential candidate for negative contrast agents for high-field imaging applications.

Water Exchange on [Ln(DO3A)(H2O)2] and [Ln(DTTA-Me)(H2O)2](-) Studied by Variable Temperature, Pressure, and Magnetic Field NMR.

Water exchange kinetics of [Ln(L)(H2O)2](x) complexes (Ln = Pr, Nd, Dy, Tm, and Yb; L = DO3A and DTTA-Me) were studied by (17)O NMR spectroscopy as a function of temperature, pressure, and frequency and by (1)H nuclear magnetic relaxation dispersion. Water exchange rate constants of both complexes show a maximum at dysprosium. Water exchange on negatively charged complexes of the acyclic DTTA-Me ligand is much faster than on the neutral complexes of the macrocyclic DO3A. Small activation volumes |ΔV(⧧)| < 1 cm(3) mol(-1) measured for water exchange on [Ln(DO3A)(H2O)2] indicate an interchange type of mechanism (I) for the lanthanide complexes studied. In the case of [Ln(DTTA-Me)(H2O)2](-), a change in mechanism is detected from a dissociative mechanism (D, ΔV(⧧) = 7 cm(3) mol(-1)) for complexes with larger ions (Pr to Gd) to an interchange mechanism (Id, I; ΔV(⧧) = +1.8 and +0.4 cm(3) mol(-1)) for complexes with smaller ions (Dy and Tm).

Gd3TCAS2: An Aquated Gd(3+)-Thiacalix[4]arene Sandwich Cluster with Extremely Slow Ligand Substitution Kinetics.

In aqueous solution, Gd3+ and thiacalix[4]arene-p-tetrasulfonate (TCAS) form the complex [Gd3TCAS2]7–, in which a trinuclear Gd3+ core is sandwiched by two TCAS ligands. Acid-catalyzed dissociation reactions, as well as transmetalation and ligand exchange with physiological concentrations of Zn2+ and phosphate, showed [Gd3TCAS2]7– to be extremely inert compared to other Gd complexes. Luminescence lifetime measurements of the Tb analogue Tb3TCAS2 allowed estimation of the mean hydration number q to be 2.4 per Tb ion. The longitudinal relaxivity of [Gd3TCAS2]7– (per Gd3+) was r1 = 5.83 mM–1 s–1 at 20 Hz (37 °C, pH 7.4); however, this relaxivity was limited by an extremely slow water exchange rate that was 5 orders of magnitude slower than the Gd3+ aqua ion. Binding to serum albumin resulted in no relaxivity increase owing to the extremely slow water exchange kinetics. The slow dissociation and water exchange kinetics of [Gd3TCAS2]7– can be attributed to the very rigid coordination geometry.

Isomerism in benzyl-DOTA derived bifunctional chelators: implications for molecular imaging.

The bifunctional chelator IB-DOTA has found use in a range of biomedical applications given its ability to chelate many metal ions, but in particular the lanthanide(III) ions. Gd(3+) in particular is of interest in the development of new molecular imaging agents for MRI and is highly suitable for chelation by IB-DOTA. Given the long-term instability of the aryl isothiocyanate functional group we have used the more stable nitro derivative (NB-DOTA) to conduct a follow-up study of some of our previous work on the coordination chemistry of chelates of these BFCs. Using a combination of NMR and HPLC to study the Eu(3+) and Yb(3+) chelates of NB-DOTA, we have demonstrated that this ligand will produce two discrete regioisomeric chelates at the point at which the metal ion is introduced into the BFC. These regioisomers are defined by the position of the benzylic substituent on the macrocyclic ring: adopting an equatorial position either at the corner or the side of the [3333] ring conformation. These regioisomers are incapable of interconversion and are distinct, separate structures with different SAP/TSAP ratios. The side isomer exhibits an increased population of the TSAP isomer, pointing to more rapid water exchange kinetics in this regioisomer. This has potential ramifications for the use of these two regioisomers of Gd(3+)-BFC chelates in MRI applications. We have also found that, remarkably, there is little or no freedom of rotation about the first single bond extending from the macrocyclic ring to the benzylic substituent. Since this is the linkage through which the chelate is conjugated to the remainder of the molecular imaging probe, this result implies that there may be reduced local rotation of the Gd(3+) chelate within a molecular imaging probe. This implies that this type of BFC could exhibit higher relaxivities than other types of BFC.

Neutron scattering from myelin revisited: bilayer asymmetry and water-exchange kinetics.

Rapid nerve conduction in the central and peripheral nervous systems (CNS and PNS, respectively) of higher vertebrates is brought about by the ensheathment of axons with myelin, a lipid-rich, multilamellar assembly of membranes. The ability of myelin to electrically insulate depends on the regular stacking of these plasma membranes and on the presence of a number of specialized membrane-protein assemblies in the sheath, including the radial component, Schmidt-Lanterman incisures and the axo-glial junctions of the paranodal loops. The disruption of this fine-structure is the basis for many demyelinating neuropathies in the CNS and PNS. Understanding the processes that govern myelin biogenesis, maintenance and destabilization requires knowledge of myelin structure; however, the tight packing of internodal myelin and the complexity of its junctional specializations make myelin a challenging target for comprehensive structural analysis. This paper describes an examination of myelin from the CNS and PNS using neutron diffraction. This investigation revealed the dimensions of the bilayers and aqueous spaces of myelin, asymmetry between the cytoplasmic and extracellular leaflets of the membrane, and the distribution of water and exchangeable hydrogen in internodal multilamellar myelin. It also uncovered differences between CNS and PNS myelin in their water-exchange kinetics.

Structure-redox-relaxivity relationships for redox responsive manganese-based magnetic resonance imaging probes.

A library of 10 Mn-containing complexes capable of switching reversibly between the Mn(II) and Mn(III) oxidation states was prepared and evaluated for potential usage as MRI reporters of tissue redox activity. We synthesized N-(2-hydroxybenzyl)-N,N′,N′-ethylenediaminetriacetic acid (HBET) and N-(2-hydroxybenzyl-N,N′,N′-trans-1,2-cyclohexylenediaminetriacetic acid (CyHBET) ligands functionalized (−H, −OMe, −NO2) at the 5-position of the aromatic ring. The Mn(II) complexes of all ligands and the Mn(III) complexes of the 5-H and 5-NO2 functionalized ligands were synthesized and isolated, but the Mn(III) complexes with the 5-OMe functionalized ligands were unstable. 1H relaxivity of the 10 isolable complexes was measured at pH 7.4 and 37 °C, 1.4 T. Thermodynamic stability, pH-dependent complex speciation, hydration state, water exchange kinetics of the Mn(II) complexes, and pseudo-first order reduction kinetics of the Mn(III) complexes were studied using a combination of pH-potentiometry, UV–vis spectroscopy, and 1H and 17O NMR measurements. The effects of ligand structural and electronic modifications on the Mn(II/III) redox couple were studied by cyclic voltammetry. The Mn(II) complexes are potent relaxation agents as compared to the corresponding Mn(III) species with [MnII(CyHBET)(H2O)]2– exhibiting a 7.5-fold higher relaxivity (3.3 mM–1 s–1) than the oxidized form (0.4 mM–1 s–1). At pH 7.4, Mn(II) exists as a mixture of fully deprotonated (ML) and monoprotonated (HML) complexes and Mn(II) complex stability decreases as the ligands become more electron-releasing (pMn for 10 μM [MnII(CyHBET–R′)(H2O)]2– decreases from 7.6 to 6.2 as R′ goes from −NO2 to −OMe, respectively). HML speciation increases as the electron-releasing nature of the phenolato-O donor increases. The presence of a water coligand is maintained upon conversion from HML to ML, but the water exchange rate of ML is faster by up to 2 orders of magnitude (kex310 for H[MnII(CyHBET)(H2O)]− and [MnII(CyHBET)(H2O)]2– are 1.2 × 108 and 1.0 × 1010 s–1, respectively). The Mn(II/III) redox potential can be tuned over a range of 0.30 V (E1/2 = 0.27–0.57 V) through electronic modifications to the 5-substituent of the aromatic ligand component. However, care must be taken in tuning the ligand electronics to avoid Mn(III)–ligand autoredox. Taken together, these results serve to establish criteria for optimizing Mn(III) versus Mn(II) relaxivity differentials, complex stability, and Mn(II/III) redox potential.