Sun Un

Tuning the redox properties of manganese(II) and its implications to the electrochemistry of manganese and iron superoxide dismutases.

Superoxide dismutases (SODs) catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. The active metal sites of iron and manganese superoxide dismutases are structurally indistinguishable from each other. Despite the structural homology, these enzymes exhibit a high degree of metal selective activity suggesting subtle redox tuning of the active site. The redox tuning model, however, up to now has been challenged by the existence of so-called cambialistic SODs that function with either metal ion. We have prepared and investigated two sets of manganese complexes in which groups of varying electron-withdrawing character, as measured by their Hammett constants sigma Para, have been introduced into the ligands. We observed that the Mn(III)/Mn(II) reduction potential for the series based on 4-X-terpyridine ligands together with the corresponding values for the iron-substituted 4-X-terpyridine complexes changed linearly with sigma Para. The redox potential of the iron and manganese complexes could be varied by as much as 600 mV by the 4-substitution with the manganese complexes being slightly more sensitive to the substitution than iron. The difference was such that in the case where the 4-substituent was a pyrrolidine group both the manganese and the iron complex were thermodynamically competent to catalytically disproportionate superoxide, making this particular ligand cambialistic. Taking our data and those available from the literature together, it was found that in addition to the electron-withdrawing capacity of the 4-substituents the overall charge of the Mn(II) complexes plays a major role in tuning the redox potential, about 600 mV per charge unit. The ion selectivity in Mn and FeSODs and the occurrence of cambialistic SODs are discussed in view of these results. We conclude that the more distant electrostatic contributions may be the source of metal specific enzymatic activity.

ON THE CALCULATION OF G TENSORS OF ORGANIC RADICALS

The results of calculations of doublet state G tensors of organic radicals often show unsatisfactory agreement with the respective experimental data. This led us to reconsider the results of the Rayleigh–Schrodinger perturbation theory approach found in the literature. We came to the conclusion that neither the expressions given for restricted nor for unrestricted Hartree–Fock functions are fully correct. In this paper new expressions for the G tensor to be used with restricted Hartree–Fock functions will be introduced and results of calculations employing these new formulas in conjunction with molecular states from semiempirical Hartree–Fock-type calculations are discussed.

pH-Dependent Structures of the Manganese Binding Sites in Oxalate Decarboxylase as Revealed by High-Field Electron Paramagnetic Resonance

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.

The relationship between the molecular structure of semiquinone radicals and their g-values

Abstract The g -values of semiquinones are analyzed using molecular orbital (MO) theory. MO calculations clearly show the importance of molecular geometry and the local electronic environment, such as hydrogen bonds, on the g -values of semiquinones. In order to assess the magnitude of the effects, g -values are calculated using a modified method of Stone and Angstl. This new method uses the unrestricted Hartree-Fock (UHF) MNDO method with PM3 parameterization, is free of adjustable parameters, but necessarily incorporates several assumptions and approximations relevant to the use of UHF wavefunctions and excited states. For a number of semiquinone radical anions, differences between measured and calculated g -value components were as small as 1–5 × 10 −4 . The effects of hydrogen bonds on g -values and spin densities are calculated. Hydrogen bonding distances obtained from the g -values are found to be in good agreement with ENDOR data.

Understanding the influence of the protein environment on the Mn(II) centers in Superoxide Dismutases using High-Field Electron Paramagnetic Resonance.

One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory activity as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs.

Activation of a unique flavin-dependent tRNA-methylating agent.

TrmFO is a tRNA methyltransferase that uses methylenetetrahydrofolate (CH2THF) and flavin adenine dinucleotide hydroquinone as cofactors. We have recently shown that TrmFO from Bacillus subtilis stabilizes a TrmFO-CH2-FADH adduct and an ill-defined neutral flavin radical. The adduct contains a unique N-CH2-S moiety, with a methylene group bridging N5 of the isoalloxazine ring and the sulfur of an active-site cysteine (Cys53). In the absence of tRNA substrate, this species is remarkably stable but becomes catalytically competent for tRNA methylation following tRNA addition using the methylene group as the source of methyl. Here, we demonstrate that this dormant methylating agent can be activated at low pH, and we propose that this process is triggered upon tRNA addition. The reaction proceeds via protonation of Cys53, cleavage of the C-S bond, and generation of a highly reactive [FADH(N5)═CH2]+ iminium intermediate, which is proposed to be the actual tRNA-methylating agent. This mechanism is fully supported by DFT calculations. The radical present in TrmFO is characterized here by optical and EPR/ENDOR spectroscopy approaches together with DFT calculations and is shown to be the one-electron oxidized product of the TrmFO-CH2-FADH adduct. It is also relatively stable, and its decomposition is facilitated by high pH. These results provide new insights into the structure and reactivity of the unique flavin-dependent methylating agent used by this class of enzymes.

Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, inu2005vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E.u2005coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements.

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.

A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.

RIDME spectroscopy on high-spin Mn2+ centers

Pulsed EPR dipolar spectroscopy is a powerful tool for determining the structure and conformational dynamics of biological macromolecules, as it allows precise measurements of distances in the range of 1.5-10 nm. Utilization of high-spin Mn2+ species as spin probes for distance measurements is of significant interest, because they are biologically compatible and endogenous in numerous biological systems. However, to date dipolar spectroscopy experiments with this kind of species have been underexplored. Here we present pulsed electron electron double resonance (PELDOR also called DEER) and relaxation-induced dipolar modulation enhancement (RIDME) experiments, which have been performed at W-band (94 GHz) and J-band frequencies (263 GHz) on a bis-MnDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) model system. The distances obtained from these experiments are in good agreement with predictions. RIDME experiments reveal a significantly higher modulation depth compared to PELDOR, which is an important consideration for biological samples. These experiments also feature higher harmonics of the dipolar coupling frequency due to effective multiple-quantum relaxation of high-spin Mn2+ as well as the multiple-component background function. Harmonics of the dipolar coupling frequency were taken into account by including additional terms in the kernel function of Tikhonov regularization analysis.

How Bonding in Manganous Phosphates Affects their Mn(II)–31P Hyperfine Interactions

Manganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn(II) electron-nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn(II)-(31)P hyperfine interactions of various Mn(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese-phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ (31)P ENDOR spectra of cellular Mn(II) complexes.