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The structure of the DNA duplex dodecamer, d(CCTCTGGTCTCC. GGAGACCAGAGG), containing the cisplatin d(GpG) 1,2-intrastrand cross-link at the position denoted by asterisks, was determined in solution by high-resolution 2D NMR spectroscopy and restrained molecular dynamics refinement. The cis-[Pt(NH3)2¿d(GpG-N7(1), N7(2))¿] lesion causes the adjacent guanine bases to roll toward one another by 49 degrees, leading to an overall helix bend angle of 78 degrees. These features are more exaggerated than those observed in the X-ray crystal structure determined for the same platinated duplex [Takahara et al. (1995) Nature 377, 649-652]. A common property of the solution and crystal structures is the widening and flattening of the minor groove opposite the platinum adduct, affording geometric parameters resembling those found in A-form DNA. This deformation is especially noteworthy for the solution structure because its sugar puckers are primarily those of B-form DNA. The unwinding of the helix at the site of platination is 25 degrees. The curvature and shape of the platinated duplex are remarkably similar to those observed in DNA duplexes complexed by the HMG-domain proteins SRY and LEF-1. The structure reveals how cisplatin binding alters DNA in such a manner as to facilitate HMG-domain protein recognition.

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Interactions of water and methanol with a mixed valence Mn(III)Mn(IV) complex are explored with 1H electron spin echo (ESE)-electron nuclear double resonance (ENDOR) and 1H and 2H ESE envelope modulation (ESEEM). Derivatives of the (2-OH-3,5-Cl2-SALPN)2 Mn(III)Mn(IV) complex are ideal for structural and spectroscopic modeling of water binding to multinuclear Mn complexes in metalloproteins, specifically photosystem II (PSII) and manganese catalase (MnCat). Using ESE-ENDOR and ESEEM techniques, 1H hyperfine parameters are determined for both water and methanol ligated to the Mn(III) ion of the complex. The protons of water directly bound to Mn(III) are inequivalent and exhibit roughly axial dipolar hyperfine interactions (T dip = 8.4 MHz and T dip = 7.4 MHz), permitting orientations and radial distances to be determined using a model where the proton experiences a point dipole interaction with each Mn ion. General equations are given for the components of the rhombic dipolar hyperfine interaction between a proton and a spin coupled dinuclear metal cluster. The observed ENDOR pattern is from water protons 2.65 and 2.74 Å from the Mn(III) which make an Mn(IV)−Mn(III)−H angle of ∼160°. For the alcohol proton in the analogous methanol bound complex, a 2.65 Å Mn(III)−H distance is observed. Three pulse 2H ESEEM gives best fit Mn(III)−2H(1H) radial distances of 3.0, 3.5, and 4.0 Å for the three methyl deuterons in this complex.

The series of complexes [Mn(2)(2-OH(Xsal)pn)(2)](2-,-,0,+) [where 2-OH(Xsal)pn represents substituted-phenyl-ring derivatives (X = H, 5-Cl, 3,5-Cl(2), 5-NO(2)) of 1,3-bis(salicylideneamino)-2-propanol] allow for the first detailed structural, magnetic, and spectroscopic study of a series of complexes that are the most active functional models for the manganese catalases. Representative examples of each oxidation state of the series (mimicking all of the known oxidation states for the enzyme's reaction chemistry) have been crystallographically characterized. The molecules presented herein are described as symmetric derivatives because they form dimers with both of the ligands spanning both Mn ions with the alkoxide on the backbone of the ligand bridging the metals. The variation in Mn-Mn separation across the four structures is 0.11 Å [Mn(II)-Mn(II) = 3.33 Å; Mn(II)-Mn(III) = 3.25 Å; Mn(III)-Mn(III) = 3.36 Å; Mn(III)-Mn(IV) = 3.25 Å], showing that the basic core structure is highly invariant. Nonetheless, significant structural changes in the polyhedra of each manganese ion can be observed across the range of metal oxidation states. These symmetric structures are distinct from the previously described asymmetric {[Mn(2)(2-OH(Xsal)pn)(2)](sol)}(0,+) structures which have only one bridging alkoxide and one monodentate solvent bound to the Mn(III) ion. These two forms (symmetric and asymmetric) are reminiscent of the carboxylate shift in metal carboxylate chemistry and illustrate how alkoxide ligands can participitate in an analogous alkoxide shift in order to generate a binding site for an incoming ligand, such as methanol, or substrate, such as hydrogen peroxide. This is the first series that allows the observation of the effect of subtle changes in geometry on the sign if not the magnitude of magnetic exchange in dimeric systems across a range of oxidation states. Regardless of the symmetric or asymmetric nature of the complex, the exchange parameter J was found to be very low; however, both ferro- and antiferromagnetic exchange can be realized with these dimers.

Indoxyl sulfate is a protein metabolite that is concentrated in the serum of patients with chronic renal insufficiency. It also is a uremic toxin that has been implicated in the progression of chronic renal disease in rodent models. We have shown previously that mesangial cell redox status is related to activation of mitogen-activated protein kinases and cell proliferation, which are factors related to glomerular damage. We used three methods to examine the ability of indoxyl sulfate to alter mesangial cell redox as a possible mechanism for its toxicity. Indoxyl sulfate increases mesangial cell reduction rate in a concentration-dependent manner as demonstrated by redox microphysiometry. Alterations occurred at concentrations as low as 100 M, with more marked alterations occurring at higher concentrations associated with human renal failure. We demonstrated that indoxyl sulfate induces the production of intracellular reactive oxygen species (ROS) in mesangial cells (EC50 ϭ 550 M) by using the ROS-sensitive fluorescent dye CM-DCF. ROS generation was only partially (ϳ50%) inhibited by the NADPH oxidase inhibitor diphenylene iodinium at low (Յ300 M) indoxyl sulfate concentrations. Diphenylene iodinium was without effect at higher concentrations of indoxyl sulfate. We also used electron paramagnetic spin resonance spectroscopy with extracellular and intracellular spin traps to show that indoxyl sulfate increases extracellular SOD-sensitive O 2

The complexes [Mn(2-OH(X-sal)pn)] 2 n-(where X ) 5-OCH 3 , H, 5-Cl, 3,5-diCl, or 5-NO 2 and where n ) 0 or 2) are shown to be excellent hydrogen peroxide disproportionation catalysts in acetonitrile. When carried out in an open vessel, the reaction can occur for over 5000 turnovers without an indication of catalyst decomposition. The disproportionation reaction cycles between the [Mn III (2-OH(X-sal)pn)] 2 and the [Mn II (2-OH(X-sal)pn)] 2 2oxidation levels. All derivatives show saturation kinetics with the highest k cat (21.9 ( 0.2 s -1 ) observed for the [Mn III (2-OH(5-Clsal)pn)] 2 dimer and the optimal k cat /K M (990 ( 60 s -1 ‚M -1 ) observed for the [Mn III (2-OHsalpn)] 2 . The first step of the reaction is proposed to be the binding of peroxide to the [Mn III (2-OH(X-sal)pn)] 2 through an alkoxide shift to form a ternary intermediate {[Mn III (2-OH(X-sal)pn)] 2 (H 2 O 2 )}. We propose that the turnoverlimiting step is the oxidation of peroxide from this intermediate. The binding efficiency of the peroxide is dependent on the phenyl-ring substitution with the derivatives donating the most electrons having the highest affinity for the substrate. Studies with isotopically labeled H 2 O 2 indicate that protons are important in the turnover-limiting step of the reaction and that the O-O bond is not cleaved during peroxide oxidation. In a closed vessel, the product dioxygen will oxidize [Mn II (2-OH(5-NO 2 sal)pn)] 2 2to [Mn II/III (2-OH(5-NO 2 sal)pn) 2 ] -, and this species can then be stoichiometrically oxidized by hydrogen peroxide to give [Mn III/IV (2-OH(5-NO 2 sal)pn) 2 (µ 2 -O) 2 ] -. This diµ 2 -oxobridged species is catalytically incompetent; however, addition of hydroxylamine hydrochloride restores catalytic activity. The relationship of this catalytic disproportionation of hydrogen peroxide and inactivation of the catalyst will be used to define a model for similar reactions observed for the Lactobacillus plantarum Mn catalase.Similar to the heme catalases, the manganese catalases disproportionate hydrogen peroxide according to eq 1.These manganese enzymes have been isolated from three different bacteria: Lactobacillus plantarum, 2,3 Thermus thermophilus, 4 and Thermoleophilium album. 5 Through activity studies, 5-7 spectroscopy, 8-11 and X-ray crystallographic structural analysis, 12-14 all three enzymes appear to be very similar.The dinuclear manganese center probably cycles between the Mn II 2 T Mn III 2 oxidation levels during the normal catalytic cycle with at least two proposed mechanisms for the catalytic disproportionation of hydrogen peroxide by the Mn catalases having been presented. 7 Evidence for this cycle has been shown by the identical steady-state kinetics for the oxidized (Mn III 2 ) and reduced (Mn II 2 ) forms of T. thermophilus 6 and by observation of changes in XANES spectra during turnover of the L. plantarum enzyme. 15 Both the L. plantarum 7 and T. thermophilus 6 enzymes exhibit substrate saturation kinetics and show no inhibition by hydrogen peroxide even at high substrate c...

Manganese catalases use a dinuclear active site to catalyze the disproportionation of hydrogen peroxide to dioxygen and water. A low-resolution structureof the Thermus thermophilus enzyme demonstrates that two manganese ions are in close proximity (~3 . 6